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Combination treatment of alkaline electrolyzed water and citric acid with mild heat to ensure microbial safety, shelf-life and sensory quality of shredded carrots

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The objective of this study was to determine the synergistic effect of alkaline electrolyzed water and citric acid with mild heat against background and pathogenic microorganisms on carrots. Shredded carrots were inoculated with approximately 6-7 log CFU/g of Escherichia coli O157:H7 (932, and 933) and Listeria monocytogenes (ATCC 19116, and 19111) and then dip treated with alkaline electrolyzed water (AlEW), acidic electrolyzed water (AcEW), 100 ppm sodium hypochlorite (NaOCl), deionized water (DaIW), or 1% citric acid (CA) alone or with combinations of AlEW and 1% CA (AlEW + CA). The populations of spoilage bacteria on the carrots were investigated after various exposure times (1, 3, and 5 min) and treatment at different dipping temperatures (1, 20, 40, and 50 °C) and then optimal condition (3 min at 50 °C) was applied against foodborne pathogens on the carrots. When compared to the untreated control, treatment AcEW most effectively reduced the numbers of total bacteria, yeast and fungi, followed by AlEW and 100 ppm NaOCl. Exposure to all treatments for 3 min significantly reduced the numbers of total bacteria, yeast and fungi on the carrots. As the dipping temperature increased from 1 °C to 50 °C, the reductions of total bacteria, yeast and fungi increased significantly from 0.22 to 2.67 log CFU/g during the wash treatment (p ≤ 0.05). The combined 1% citric acid and AlEW treatment at 50 °C showed a reduction of the total bacterial count and the yeast and fungi of around 3.7 log CFU/g, as well as effective reduction of L. monocytogenes (3.97 log CFU/g), and E. Coli O157:H7 (4 log CFU/g). Combinations of alkaline electrolyzed water and citric acid better maintained the sensory and microbial quality of the fresh-cut carrots and enhanced the overall shelf-life of the produce.
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Combination treatment of alkaline electrolyzed water and citric acid with mild
heat to ensure microbial safety, shelf-life and sensory quality of shredded carrots
S.M.E. Rahman
a
, Yong-Guo Jin
b
, Deog-Hwan Oh
a
,
*
a
Department of Food Science and Biotechnology, Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea
b
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
article info
Article history:
Received 28 July 2010
Received in revised form
4 October 2010
Accepted 19 October 2010
Available online 27 October 2010
Keywords:
Alkaline electrolyzed water
Citric acid
Mild heat
Combination treatment
Microbial safety
Shelf-life and sensory quality
Shredded carrots
abstract
The objective of this study was to determine the synergistic effect of alkaline electrolyzed water and
citric acid with mild heat against background and pathogenic microorganisms on carrots. Shredded
carrots were inoculated with approximately 6e7 log CFU/g of Escherichia coli O157:H7 (932, and 933) and
Listeria monocytogenes (ATCC 19116, and 19111) and then dip treated with alkaline electrolyzed water
(AlEW), acidic electrolyzed water (AcEW), 100 ppm sodium hypochlorite (NaOCl), deionized water
(DaIW), or 1% citric acid (CA) alone or with combinations of AlEW and 1% CA (AlEW þCA). The pop-
ulations of spoilage bacteria on the carrots were investigated after various exposure times (1, 3, and
5 min) and treatment at different dipping temperatures (1, 20, 40, and 50
C) and then optimal condition
(3 min at 50
C) was applied against foodborne pathogens on the carrots. When compared to the
untreated control, treatment AcEW most effectively reduced the numbers of total bacteria, yeast and
fungi, followed by AlEW and 100 ppm NaOCl. Exposure to all treatments for 3 min signicantly reduced
the numbers of total bacteria, yeast and fungi on the carrots. As the dipping temperature increased from
1
Cto50
C, the reductions of total bacteria, yeast and fungi increased signicantly from 0.22 to
2.67 log CFU/g during the wash treatment (p0.05). The combined 1% citric acid and AlEW treatment at
50
C showed a reduction of the total bacterial count and the yeast and fungi of around 3.7 log CFU/g, as
well as effective reduction of L. monocytogenes (3.97 log CFU/g), and E. Coli O157:H7 (4 log CFU/g).
Combinations of alkaline electrolyzed water and citric acid better maintained the sensory and microbial
quality of the fresh-cut carrots and enhanced the overall shelf-life of the produce.
Ó2010 Elsevier Ltd. All rights reserved.
1. Introduction
The consumption of fresh fruits and vegetables is increasing
rapidly in Korea as well as throughout the world due to their health
effects (Huxley et al., 2004). In addition, the demand for ready-to-
use shredded and sliced carrots has become widespread. Accord-
ingly, the minimally processed fresh-cut produce industry has been
growing rapidly over the past decade, stimulated by strong
consumer demand for ready-to-eat produce that is convenient and
nutritious (IFPA, 2000). However, the frequency of outbreaks of
foodborne illnesses associated with consumption of fresh fruits and
vegetables has increased in recent years, in part due to the
increased demand for fresh produce (Nguyen-the and Carlin, 1994;
Beuchat, 1996; Roever,1998; Francis et al.,1999). This is because the
processing steps (peeling and cutting) enhance the susceptibility of
these products to microbial growth (Tournas, 2005). Indeed,
various bacterial pathogens have been reported to survive and
grow on fruits and vegetables (Nguyen-the and Carlin, 1994).
Washing is one of the most important steps during the pro-
cessing of produce since it removes soil and microorganisms from
the surface. The use of effective sanitizing agents during the
washing of produce is necessary to ensure product safety. In mini-
mally processed vegetables, such as shredded carrot, chlorine
solutions have been widely employed by the industry for sanitiza-
tion purposes. However, reduced microbiological efciency coupled
with sensorial changes and the eventual formation of carcinogenic
chlorinated compounds have demonstrated the need for alternative
decontamination methodologies. Sanitizing treatments reported to
reduce bacteria on carrots include rinsing in lemon juice and lemon
juice vinegar (Sengun and Karapinar, 2004, 2005) and anolyte water
and chlorinated water (Workneh et al., 2003). Chlorine dioxide,
ozone, and thyme essential oil have also been used to decontami-
nate carrots (Singh et al., 2002). Moreover, a 3 log decrease in
microbial load was observed in response to heat treatment (Alegria
et al., 2009, 2010). Other methods of sterilization that have been
*Corresponding author. Tel.: þ82 33 250 6457; fax: þ82 33 250 6457.
E-mail addresses: ehsan_bau@yahoo.com (S.M.E. Rahman), deoghwa@kangwon.
ac.kr (D.-H. Oh).
Contents lists available at ScienceDirect
Food Microbiology
journal homepage: www.elsevier.com/locate/fm
0740-0020/$ esee front matter Ó2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fm.2010.10.006
Food Microbiology 28 (2011) 484e491
Author's personal copy
tested include acidied sodium chlorite (Ruiz-Cruz et al., 2006,
2007), peroxyacetic acid (Vandekinderen et al., 2009), and irradia-
tion (Chaudry et al., 2004) treatment. Warm water (Klaiber et al.,
2005) and electrolyzed water (Izumi, 1999) have also been found
to decrease the populations of bacteria on carrots.
The combined use of several disinfectant agents has been widely
reported in the last few years (Beltrán et al., 2005; Ukuku et al.,
2005; Uyttendaele et al., 2004). Combinations of lactic acid, chlo-
rinated water, thyme essential oil solution, sodium lactate, citric
acid, hydrogen peroxide, ozone and peroxyacetic acid have previ-
ously been tested and combinations of chemical disinfectants have
generally been found to maintain better sensory and microbial
quality of the product. Additionally, a novel produce-washing
procedure using a combination of alkaline electrolyzed water
(AlEW), AcEW and mild heat induced a signicant bactericidal
effect when compared with treatment at ambient temperature
(Koseki and Isobe, 2007). Recently, combinations of alkaline elec-
trolyzed water and citric acid showed a strong synergistic antimi-
crobial effect that reduced background ora and foodborne
pathogens on fresh-cut produce and cereal grains (Park et al., 2004,
2009; Rahman et al., 2010).
Minimally processed (MP) carrots constitute one of the major
minimally processed vegetables (MPV). The main problems that
limit the shelf-life of MP carrots are white blush discolouration
caused by tissue dehydration and microbial spoilage (Emmambux
and Minnaar, 2003). Therefore, treatments that can inactivate the
natural microora while keeping the tissue hydrated may prolong
their shelf-life. Thus the objective of this study was to investigate
the synergistic effect of alkaline electrolyzed water and citric acid
with mild heat on the microbiological quality of shredded carrots
measured immediately after treatment, as well as to study the
shelf-life and sensory attributes during subsequent storage at 4 and
25
C.
2. Materials and methods
2.1. Preparation of inocula
The two strains of Listeria monocytogenes (ATCC 19116 and ATCC
19111) a n d Escherichia coli O157:H7 (932 and 933) used in this
experiment were obtained from Korean National Institute of Health
(Seoul, Korea) and Department of Food Science, University of
Georgia (Grifn, GA, USA), respectively. Stock cultures were main-
tained on tryptic soy agar (TSA) (BD Diagnostics, Franklin Lakes, NJ,
USA) slants at 4
C. Prior to use, all strains were separately grown in
tryptic soy broth (TSB) (BD Diagnostics) at 37
C with two consec-
utive transfers after 24 h periods for a total of 48 h of incubation. All
working cultures (approximately 10
9
CFU/mL) grown in TSB were
separately centrifuged at 4000gfor 15 min at 4
C and the
supernatants were discarded. The cell pellets were washed twice
with 0.1% peptone water (pH 7.1) and resuspended in 10 mL of the
same solution to obtain a nal cell concentration of 10
9
CFU/mL.
After this, two strains of E. coli O157:H7 and L. monocytogenes were
combined in a cocktail with approximately equal numbers in the
nal population. These culture cocktails were used in subsequent
experiments. The bacterial population in each cocktail culture was
conrmed 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.
2.2. Preparation of carrot samples
Fresh carrot (Daucus carota L.) roots were purchased from a local
wholesale market in Chuncheon, Korea and then transported to the
laboratory and used within 24 h following storage at 4
C. The
carrot root tips and leaf ends were removed and roots with visible
damage were discarded. Carrots were washed with tap water to
remove the soil and then hand peeled and shredded into 3 3cm
pieces (2e5 mm wide) using a knife that had been dipped into 70%
(v/v) alcohol and amed before use. Aliquots with a weight of
10.0 0.3 g were then used for subsequent analyses.
2.3. Inoculation of carrot samples
To destroy the background microora, 10 g of carrots were
placed on a sterile perforated tray and treated with UV light (Phi-
lips, TUV 15W) in a UV cabinet (Entkeimungsschrank, 220 V, Ernst
Schuttjun Laborgerotebau, 3400, Gottingen) for 50 min (25 min for
each side) (Sengun and Karapinar, 2005; Singh et al., 2002). After
applying this treatment, the naturally existing bacterial population
was reduced to an undetectable level (with 10 CFU/g detection
limit). Accordingly, pieces of carrots (10 g each) were placed into
sterile bags and then sprinkle inoculated with the mixed inocula
(1 mL from each) of E. coli O157:H7 and L. monocytogenes to obtain
an initial level of 10
7
log CFU/g. The samples were then shaken
vigorously for 3 min so that the inoculum would be evenly
distributed. To allow attachment of bacteria, inoculated carrots
were air-dried in a laminar ow hood for 1 h at 22 2
C and then
stored in a refrigerator at 4
C for 24 h before exposure to various
sanitizing treatments.
2.4. Preparation of sanitizers
Electrolyzed water (EW) was produced from 0.1% NaCl solution
using a ow-type electrolysis (A2-1000, Korean E & S Fist Inc.,
Seoul, Korea) set at 16 A. When a stable amperage was reached after
15 min, AcEW (pH of 2.4e2.6, ORP of 1000 mVe1100 mV, and an
available chlorine concentration of 50e60 mg/L) and AlEW (pH of
11e11.2 and ORP of 830 mV to 850 mV) were collected from the
anode compartment and the cathode respectively, separated by
a membrane. The sodium hypochlorite solution (NaOCl: pH 9.8,
100 mg/L available 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 (CA) (Yakuri Pure Chem-
icals Co., Kyoto, Japan) was dissolved in 1 L of sterile distilled water
to give a nal concentration of 1% CA solution (w/v). Sterile
deionized water (DIW) was used as a control. The pH, ORP and
available chlorine concentration of treatment solutions were
measured immediately before treatment with a dual-scale pH
meter (Accumet model 15, Fisher Scientic 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 0e300 mg/L.
2.5. Sanitization treatment
The following sanitizer treatments were evaluated for their
efcacy in killing or reducing E. coli O157:H7, L. monocytogenes and
background ora on shredded carrots. Inoculated and uninoculated
carrot samples (10 g) were placed in sterile containers and
immersed in each sanitizing agent. The carrot to solution ratio in all
treatments was 1:10 (w/v). Both DIW washed and unwashed carrot
samples were used as controls. To evaluate the effect of dipping
time on the reduction of microorganisms, each piece (10 g) of
uninoculated samples was dipped for 3, 5, and 10 min and the
samples were then immersed at 1, 20, 40, and 50
C to evaluate the
effect of temperature. The following dipping solutions were used
either alone or in combination with AlEW as dipping agents: DIW,
100 ppm NaOCl, electrolyzed water (AcEW and AlEW) and 1% CA
S.M.E. Rahman et al. / Food Microbiology 28 (2011) 484e491 485
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(Fisher Scientic Co., Norcross, GA, USA). After dipping each carrot,
the moisture on each piece was drained using sterile gauze. All
individual experiments were replicated three times.
2.6. Microbiological analyses
Following treatments, all samples (each of 10 g) were aseptically
excised and immediately placed in a stomacher bag (Nasco Whirl-
Pak, Janesville, WI, USA) containing 90 mL of buffered peptone
water (BPW; Difco, Sparks, Md., USA) and then homogenized for
2 min using a Seward stomacher (400 Circulator, Seward, London,
UK).
Following homogenization, 1-mL aliquots of the sample were
serially diluted in 9 mL of sterile BPW and 0.1 mL of sample or
diluent was spread-plated onto each selective medium (in this case,
the limit of detection was 100 CFU/g, which is equivalent to
2 log CFU/g). In the case of 1 mL placed on the plates, the limit of
detection was 1 log CFU/g. The selective media, Sorbitol MacConkey
agar (Difco) and Oxford Agar Base (Difco), were used for enumer-
ation of E. coli O157:H7 and L. monocytogenes, respectively. For
uninoculated carrot samples, the mixtures were plated onto plate
count agar (Difco) for the total counts and potato dextrose agar
(Difco with 10% tartaric acid solution) to enumerate the yeast and
fungi. All plates were incubated at 37
C for 24 h, except for yeast
and fungi plates, which were incubated at 25
C for 3e5 days. The
results were expressed as the log CFU/g.
2.7. Shelf-life study
For the shelf-life study, uninoculated carrot (untreated and
treated) samples were packaged using Ziploc
Ò
vegetable bags
(Howard et al., 1999) and then marked carefully before storage at
different temperatures (4, and 25
C). During storage, sampling was
conducted at 2-day intervals for 4
C, while samples were only
collected at 3 to 12-h intervals for samples stored at 25
C. At each
sampling interval, 90 mL of 0.1% sterile peptone water were poured
into the Stomacher bag (Nasco Whirl-Pak, Janesville, WI, USA)
containing the sample. The samples were then homogenized for
2 min using a Seward stomacher (400 Circulator, Seward, London,
UK), after which 0.1 mL aliquots of the appropriate dilution were
spread onto the surface of the duplicate plates of plate count agar
(Difco) for the total counts and potato dextrose agar (Difco with 10%
tartaric acid solution) for enumeration of the yeast and fungi. The
bacterial plates were then incubated at 37
C for 24 h, while the
yeast and fungi plates were incubated at 25
C for 3e5 days and
expressed as log CFU/g. Each experiment was replicated three
times, and the means of microbial populations (log CFU/g) from
different storage temperatures were calculated. The end of the
shelf-life arrived when the population of a group of microorgan-
isms reached an unacceptable level or when the sensory panel
rejected the samples.
2.8. Evaluation of the sensory quality during the shelf-life
Sensory evaluation of shredded carrots (treated and untreated)
was conducted for 3-day intervals during storage (15 days) at 4
C.
A panel of ve trained judges (members of our department) was
then assigned to distinguish the following sensory attributes: color,
rmness, fresh-like aroma and general acceptance. The attributes
were scored using a 5-point numeric rating scale (Alegria et al.,
2009) as follows:
Color:1¼very light orange; 2 ¼light orange; 3 ¼orange;
4¼dark orange; 5 ¼very dark orange, where 3 is an anchor
corresponding to the initial carrot fresh appearance immedi-
ately after cutting;
Firmness:1¼very rm; 2 ¼rm; 3 ¼moderate; 4 ¼soft; and
5¼very soft, where 1 is an anchor corresponding to the
perception of the fresh-cut carrot immediately after cutting;
Fresh-like aroma:1¼very intense; 2 ¼intense; 3 ¼moderate;
4¼low; and 5 ¼absent, where 1 is an anchor corresponding to
the perception of the carrots aroma immediately after cutting.
Panelists were also asked to identify the presence of any off-
odors in the comments section;
General acceptance:1¼excellent freshly cut; 2 ¼good; 3 ¼limit
of marketability; 4 ¼fair limit of usability; and 5 ¼poor
unusable.
Panelists were also asked to provide any relevant comments.
The mean scores of the attribute intensity and acceptance were
calculated and the end of the shelf-life from the sensory point of
view was reached when at least one of the mean scores was above
the middle point of the respective scale. The applied cut-off for the
general acceptance was xed at 3 (limit of marketability), and
scores above 3 indicated unacceptable samples.
2.9. Statistical analyses
All analyses were conducted in duplicates with 3 replicates of
each experiment. Data were expressed as the means standard
errors. The results were analyzed using the SPSS statistical package
(SPSS Inc., Chicago, IL). An analysis of variance (ANOVA) was used to
evaluate the treatment, dipping time, and temperature as xed
effects. Tukeys multiple range tests were used to determine the
signicant difference at p0.05.
3. Results and discussion
The properties (pH, ORP, and residual chlorine) of treatment
solutions (AlEW, AcEW, NaOCl, 1% CA, AlEW þ1% CA and DIW) used
in this study are presented in Table 1.The washing effect of DIW,
NaOCl, AlEW and AcEW in shredded carrots against background
ora was observed in this study. When compared to unwashed
controls, different wash solutions reduced the total bacteria by
0.46e2.25 log CFU/g (Fig. 1A) and the yeast and fungi by
0.30e2.25 log CFU/g (Fig. 1B) after different dipping times (1, 3 and
5 min). For all dipping times, DIW treatment reduced the pop-
ulations by less than 0.9 log CFU/g, while AcEW treatment showed
the greatest reduction of 2.25 log CFU/g for the total bacteria and
yeast and fungi count, respectively compared to the untreated
control. Three and 5 min dipping time from each treatment except
DIW showed better effect than 1 min on the reduction of total
Table 1
Physicochemical properties of washing solutions.
a
Washing solutions pH ORP (mV) ACC
b
(mg/L)
AlEW
c
11.3 0.2 810 15 Not measured
AcEW
d
2.54 0.3 1100 20 50 2.2
NaOCl
e
9.8 0.2 690 25 100 4.5
1% CA
f
2.23 0.04 970 10 Not measured
AlEW þ1% CA
g
2.48 0.02 550 15 Not measured
DIW
h
7.0 0.1 412 18 0.3 0.1
a
Values are mean standard deviation, n¼3.
b
Available chlorine concentration.
c
Alkaline electrolyzed water.
d
Acidic electrolyzed water.
e
Sodium hypochlorite solution.
f
Citric acid (1%).
g
Alkaline electrolyzed water þ1% Citric acid.
h
Deionized water.
S.M.E. Rahman et al. / Food Microbiology 28 (2011) 484e491486
Author's personal copy
counts on carrot (Fig. 1A). Similar results were observed in the
reduction of yeast and fungi on carrot (Fig. 1B). From these results,
there was no any remarkable difference (p0.05) found in
microbial reduction between 3 and 5 min dipping. Also 5 min
dipping resulted in changing color (Visual estimation) of shredded
carrots. According to these results, 3 min exposure would be the
most appropriate to minimize the quality deterioration (color),
thus it was chosen for following treatments. Washing with sani-
tizers also signicantly (p0.05) reduced the background ora
when compared to the unwashed control. The relative bacterial
reduction potency occurred in the order AcEW >AlEW >
NaOCl >DIW. The sanitizing effect of electrolyzed water against
total microora on fresh-cut vegetables was rst reported by Izumi
(1999), who found that fresh-cut carrots, bell peppers, spinach,
Japanese radish, and potatoes treated by dipping in electrolyzed
water (pH 6.8, 20 ppm available chlorine) for 3 min showed
a reduction in the total microbial count of 0.6e2.6 log CFU/g. He
also found that the electrolyzed water did not inuence the tissue
pH, surface color, or general appearance of the fresh-cut vegetables.
González et al. (2004) reported that shredded carrots treated with
1000 ppm acidied sodium chlorite (ASC) showed at least
a 3.27 log CFU/g reduction in the total aerobic count. However, the
concentrations used by these authors were not able to maintain the
quality of shredded carrots (shelf-life 4e6 days at 5
C).
Three minutes of dipping was nally selected to measure the
effects of different dipping temperatures (1, 20, 40 and 50
C).
Regardless of the dipping temperature, a reduction of 0.29e2.67
log CFU/g (Fig. 2A) and 0.22e2.60 log CFU/g (Fig. 2B) were achieved
for total bacteria and yeast and fungi, respectively, in carrots treated
with the aforementioned sanitizers. Table 2 also showed the effects
of dipping temperatures on the efcacy of AlEW and CA alone or in
combination in the reduction of the total bacteria, yeast and fungi
on carrot. Washing with AlEW (1.17 log reduction of total counts)
and 1% citric acid (1.7 log reduction of total counts) at 1
C for 3 min
showed signicantly greater (p0.05) sanitizing effect than DIW
treatment (0.29 log reduction of total counts) compared to control,
while sanitizing effect with AlEW (1.78 log reduction of total
counts) and 1% citric acid (2.37 log reduction of total counts) was
much more enhanced than DIW treatment (0.76 log reduction of
total counts) at 20
C. Similar results were observed in yeast and
fungi at each treatment. The reductions in microora signicantly
increased (p0.05) as the dipping temperature increased from 1 to
50
C for each treatment. It has been reported that mild heat
treatment improved the efcacy of a sanitizer at killing or removing
microorganisms on produce. These mild heat treatments consisted
of subjecting the products to temperatures of 50e90
C for periods
of time not exceeding 1e5 min (Orsat et al., 2001). Klaiber et al.
(2005) found that washing uncut carrots with cold chlorinated
water (200 mg/L, 4
C) and warm tap water (50
C) reduced aerobic
mesophilic bacteria by 1.7 and 2.0 log CFU/g, respectively, while
A
B
Fig. 1. Inactivation effect of electrolyzed water on the total counts (A), and yeast and
fungi (B) in carrots by different dipping times. Vertical bars represent mean standard
error of mean, n¼6. Bars labeled with different letters in the same treatment indicate
a signicant difference (p0.05). (Control: not treated; DIW: deionized water; NaOCl:
100 mg/L sodium hypochlorite; AlEW: alkaline electrolyzed water; AcEW: acidic
electrolyzed water).
A
B
Fig. 2. Inactivation effect of electrolyzed water on the total counts (A), and yeast and
fungi (B) in carrots treated at different dipping temperatures for 3 min. Vertical bars
represent mean standard error of mean, n¼6. Bars labeled with different letters in
the same treatment indicate a signicant difference (p0.05). (Control: not treated;
DIW: deionized water; NaOCl: 100 mg/L sodium hypochlorite; AlEW: alkaline elec-
trolyzed water; AcEW: acidic electrolyzed water).
S.M.E. Rahman et al. / Food Microbiology 28 (2011) 484e491 487
Author's personal copy
washing with warm chlorinated water (200 mg/L, 50
C) resulted in
a 2.3 log CFU/g reduction. Shredded lettuce treated with chlori-
nated water for 3 min at 47
C showed a reduction in the aerobic
bacterial count of 3 log CFU/g, while a reduction of approximately
1 log CFU/g was observed in response to the same treatment at 4
C
(Delaquis et al., 1999). Koseki et al. (2004) found that the appear-
ance of mildly heated (50
C) lettuce did not deteriorate after
treatment. They also found that the application of AlEW as a pre-
wash agent was useful, and that combined treatment with AlEW
and AcEW was effective. Mild heat treatment of fresh produce has
been reported to enhance the bactericidal effect of sanitizers and
the physiological and sensory quality of the produce. A signicantly
more enhanced anti-browning effect was observed (Jin et al., 2009)
when the lettuce leaves were subjected to both 1% citric acid and
AlEW treatment at temperatures greater than 40
C. Conversely,
our results revealed that combined 1% citric acid and AlEW treat-
ment at 50
C resulted in a signicant (p0.05) reduction of the
total bacterial counts (3.71 log CFU/g) and yeast and fungi
(3.69 log CFU/g) on shredded carrots when compared to each single
treatment alone (Table 2).
Based on our experimental data derived from the combined
effect of AlEW and 1% CA against background microora (Table 2),
the optimal condition (3 min at 50
C) was selected to determine
the combined effect on the reduction of L. monocytogenes and E. coli
O157:H7 inoculated on shredded carrots (Fig. 3A and B). Dipping
shredded carrots in DIW for 3 min at 50
C for L. monocytogenes
reduced the populations by 1.33 log CFU/g, while treatment with
100 ppm NaOCl, AlEW, and 1% CA alone decreased these pop-
ulations by 2.30, 2.70, and 2.81 log CFU/g, respectively (Fig. 3A).
Combined treatment with AlEW and 1% CA signicantly (p0.05)
Table 2
Effect of alkaline electrolyzed water and citric acid, either alone or in combination,
on the inactivation of total counts, and yeast and fungi on carrots at different dipping
temperatures for 3 min.
Treatment Temperature
(
C)
Total count
(log CFU/g)
Yeast and fungi
(log CFU/g)
Control e4.99 0.16Aa
a
3.69 0.17Aa
DIW 1 4.70 0.18ab 3.47 0.17ab
20 4.23 0.21b 3.16 0.21b
40 3.97 0.18bc 2.82 0.18bc
50 3.68 0.16Bc 2.61 0.19Bc
AlEW 1 3.82 0.14b 2.46 0.11b
20 3.21 0.10c 1.81 0.10c
40 2.97 0.15cd 1.58 0.12cd
50 2.62 0.12Cd 1.32 0.14Cd
CA 1 3.29 0.11b 2.02 0.18b
20 2.62 0.16c 1.45 0.10c
40 2.31 0.15cd 1.19 0.21cd
50 2.03 0.21Cd <1.0
AlEW þCA 1 2.49 0.18b 1.23 0.17b
20 1.82 0.19c <1.0
40 1.53 0.11cd <1.0
50 1.28 0.13Dd NE
Control (not treated), DIW (deionized water), AlEW (alkaline electrolyzed water), CA
(1% citric acid), and AlEW þCA (alkaline electrolyzed water þ1% citric acid).
<1.0 eNot detectable on direct plate count, but positive on enrichment media.
NE eNegative on enrichment.
a
Values are mean standard error of mean, n¼6. Means within a column for
each treatment bearing different lowercase letters are signicantly different
(p0.05). Means within the same column with different capital letters are signif-
icantly different (p0.05) between all treatments at 50
C compared to control.
A
B
Fig. 3. Effect of alkaline electrolyzed water and citric acid either alone or in combi-
nation on the inactivation of L. monocytogenes (A) and E. coli O157:H7 (B) on carrots
when applied at 50 C for 3 min. Vertical bars represent mean standard error of
mean, n¼6. Bars labeled with different letters among treatments indicate a signicant
difference (p0.05) (Control: not treated; DIW: deionized water; NaOCl: 100 mg/L
sodium hypochlorite; AlEW: alkaline electrolyzed water; CA: 1% citric acid;
AlEW þCA: alkaline electrolyzed water þ1% citric acid).
A
B
Fig. 4. Effect of combined treatment with alkaline electrolyzed water (AlEW) and
Citric acid (CA) with or without mild heat (H) on the total aerobic counts (A) and yeast
and fungi (B) on carrots during storage at 4 C. Values shown are mean standard
error of mean, n¼6. The error bars indicate 95% condence intervals.
S.M.E. Rahman et al. / Food Microbiology 28 (2011) 484e491488
Author's personal copy
decreased the populations of L. monocytogenes by 3.97 log CFU/g
when compared with each treatment alone. Similar reductions in
the levels of E. coli O157:H7 on carrots were observed under the
same conditions (Fig. 3B). Specically, treatment with DIW, NaOCl,
AlEW, and 1% CA alone decreased the populations by 1.35, 2.27,
2.66, and 2.78 log CFU/g, respectively, but combined treatment
with AlEW and 1% CA showed the strongest (p0.05) inactivation
of 4.0 log CFU/g compared to other treatment alone. Many sani-
tizers have been found to effectively reduce pathogens, including
E. coli O157:H7, Salmonella spp. and L. monocytogenes, on fresh-cut
carrot shreds (Singh et al., 2002; Ruiz-Cruz et al., 2007; González
et al., 2004). Neutral electrolyzed water (NEW) has also been
used (Abadias et al., 2008) against native microbiota on grated
carrot and no changes in carrot appearance were noted. Acidied
sodium chlorite (ASC) was found to be the most effective treatment
for reducing pathogens on fresh-cut carrots at all concentrations
evaluated (Ruiz-Cruz et al., 2007). Specically, they found that ASC
reduced the three pathogen populations to undetectable levels
(with a 10 CFU/g detection limit), achieving reductions of 4.81, 4.84
and 2.5 log CFU/g for E. coli,Salmonella and L. monocytogenes,
respectively. In addition, Park and Beuchat (1999) showed that
treatment with 40 or 80 ppm peroxyacetic acid (PA) reduced the
population of Salmonella by 2.6 log CFU/g. Many combinations of
physical and chemical treatments have been tested to determine if
they showed enhanced antimicrobial action in recent years. Effec-
tive combined use of AlEW and AcEW with mild heat was reported
by Koseki et al. (2004). Conversely, AcEW combined with ozone
was applied using sequential washes (Wang et al., 2004). Bari et al.
(2005) investigated the efcacy of nisin and pediocin treatments in
combination with EDTA, citric acid, sodium lactate, potassium
sorbate and phytic acid for reducing L. monocytogenes on fresh-cut
produce. Yuk et al. (2007) showed that ozone treatment produced
reductions of E. coli O157:H7 and L. monocytogenes that were less
than 1.0 and 0.5 log, respectively, on enoki mushroom. The efcacy
was improved using combined application of 3 ppm ozone and 1%
citric acid, which resulted in reductions of 2.26 and 1.32 log,
respectively. It would be expected that combinations of sanitizers
and/or other intervention methods would have additive, syner-
gistic or antagonistic interactions (Parish and Davidson, 1993;
Rajkowski and Baldwin, 2003).
The changes in microbial populations during the shelf-life study
are shown in Figs. 4 and 5. Combined treatment with AlEW and 1%
CA with mild heat treatment was found to effectively control the
growth of aerobic bacteria, yeast and fungi during storage at 4 and
25
C for 15 days and 48 h, respectively, when compared to
unwashed controls. The end of the shelf-life was considered to
occur when the total aerobic count was 7e8 log CFU/g and the total
yeast count was 5 log CFU/g (Debevere, 1996; Gómez-López et al.,
2007a; Alegria et al., 2010). The results of our study revealed that
no microbial counts reached unacceptable levels after 15 days at
4
C and 48 h of storage at 25
C for treated carrots. The shelf-life of
controls (untreated carrots) stored at 4
C was limited to 10 days
and 24 h stored when stored at 25
C due to the occurrence of
unacceptable counts of bacteria (10
7
CFU/g) and yeast and fungi
(10
5
CFU/g) (Figs. 4A and B, 5A and B). This could have resulted in
shelf-life extensions of ve days and 24 h for treated carrots stored
at 4 and 25
C, respectively. Similarly, Gómez-López et al. (2007a)
found a shelf-life extension of 4 days in response to the applica-
tion of gaseous ClO
2
to minimally processed carrots. Conversely,
a shelf-life extension of at least ve and three days in samples
stored at 4 and 7
C, respectively, can be achieved by treating
A
0
1
2
3
4
5
6
7
8
9
0 6 12 18 24 30 36 42 48
Storage time (h)
)g/ufcgol(tnuocciborealatoT
control AlEW+CA AlEW+CA+H
control AlEW+CA AlEW+CA+H
B
0
1
2
3
4
5
6
0 6 12 18 24 30 36 42 48
Stora
g
e time (h)
)g/ufcgol(ignufdnatsaeY
Fig. 5. Effect of combined treatment with alkaline electrolyzed water (AlEW) and citric
acid (CA) with or without mild heat (H) on the total aerobic counts (A), and yeast and
fungi (B) on carrots during storage at 25 C. Values shown are mean standard error of
mean, n¼6. The error bars indicate 95% condence intervals.
Table 3
Sensory evaluation of shredded carrots treated with AlEW þCA with mild heat and stored at 4
C for 15 days.
Quality attributes Storage (days)
0 3 6 9 12 15
Color Untreated 2.7 0.3 2.5 0.2 2.2 0.1 1.9 0.1 1.5 0.2 1.2 0.3
Treated 3.0 0.2 2.8 0.2 2.6 0.3 2.4 0.1 2.2 0.2 2.0 0.1
Firmness Untreated 1.3 0.3 1.7 0.2 2.3 0.3 3.0 0.4 3.5 0.3 4.0 0.6
Treated 1.0 0.2 1.3 0.1 1.7 0.4 2.1 0.3 2.5 0.2 3.0 0.5
Fresh-like aroma Untreated 1.3 0.4 1.8 0.3 2.2 0.4 2.9 0.5 3.2 0.3 3.5 0.4
Treated 1.2 0.3 1.5 0.2 1.8 0.3 2.2 0.4 2.6 0.5 2.9 0.3
General acceptance Untreated 1.2 0.2 1.8 0.3 2.4 0.4 3.0 0.5 3.4 0.4 3.9 0.6
Treated 1.0 0.1 1.3 0.2 1.6 0.3 2.0 0.4 2.5 0.5 3.0 0.4
Values are the average of ve observations (Mean SD). Numbers in bold are scores above the acceptability limit. Colour scoring system: 1 ¼very light orange; 2 ¼light
orange; 3 ¼orange; 4 ¼dark orange; 5 ¼very dark orange, where anchor 3 corresponds to the initial carrot fresh appearance immediately after cutting.
Firmness scoring system: 1 ¼very rm, 2 ¼rm; 3 ¼moderate; 4 ¼soft; and 5 ¼very soft, where anchor 1 corresponds to the perception of the fresh-cut carrot immediately
after cutting.
Fresh-like aroma scoring system: 1 ¼very intense, 2 ¼intense; 3 ¼moderate; 4 ¼low; and 5 ¼absent, where anchor 1 corresponds to the perception of the carrots aroma
immediately after cutting.
General acceptance scoring system: 1 ¼excellent freshly cut, 2 ¼good, 3 ¼limit of marketability, 4 ¼fair limit of usability and 5 ¼poor unusable.
S.M.E. Rahman et al. / Food Microbiology 28 (2011) 484e491 489
Author's personal copy
shredded cabbage with neutral electrolyzed oxidizing water
(Gómez-López et al., 2007b).
The sensorial quality of the shredded carrots was evaluated by
a sensory panel throughout the storage period. The mean scores of
sensory attributes (color, rmness, fresh-like aroma, general
acceptance) for untreated and treated carrots are summarized in
Table 3. Sensory attributes were distinguished by a panel using
a 5-point numeric rating scale (Alegria et al., 2009). The general
acceptance was xed at a score of 3 (limit of marketability), and
scores greater than 3 indicated unacceptable samples. The end of
the shelf-life from the sensory point of view was attained when at
least one of the mean scores was above the middle point of the
respective scale. In the case of untreated samples and the rmness,
fresh-like aroma and general acceptance reached a score of 3 after
storage for nine days at 4
C. These ndings indicated that the shelf-
life of untreated carrots was limited to nine days from the sensorial
point of view. Moreover, the sensory attributes were acceptable for
treated carrots, with the shelf-life being extended up to 15 days at
refrigerated temperature. These results are in agreement with
those of Chaudry et al. (2004), who noted that minimally processed
carrots may be treated with a 2 kGy dose of gamma radiation to
keep the appearance and avor quality acceptable and extend the
shelf-life up to 14 days at refrigerated temperature. Farkas et al.
(1997) reported that a 1 kGy dose of gamma radiation was suf-
cient to reduce bacterial load and improve microbiological shelf-
life, and that it extended the sensorial quality of precut peppers and
carrots.
Based on the results of these studies, combined treatment with
AlEW and 1% CA with mild heat appears to be a promising method
to ensure microbial safety as well as to prolong the shelf-life of
treated shredded carrots without impairing sensorial quality. The
application of combined AlEW and CA for controlling microorgan-
isms on carrots has not previously been reported. Therefore, the
synergistic effect of AlEW and CA may provide valuable insight into
the reduction of foodborne pathogens on fresh-cut produce. In
general, combinations of chemical disinfectants maintain better
sensory and microbial quality of the product. Therefore, more
studies should be conducted to determine the synergistic effects of
combining disinfection technologies.
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... It has been reported that an increasing amperage improves the sanitizing efficiency of EW, for example, by increasing the current from 1.15 to 1.45 A, a log reduction between 4.9 and 5.6 CFU/mL for Listeria monocytogenes and E. Coli O157:H7 was found. Moreover, the ORP, ACC, and pH also increased by increasing amperage [78]. ...
... It is also hypothesized that EW does not generate antimicrobial resistance in bacteria. As far as the sensory or organoleptic parameters of food are concerned, these remain unaffected while utilizing acidic electrolyzed water (AEW), neutral electrolyzed water (NEW), slightly acidic electrolyzed water (SAEW), and strongly acidic electrolyzed water (StAEW) [66,71,73,77,78]. Moreover, the cost of EW is negligible compared to its counterparts. ...
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... Similarly, the effect of AEW was found to be very effective for reducing V. parahaemolyticus and E. coli O157:H7 on tilapia skin [96]. Although the application of EW on fish products appears a promising technique for reducing the total count of pathogenic and spoilage bacteria, at the same time, it has shown some undesirable effects on the organoleptic quality and nutritional value of food [97][98][99][100]. Since food safety must be accompanied by sensory quality, to overcome these limitations, a combination of two or more preservative and sanitizing technologies in low quantities is suggested. ...
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Fish products are highly perishable, requiring proper processing to maintain their quality and safety during the entire storage. Different from traditional methods used to extend the shelf-life of these products (smoking, salting, marinating, icing, chilling, freezing, drying, boiling, steaming, etc.), in recent years, some alternative methods have been proposed as innovative processing technologies able to guarantee the extension of their shelf-life while minimally affecting their organoleptic properties. The present review aims to describe the primary mechanisms of some of these innovative methods applied to preserve quality and safety of fish products; namely, non-thermal atmospheric plasma (NTAP), pulsed electric fields (PEF), pulsed light (PL), ultrasounds (US) and electrolyzed water (EW) are analysed, focusing on the main results of the studies published over the last 10 years. The limits and the benefits of each method are addressed in order to provide a global overview about these promising emerging technologies and to facilitate their greater use at industrial level. In general, all the innovative methods analysed in this review have shown a good effectiveness to control microbial growth in fish products maintaining their organoleptic, nutritional and sensory characteristics. Most of the technologies have also shown the great advantage to have a lower energy consumption and shorter production times. In contrast, not all the methods are in the same development stage; thus, we suggest further investigations to develop one (or more) hurdle-like non-thermal method able to meet both food production requirements and the modern consumers’ demand.
... The extremely low microbial loads observed on apple slices after processing minimized the potential microbial impact on VOCs profiles. These low microbial loads were probably due to the low initial contaminations of intact apples and the sanitising effects of the acid treatment (Chen, Hu, He, Jiang, & Zhang, 2016;Rahman, Jin, & Oh, 2011). ...
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Chapter
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This study was conducted to determine the inactivation effect of electrolyzed water and organic acids either alone or in combination on L. monocytogenes or natural microflora on lettuce. Acidic electrolyzed water completely inactivated L. monocytogenes in broth system within 60 sec, but alkalin electrolyzed water caused approximate 1.7 log CFU/g reduction. However, acidic electrolyzed water reduced only 2.5 log CFU/g of L. monocytogenes on lettuce, and similar antimicrobial effect was observed with alkalin electrolyzed water. In the meantime, acidic and alkaline electrolyzed water caused approximately 2 log CFU/g reduction compared to control, whereas both electrolyzed water combined with organic acids ranged from 2.6 to 3.7 log CFU/g reduction. Among the organic acids, both electrolyzed water combined with citric acid showed the strongest synergistic antimicrobial effect to reduce L. monocytogenes on lettuce as well as total counts, yeast and molds. When antimicrobials, alone or in combination were treated into L. monocytogenes inoculated lettuce at for designed periods, the combined alkalin electrolyzed water with citric acid showed the greatest potential to inhibit growth of the bacteria. According to Scanning Electron Microscopy(SEM), the treatment of electrolyzed alkali water in combination with citric acid highly reduced the growth of the L. monocytogenes compared to single treatment and resulted in causing the destruction of cell membrane.
Chapter
Although the incidence of food-borne illnesses linked to fresh produce is low, there is increased awareness that fruits and vegetables can be contaminated with microbiological pathogens. For its microbiological sampling program of certain fresh fruits and vegetables, the U.S. Food and Drug Administration (FDA) conducted surveys of both imported and domestic produce. A 4% (44 of 1003 sampled) contamination rate was reported in published results for imported product (http://www.cfsan.fda. gov/dms/prodsur6.html). In the interim report on domestic product, there was a 1.6% violation rate (http://www.cfsan.fda.gov/dms/prodsur9.html). Two microbiological pathogens that can cause food-borne illnesses were present on the produce: Salmonella and Shigella.
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This study was conducted to determine the effects of alkaline electrolyzed water (AIEW), acidic electrolyzed water (AcEW), 1% citric acid, and 100 ppm sodium hypochlorite, either alone or in combination with citric acid, in reducing the populations of spoilage bacteria and foodborne pathogens (Listeria monocytogenes and Escherichia coli O157:H7) on lettuce at various exposure times (3, 5, and 10 min) with different dipping temperatures (1, 20, 40, and 50°C). In addition, the inhibitory effect of alkaline electrolyzed water combined with citric acid on the browning reaction during storage at 4°C for 15 days was investigated. Compared to the untreated control, electrolyzed water more effectively reduced the number of total bacteria, mold, and yeast than 100 ppm sodium hypochlorite under the same treatment conditions. All treatments exposed for 5 min significantly reduced the numbers of total bacteria, yeast, and mold on head lettuce. The inactivation effect of each treatment on head lettuce was enhanced as the dipping temperature increased from 1 to 50°C, but there was no significantly difference at temperatures greater than 40°C (p<0.05). The total counts of yeast and mold in head lettuce were completely eliminated when a combination of 1% citric acid and AlEW treatment was used at temperatures greater than 40°C. However, decreased reduction in L. monocytogenes (2.81 log CFU/g), and E. coli O157:H7 (2.93 log CFU/g) on head lettuce was observed under these treatment conditions. In addition, enhanced antibrowning effect was observed when the samples were subjected to both 1% citric acid and AlEW treatment at temperatures greater than 40oC compared to when single treatments alone were used. Thus, this combined treatment might be considered a potentially beneficial sanitizing method for improving the quality and safety of head lettuce.
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A monoterpenoid compound, benzylideneacetone (BZA), is a metabolite of an entomopathogenic bacterium, Xenorhabdus nematophila. Its primary biological activity is an inhibitor of phospholipase , which catalyzes the committed step of biosynthesis of various eicosanoids that are critically important to mediate insect immune responses. When BZA was applied to two-spotted spider mite, Tetranychus urticae, it exhibited a dose-dependent mortality in leaf-disc assay. Subsequently BZA was tested against T. urticae infesting apples in a field orchard, in which it showed a significant control efficacy, which was not statistically different with that of a commercial acaricide. BZA also had significant antibacterial activities against three species of plant pathogenic bacteria when it was added to the bacterial cultures, in which it showed the highest inhibitory activity against a bacterial wilt-causing pathogen, Ralstonia solanacearum. The bacterial pathogen caused significant disease symptom to young potato plants. However, BZA significantly suppressed the disease occurrence. This study suggests that BZA can be used to develop a novel crop protectant to control mite and bacterial pathogen.
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Chemicals containing SH-groups as sulfites and chlorine-based agents are commonly employed in the fresh-cut process of vegetables such as potatoes to prevent browning and to sanitize produce. However, there is a concern over the application of these compounds in fresh-cut commodities as they might affect human and environmental safety and this has created the need to investigate alternatives. In the present work, the effectiveness of different traditional and non-traditional sanitizers on the sensory and microbial quality of fresh-cut potatoes stored under passive modified atmosphere packaging (MAP) and vacuum packaging was investigated. Six different washing treatments consisting of water, sodium sulfite, sodium hypochlorite, Tsunami, ozone and the combination of ozone–Tsunami were evaluated. Browning and growth of aerobic mesophilic bacteria, psychrotrophic bacteria, coliforms, lactic acid bacteria (LAB), anaerobic bacteria, moulds and yeasts were studied. In general, vacuum packaging preserved the appearance better than MAP. Under MAP only sodium sulfite prevented browning although it conferred off-odors. After 14 days of storage, there was no evidence of browning in fresh-cut potatoes dipped in ozonated water or ozone–Tsunami and stored under vacuum and these treatments maintained initial texture and aroma. However, the use of ozonated water alone was not effective in reducing total microbial populations. Ozone–Tsunami resulted in the most effective treatment to control microbial growth achieving 3.3, 3.0 and 1.2 log-reductions for LAB, coliforms and anaerobic bacteria, respectively. Therefore, although microbial growth was not slowed down by ozone alone, the combination of ozone–Tsunami resulted an efficient and promising treatment for controlling microbial growth and maintaining sensory quality of potato strips under vacuum.
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The demand for fresh salad vegetables, such as iceberg lettuce, has increased worldwide in recent years. Numerous sanitizers have been examined for their effectiveness in killing or removing pathogenic bac- teria on fresh produce. However, most of the sanitizers are made from the dilution of condensed solu- tions which involves some risk in handling and is troublesome. A sanitizer that is not produced from the dilution of a hazardous condensed solution is required for practical use. Electrolyzed water and ozonated water were investigated for bactericidal effects on fresh-cut produce as a convenient and safe alternative sanitizer. Although the efficacy of acidic electrolyzed water (AcEW) as a sanitizing agent was dependent on the kind of produce treated, AcEW could be sufficiently effective to offer an alterna- tive solution to conventional sanitizers, such as sodium hypochlorite solution (150 ppm). A novel pro- duce-washing procedure using a combination of alkaline electrolyzed water (AlEW), AcEW and mild heat demonstrated significant bactericidal effect compared with the treatment with ambient tempera- ture. Besides the bactericidal effect, the progress of browning on lettuce was suppressed by using mild heat treatment. Furthermore, as a novel usage of AcEW, we examined the use of AcEW-ice for preserv- ing vegetables. AcEW-ice inactivated the spoilage and pathogenic bacteria on lettuce and reduced the temperature of lettuce during storage.
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Cut lettuce dip-inoculated with Escherichia coli O157:H7 and Salmonella was treated with alkaline electrolyzed water (AlEW) at 20°C for 5min, and subsequently washed with acidic electrolyzed water (AcEW) at 20°C for 5min. Pre-treatment with AlEW resulted in an approximate 1.8log10cfu/g reduction of microbial populations, which was significantly (p⩽0.05) greater than microbial reductions resulting from other pre-treatment solutions, including distilled water and AcEW. Repeated AcEW treatment did not show a significant bacterial reduction. Mildly heated (50°C) sanitizers were compared with normal (20°C) or chilled (4°C) sanitizers for their bactericidal effect. Mildly heated AcEW and chlorinated water (200ppm free available chlorine) with a treatment period of 1 or 5min produced equal reductions of pathogenic bacteria of 3log10 and 4log10cfu/g, respectively. The procedure of treating with mildly heated AlEW for 5min, and subsequent washing with chilled (4°C) AcEW for period of 1 or 5min resulted in 3–4log10cfu/g reductions of both the pathogenic bacterial counts on lettuce. Extending the mild heat pre-treatment time increased the bactericidal effect more than that observed from the subsequent washing time with chilled AcEW. The appearance of the mildly heated lettuce was not deteriorated after the treatment. In this study, we have illustrated the efficacious application of AlEW as a pre-wash agent, and the effective combined use of AlEW and AcEW.