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Inactivation effect of newly developed low concentration electrolyzed water
and other sanitizers against microorganisms on spinach
S.M.E. Rahman, Tian Ding, Deog-Hwan Oh
*
Department of Food Science and Biotechnology and Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea
article info
Article history:
Received 22 September 2009
Received in revised form 23 March 2010
Accepted 30 March 2010
Keywords:
Low concentration electrolyzed water
Spinach
Natural microflora
Listeria monocytogenes
Escherichia coli O157:H7
abstract
The efficacy of newly developed low concentration electrolyzed water (LcEW) was investigated to inacti-
vate the pathogens on spinach leaves as a convenient and safe alternative sanitizer and it was compared to
other sanitizers. Spinach leaves were inoculated with Escherichia coli O157:H7 and Listeria monocytogenes
and dip treated with deionized water (DIW), LcEW, strong acid electrolyzed water (SAEW), aqueous ozone
(AO), 1% citric acid (CA) and sodium hypochlorite solution (NaOCl) for 3 min at room temperature
(23 ± 2 °C). For all pathogens, the similar pattern of microbial reduction on spinach was apparent with
LcEW and SAEW washing. In the present study, it was found that LcEW inactivated, at maximum, 1.64–
2.80 log cfu/g and DIW resulted in lowest reduction, 0.31–0.95 log cfu/g of background or pathogenic
microflora present on spinach leaves compared to the unwashed control. The findings of this study indi-
cate that LcEW and SAEW did not differ significantly (P> 0.05) in reducing background or pathogenic
microflora on spinach and LcEW may be a promising sanitizer for washing vegetables without environ-
mental pollution instead of using electrolyzed oxidizing (EO) water or SAEW.
Ó2010 Elsevier Ltd. All rights reserved.
1. Introduction
Spinach has been consumed throughout the world for many
years and is an excellent source of iron, calcium, chlorophyll, beta
carotene (needed for the production of vitamin A), vitamin C, ribo-
flavin, sodium and potassium. Fresh produce is an important part
of a healthy diet for people around the world, but eating fresh un-
cooked produce is not risk free. During the last three decades, the
number of outbreaks caused by foodborne pathogens associated
with fresh produce consumption reported to the centers for dis-
ease control and prevention has increased. Fresh-cut vegetables
are highly susceptible to microbial spoilage during processing
procedures that require the use of shredders and slicers, which
may be principal sites of microbial contamination (Garg, Churey,
& Splittstoesser, 1990). The use of shredders and slicers cause the
inner tissues to be exposed to microbial contamination during
cutting (Brackett, 1987).
Sanitization of produce plays an important role in the preserva-
tion of food quality and safety of consumption. Washing produce
with tap water cannot be relied upon to completely remove patho-
genic and naturally occurring bacteria (Brackett, 1992; Nguyen-
The & Carlin, 1994). Chemical compounds such as sodium
hypochlorite (Adams, Hartley, & Cox, 1989; Bolin, Stafford, King, &
Huxsoll, 1977; Zhang & Farber, 1996), chlorine dioxide (Kim, Kim,
& Song, 2009; Zhang & Farber, 1996), sodium bisulfite (Krahn,
1977), sulfur dioxide (Bolin et al., 1977), organic acids (Adams
et al., 1989; Zhang & Farber, 1996), calcium chloride (Izumi &
Watada, 1994, 1995), acidified sodium chlorite (Allende, McEvoy,
Tao, & Luo, 2009; Liao, 2009) and ozone (Nagashima & Kamoi,
1997) have been shown to reduce microbial populations on fresh-
cut vegetables. Moreover, most of these sanitizers are made from
the dilution of condensed solutions, which in handling involves
some risk and is troublesome. A sanitizer that is not produced from
the dilution of a hazardous condensed solution is required for
practical use.
Strong acid electrolyzed water (SAEW), also named electrolyzed
oxidizing (EO) water, is one of the potential alternatives for envi-
ronmentally friendly broad spectrum microbial decontamination
that has been proven to exhibit strong bactericidal activity for
inactivating many pathogens (Fabrizio & Cutter, 2005; Kim, Hung,
& Brackett, 2000; Park, Hung, & Brackett, 2002; Venkitanarayanan,
Ezeike, Hung, & Doyle, 1999). Several studies have demonstrated
that SAEW could be used as a disinfectant in food processing
(Fabrizio & Cutter, 2004; Huang et al., 2006; Huang, Hung, Hsu,
Huang, & Hwang, 2008). However, the potential application of
SAEW is limited because of its lower pH values (62.7). At this
low pH, dissolved Cl
2
gas can be rapidly lost due to volatilization,
decreasing the bactericidal activity of the solution with time
(Len, Hung, Erickson, & Kim, 2000) and adversely affecting human
health and the environment. Moreover, the high acidity of SAEW
may cause the corrosion of equipment and consequently limit its
0956-7135/$ - see front matter Ó2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2010.03.011
*Corresponding author. Tel.: +82 33 250 6457; fax: +82 33 241 0508.
E-mail address: deoghwa@kangwon.ac.kr (D.-H. Oh).
Food Control 21 (2010) 1383–1387
Contents lists available at ScienceDirect
Food Control
journal homepage: www.elsevier.com/locate/foodcont
Author's personal copy
practical application. Our newly developed low concentration elec-
trolyzed water (LcEW) with a pH value of 6.2–6.5 (nearly neutral)
which contains low concentration free chlorine (2–5 mg/L) is pro-
duced by electrolysis of a dilute NaCl solution (0.9%) in a chamber
without a membrane. At a pH of 6.0–6.5, the effective form of chlo-
rine compounds in LcEW is almost the hypochlorous acid (HOCl)
having strong antimicrobial activity (Cao, Zhu, Shi, Wang, & Li,
2009; Ding, Rahman, Purev, & Oh, 2010; Yoshifumi, 2003). The
application of LcEW may improve the bactericidal activity by max-
imizing the use of hypochlorous acid, reducing corrosion of sur-
faces, and minimizing human health and safety issues from Cl
2
off-gassing (Guentzel, Lam, Callan, Emmons, & Dunham, 2008).
The objective of this study was to investigate the decontamination
efficacy of LcEW against natural microflora, Listeria monocytogenes
and Escherichia coli O157:H7 on spinach leaves in comparison with
other commercial sanitizers.
2. Materials and methods
2.1. Sample preparation
Spinach was purchased from a local supermarket in Chuncheon,
South Korea. The stems and defected leaves were discarded. Fresh
spinach leaves were then trimmed to approximately 3 3cm in
size and packed in a polyethylene bag and stored at 4 °C to use
for the experiment within 24 h.
2.2. Bacterial cultures
The three strains each of E. coli O157:H7 (B0259, B0297 and
B0299) and L. monocytogenes (ATCC 19115, ATCC 19111 and Scott
A) used in this experiment were obtained from Department of Food
Science, University of Georgia (Griffin, GA, USA), and Health Re-
search Department (Gyeonggi-do, Republic of Korea), respectively.
Stock cultures of each pathogen were transferred into tryptic soy
broth (TSB) and incubated for 24 h at 35 °C. Following incubation,
10 mL of each culture was sedimented by centrifugation (3000gfor
10 min at 4 °C), washed and resuspended in 10 mL of 0.1% peptone
water (pH 7.1) to obtain a final cell concentration of 10
9
cfu/mL.
Subsequently, suspended pellets of each strain of the two patho-
gens were combined to construct culture cocktails. These culture
cocktails were used in subsequent experiments. The bacterial pop-
ulation in each cocktail culture was confirmed by plating 0.1 mL
portions of appropriately diluted culture on tryptic soy agar
(TSA) (Difco Laboratories) plates and incubating the plates at
35 °C for 24 h.
2.3. Inoculation
Each trimmed leaf was placed on sterile aluminum foil in a bio-
safety hood. For inoculation, 0.1 mL of each pathogen cocktail
(10
9
cfu/mL) was applied to the abaxial-side of each leaf surface
by depositing droplets at 20 locations with a micropipettor fol-
lowed by drying in a laminar flow hood for 30 min at room temper-
ature (23 ± 2 °C) to allow for bacterial attachment to the leaf
surface. This procedure resulted in initial pathogen inocula levels
of approximately 6–7 log cfu/g.
2.4. Preparation of treatment solutions
Low concentration electrolyzed water (LcEW), also named as
slightly acidic electrolyzed water, with a pH of 6.2, ORP of 500–
520 mV and available chlorine concentration of 5 mg/L used in this
study was produced by electrolysis of a dilute NaCl solution (0.9%)
in a chamber without a membrane using a electrolysis device
(model D-7, sl No. 001171, Dolki Co., Ltd., Wonju, Korea) at a set-
ting of 3 V and 1.15 A. For a comparison test with LcEW, SAEW
with a pH of 2.54, ORP of 1100–1120 mV was generated using
EO generator (A2-1000, Korean E & S Fist Inc., Seoul, Korea) includ-
ing a small amount of salt solution and tap water at a setting of
12 A. with a residual chlorine concentration of about 50 mg/L.
The sodium hypochlorite solution (NaOCl: pH 9.8, 100 mg/L avail-
able chlorine) was prepared with the addition of 0.1 g of NaOCl (DC
Chemical Co., Seoul, Korea) in 1 L of sterile distilled water. The
crystalline citric acid (Yakuri Pure Chemicals Co., Kyoto, Japan)
was dissolved in 1 L of sterile distilled water to obtain a final con-
centration of 1% CA solution (w/v). Aqueous ozone (5 ppm) was
produced on site by an electrochemical process using a green
water ozone generator (GW-1000, Youl chon, Korea). Sterile deion-
ized water (DIW) was used as control. The pH, ORP and available
chlorine concentration of treatment solutions (LCEW and AEW)
were measured immediately before treatment with a dual-scale
pH meter (Accumet model 15, Fisher Scientific Co., Fair Lawn, NJ)
with pH and ORP electrodes. The residual chlorine was determined
by a colorimetric method using a digital chlorine test kit (RC-3F,
Kasahara Chemical Instruments Co., Saitama, Japan). The detection
limit is 0–300 mg/L.
2.5. Dip wash treatments and microbiological analysis of leaf samples
Inoculated and uninoculated spinach samples (10 g) were
placed in sterile containers and immersed in treatment solutions
(DIW, LcEW, SAEW, AO, 1% CA and NaOCl) at room temperature
(23 ± 2 °C). Unwashed spinach leaves were used as control. To eval-
uate the effect of dipping time on the reduction of microorganisms,
each 10 g piece of uninoculated spinach leaves was dipped for 0.5,
1, 3, 5, and 7 min, respectively. Following treatments, all samples
were aseptically excised and immediately placed in a stomacher
bag (Nasco Whirl-Pak, Janesville, WI, USA) containing 90 mL of
BPW and homogenized for 2 min with a Seward stomacher (400
Circulator, Seward, London, UK). After homogenization, 1 mL ali-
quots of the sample were serially diluted in 9 mL of sterile buffered
peptone water and 0.1 mL of sample or diluent was spread-plated
onto each selective medium. Total bacterial counts were deter-
mined by plating appropriately diluted samples onto tryptic soy
agar (TSA, Difco Co.). Yeasts and molds were plated on potato dex-
trose agar (PDA, Difco Co.) Two selective media of Sorbitol Mac-
Conkey agar (SMAC; Difco), and oxford agar base (OAB; Difco)
were used for the enumeration of E. coli O157:H7, and L. monocyt-
ogenes, respectively. All plates were incubated at 37 °C for 24 h, ex-
cept for yeasts and molds at 25 °C for 48 h and expressed as
log cfu/g. All independent trials were replicated three times.
2.6. Statistical analysis
Means of bacterial populations (log
10
cfu/g) from each treat-
ment were calculated from three replications for each experiment.
Data was expressed as the means ± standard errors. The results
were analyzed using SPSS statistical package (SPSS Inc., Chicago,
IL) and the significance of difference was defined at P< 0.05.
3. Results and discussion
The properties (pH, ORP, and residual chlorine) of treatment
solutions (LcEW, SAEW, NaOCl, AO, 1% CA, and DIW) used in this
study are presented in Table 1. The effect of dipping times (0.5,
1, 3, 5, and 7 min) on the reduction of total counts on spinach
leaves treated with DIW, LcEW, and SAEW are shown in Fig. 1.
From these results, treatments exposed for 3 min showed signifi-
cant difference (P< 0.05) in reducing total bacteria from the
1384 S.M.E. Rahman et al. / Food Control 21 (2010) 1383–1387
Author's personal copy
dipping times of 0.5 and 1 min, but there was no significant differ-
ence (P> 0.05) from the dipping times of 5 and 7 min. Thus, the
3 min dipping time was chosen for the following treatments which
were done at room temperature. After sanitizing treatments in
3 min dipping, the populations of total aerobic bacteria in the spin-
ach leaves were 5.55, 5.03, 3.62, 3.61, 4.48, 4.16 and 3.94 log cfu/g
for the control, DIW, LcEW, SAEW, AO, 1% CA and NaOCl treat-
ments, respectively (Fig. 2). The greatest reduction was achieved
with LcEW and SAEW. The LcEW and SAEW treatment reduced
the microbial load in the spinach leaves by 1.93 and 1.94 log cfu/
g, respectively, compared to the unwashed control, whereas wash-
ing with DIW produced the lowest reduction by 0.52 log cfu/g.
Yeasts and molds had a more or less similar pattern to that of total
aerobic bacteria (Fig. 3). Initial populations of yeasts and molds in
the spinach leaves were 4.05, 3.74, 2.41, 2.48, 3.17, 3.00 and
2.67 log cfu/g for the control, DIW, LcEW, SAEW, AO, 1% CA and
NaOCl treatments, respectively. The LcEW treatment caused the
most effective reduction of yeasts and molds in the spinach leaves
by 1.64 log cfu/g compared to the unwashed control, whereas
washing with DIW only showed a reduction of 0.31 log cfu/g. These
results demonstrate that LcEW is an effective sanitizer for micro-
bial decontamination. Similar work was done by Koide, Takeda,
Shi, Shono, & Atungulu, 2009; they found that slightly acidic elec-
trolyzed water (SlAEW, pH 6.1, 20 mg/L available chlorine) reduced
about by 1.5 log cfu/g for total aerobic bacteria and 1.3 log cfu/g for
molds and yeasts, compared to fresh cut cabbage before dipping.
Spinach leaves were inoculated with approximately 6–7 log cfu/
gofE. coli O157:H7 and L. monocytogenes and treated with DIW,
LcEW, SAEW, AO, 1% CA and NaOCl. Sanitizing treatment of the
inoculated spinach leaves significantly reduced the microbial pop-
ulations. After treatment, the initial populations of E. coli O157:H7
in the spinach leaves were 6.77, 5.97, 4.37, 4.47, 5.55, 5.27 and
4.76 log cfu/g for the control, DIW, LcEW, SAEW, AO, 1% CA and
NaOCl treatments, respectively (Fig. 4). In fact, the LcEW showed
the greatest reduction of the population of E. coli O157:H7 by
2.40 log cfu/g compared to the unwashed control, whereas wash-
ing with DIW produced the lowest reduction by 0.80 log cfu/g.
Table 1
Physicochemical properties of treatment solutions.
Treatment solutions pH ORP (mV) ACC
a
(mg/L)
LcEW
b
6.3 ± 0.2 520 ± 20 5 ± 0.1
SAEW
c
2.54 ± 0.3 1130 ± 20 50 ± 2.2
NaOCl
d
10.6 ± 0.2 630 ± 15 100 ± 4.5
AO
e
6.6 ± 0.1 1245 ± 25 5.2 ± 0.2 mg O
3
/L of water
1% CA
f
2.6 ± 0.2 1120 ± 10 Not measured
DIW
g
7.0 ± 0.1 412 ± 18 0.3 ± 0.1
a
Available chlorine concentration.
b
Low concentration electrolyzed water.
c
Strong acid electrolyzed water.
d
Sodium hypochlorite solution.
e
Aqueous ozone.
f
Citric acid.
g
Deionized water.
c
c
c
b
ab
a
c
c
c
b
b
a
c
c
c
b
b
a
0
1
2
3
4
5
6
7
0 0.5 1 3 5 7
Di
pp
in
g
time (min)
log cfu g-1
DIW LcEW SAEW
Fig. 1. Surviving population of total bacteria on fresh-cut spinach after washing
with deionized water (DIW), low concentration electrolyzed water (LcEW) and
strong acid electrolyzed water (SAEW) at different dipping times (min). Vertical
bars represent means of three replications ±SE. Bars labeled with different letters
indicate significant difference at P< 0.05.
Total bacteria
de
cd
c
ee
b
a
0
1
2
3
4
5
6
Control DIW LcEW SAEW AO 1% CA NaOCl
Treatments
log cfu g-1
Fig. 2. Surviving population of total bacteria on fresh-cut spinach after washing
with deionized water (DIW), low concentration electrolyzed water (LcEW), strong
acid electrolyzed water (SAEW), aqueous ozone (AO), 1% citric acid (CA) and sodium
hypochlorite (NaOCl), relative to an unwashed control. Vertical bars represent
means of three replications ±SE. Bars labeled with different letters indicate
significant difference at P< 0.05.
Yeasts and moulds
cd
cd
bc
dd
ab
a
0
1
2
3
4
5
6
Control DIW LcEW SAEW AO 1% CA NaOCl
Treatments
log cfu g-1
Fig. 3. Surviving population of yeasts and molds on fresh-cut spinach after washing
with deionized water (DIW), low concentration electrolyzed water (LcEW), strong
acid electrolyzed water (SAEW), aqueous ozone (AO), 1% citric acid (CA) and sodium
hypochlorite (NaOCl), relative to an unwashed control. Vertical bars represent
means of three replications ±SE. Bars labeled with different letters indicate
significant difference at P< 0.05.
E. coli O157:H7
d
c
bc
d
d
b
a
0
1
2
3
4
5
6
7
8
Control DIW LcEW SAEW AO 1% CA NaOCl
Treatments
log cfu g-1
Fig. 4. Surviving population of E. coli O157:H7 on fresh-cut spinach after washing
with deionized water (DIW), low concentration electrolyzed water (LcEW), strong
acid electrolyzed water (SAEW), aqueous ozone (AO), 1% citric acid (CA) and sodium
hypochlorite (NaOCl), relative to an unwashed control. Vertical bars represent
means of three replications ±SE. Bars labeled with different letters indicate
significant difference at P< 0.05.
S.M.E. Rahman et al. / Food Control 21 (2010) 1383–1387 1385
Author's personal copy
However, L. monocytogenes had a similar pattern to E. coli O157:H7.
After treatment, the populations of L. monocytogenes in spinach
leaves were 6.96, 6.01, 4.16, 4.26, 5.56, 5.26 and 4.76 log cfu/g for
the control, DIW, LcEW, SAEW, AO, 1% CA and NaOCl treatments,
respectively (Fig. 5). LcEW significantly reduced the populations
of L. monocytogenes by 2.80 log cfu/g compared to the unwashed
control, while washing with DIW reduced the populations by
0.95 log cfu/g. Guentzel et al. (2008) reported a similar study in
which they also used dip (10 min) treatment of spinach leaves in
neutral electrolyzed water (NEW) (pH: 6.3–6.5; ORP: 800–
900 mV and residual chlorine of 100–120 mg/L) resulted in a
reduction of 4.0–5.0 log cfu/mL for E. coli,Salmonella typhimurium,
Staphylococcus aureus,L. monocytogenes and Enterococcus faecalis.
In contrast, Yang, Swem, and Li (2003) found a 2.0 log cfu/g reduc-
tion in S. typhimurium,E. coli and L. monocytogenes on the surfaces
of romaine lettuce after a 5 min dip in neutral EO water (pH 7;
300 mg/L residual chlorine). Park, Alexander, Taylor, Costa, and
Kang (2008) also reported that after 3 min of AcEW treatment,
E. coli O157:H7 was reduced by more than 3.50 log cfu/g on spin-
ach. In our experiment, populations of E. coli O157:H7 and L. mon-
ocytogenes on spinach was reduced by 1.22 and 1.40 log cfu/g for
5 ppm AO; 1.5 and 1.70 log cfu/g for 1% CA treatment, respectively.
On the other hand, Yuk et al. (2006) demonstrated that treatment
with 3 ppm ozone combined with 1% citric acid for a 1 min immer-
sion resulted in 2.31 and 1.84 log cfu/g reductions on lettuce for
E. coli O157:H7 and L. monocytogenes, respectively.
Non-thermal processing is a means of enhancing food safety
without compromising quality of food products. Recently the use
of ozone gas (Klockow & Keener, 2009), X-ray (Mahmoud, Bach-
man, & Linton, 2010), and chemical sanitizer combined with mod-
ified atmosphere packaging (Lee & Baek, 2008) have been reported
as non-thermal technology those have shown promise for reducing
pathogenic and spoilage bacteria on spinach leaves. Numerous
sanitizers have been examined for their effectiveness in killing or
removing pathogenic bacteria on fresh produce, such as E. coli
O157:H7, Salmonella spp. and L. monocytogenes (Beuchat, 1998).
Zhang and Farber (1996) examined the effects of various disinfec-
tants (chlorine, chlorine dioxide, trisodium phosphate, Salmide
Ò
,
organic acids) against L. monocytogenes on fresh-cut vegetables at
4 and 22 °C with a 10 min exposure and observed an average
reduction of 0.2–1.8 log cfu/g. Although many studies on disinfec-
tion efficacy of sodium hypochlorite solution and AcEW on vegeta-
bles were reported and reviewed (Al-Haq, Sugiyama, & Isobe, 2005;
Hricova, Stephan, & Zweifel, 2008; Huang et al., 2008; Koseki, Yos-
hida, Isobe, & Itoh, 2001), few researches have been conducted on
disinfection efficacy of SlAEW (Koide et al., 2009) and of NEW
(Abadias, Usall, Oliveira, Alegre, & Viñas, 2008; Guentzel et al.,
2008; Gómez-López et al., 2007) on vegetables and no report has
been published yet on the use of LcEW.
In conclusion, LcEW (pH 6.2–6.5, 5 mg/L available chlorine)
seems to be a promising and environmentally-friendly non-ther-
mal disinfection method as it would allow reduction of the amount
of free chlorine used for the disinfection of fresh-cut produce by
the food industry at the same or a little more than microbial reduc-
tion levels obtained with the use of SAEW (pH 2.5–2.7, 50 mg/L
available chlorine). Future studies should be elucidated with differ-
ent contact time to simulate typical commercial conditions in addi-
tion to other types of fruits, vegetables and microbial pathogens.
Acknowledgement
This study was financially supported by Kangwon BIO-NURI,
South Korea.
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L. monocytogenes
d
c
c
e
e
b
a
0
1
2
3
4
5
6
7
8
Control DIW LcEW SAEW AO 1% CA NaOCl
Treatments
log cfu g-1
Fig. 5. Surviving population of L. monocytogenes on fresh-cut spinach after washing
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