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Ó 2020 Published by Elsevier Inc. on behalf of Poultry Science Association Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Effect of neutral electrolyzed water as antimicrobial

intervention treatment of chicken meat and on
trihalomethanes formation
V. M. Hernández-Pimentel,* C. Regalado-González,* G. M. Nava-Morales,*
Y. Meas-Vong,† M. P. Castañeda-Serrano,‡ and B. E. García-Almendárez*,1
*
DIPA, PROPAC, Faculty of Chemistry, Autonomous University of Queretaro, C. U. Cerro
de las Campanas s/n, Col. Las Campanas, 76010 Querétaro, Qro, México; †Center for
Research and Development in Electrochemistry (CIDETEQ), Parque Tecnológico
Querétaro, 76703 Pedro Escobedo, Querétaro, México; and ‡Faculty of Veterinary
Medicine and Zootechnics, National Autonomous University of Mexico, Ciudad
Universitaria, 04510 CDMX, México

Primary Audience: Poultry Processors, Food Safety Professionals, Poultry Researchers

SUMMARY
Salmonella is the most common pathogen in poultry associated to foodborne outbreaks.
There is concern about the use of sodium hypochlorite (NaClO) during antimicrobial in-
terventions in broiler chicken processing because of trihalomethane (THM) generation through
oxidation of organic matter. In this study, we showed the effectiveness of neutral electrolyzed
water (NEW) in broiler chickens, and their physicochemical properties after antimicrobial
intervention. Neutral electrolyzed water showed 10 times higher antimicrobial effect on Sal-
monella pure culture than NaClO treatments but similar results were achieved on inoculated
chicken carcasses. The mechanism of action of NEW was revealed by microscopic studies,
showing cell swelling and morphologic changes on membrane. Neutral electrolyzed water was
effective in reducing total viable counts and coliforms on broiler chicken carcasses, without
color and pH modification. No THM were detected on chicken meat using 50 mg/L of either
antimicrobial agents but when using higher concentration, THM were generated. Only
0.037 6 0.001 mg/kg of chloroform was detected after $100 mg/L NEW treatments, whereas
bromoform and chlorodibromomethane were generated after similar concentration of NaClO
treatments. Neutral electrolyzed water represents a safe alternative for antimicrobial broiler
chicken intervention owing to competitive production cost, safe handling, low corrosion ca-
pacity, and low toxicity because of lack of by-product generation.

Key words: neutral electrolyzed water, trihalomethane, antimicrobial intervention

2020 J. Appl. Poult. Res. 29:622–635
https://doi.org/10.1016/j.japr.2020.04.001

1
Corresponding author: blancag@uaq.mx
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HERNÁNDEZ-PIMENTEL ET AL: ANTIMICROBIALS ON CHICKEN
DESCRIPTION OF PROBLEM
The Centers for Disease Control and Pre-
vention (CDC, 2019) reported that Salmonella
is the most common pathogen related to
broiler chicken outbreaks. Nowadays, sani-
tizing agents commonly used for fresh poultry
decontamination interventions involve chlo-
rine compounds, trisodium phosphate, and
organic acids. Neutral electrolyzed water
(NEW) is gaining importance in the food in-
dustry mainly owing to its effective antimi-
crobial activity, noncorrosiveness,
environment-friendly properties, in situ pro-
duction at low cost, and safe handling (Aider
et al., 2012; Jiménez-Pichardo et al., 2016).
Neutral electrolyzed water was obtained from
an electrolytic process using sodium chloride
(NaCl) solution (0.1–1% w/v) that generates
highly oxidizing chlorine species in automated
dispensers, whose titer is usually measured as
total available chlorine (TAC) (Rahman et al.,
2010). Several compounds are produced, and
the most abundant species are hypochlorous
acid (HOCl), hypochlorite ions (ClO2), chlo-
rine dioxide (ClO2), and ozone (O3) (Al-Haq
et al., 2005). The mechanism of action of
NEW has been associated with membrane
damage, enzyme inhibition, weakening of
membrane transport capacity, and high
oxidation–reduction potential (ORP) (Rahman
et al., 2016; Ye et al., 2017).
Neutral electrolyzed water is permitted in
the United States for poultry processing at
doses #50 mg/L (FSIS, 2019), but reports
have mainly focused on its antimicrobial ac-
tivity (Rasschaert et al., 2013; Al-Holy et al.,
2015) without evaluation of trihalomethanes
(THM) formation in treated broiler chickens.
However, nowadays other countries do not
have legislation concerning its use. Acidic
electrolyzed water (AEW) has been previ-
ously used to prevent the presence of food-
borne pathogens in chicken carcasses
(Fabrizio et al., 2002) and chicken legs (Al-
Holy et al., 2015). However, this oxidizing
acidic water is becoming less popular because
of potential corrosion of stainless steel sur-
faces (Len et al., 2002). On the other hand,
there are few reports of NEW antimicrobial
interventions on broiler chickens, using
623
slightly AEW to reduce the population of
inoculated bacterial pathogens on chicken
breasts (Rahman et al., 2012) and carcasses
(Rasschaert et al., 2013). Oxidation–reduction
potential has been considered as one of the
major factors affecting antimicrobial activity
(Aider et al., 2012). The safety of antimicro-
bial interventions has been extensively dis-
cussed in the European Union owing to
lingering toxic residues at the consumption
stage (EFSA, 2005; Hugas and Tsigarida,
2008; FAO/WHO, 2018). The use of NaClO
in the presence of organic matter promotes
THM formation by an oxidation reaction, and
thus, it has been considered a great disad-
vantage. Chloroform is considered as an in-
dicator of these toxic compounds and has
been detected after NaClO treatments in
broiler chickens, which represents a chemical
hazard because of its carcinogenic properties
(Robinson et al., 1981; Vizzier-Thaxton et al.,
2010). No evidence has been found about
THM formation resulting from the use of
electrolyzed water. This work was aimed to
study the effectiveness of NEW antimicrobial
intervention in broiler chickens, their physi-
cochemical properties after treatments, and
THM generation.
MATERIALS AND METHODS
Chemicals
Neutral electrolyzed water was obtained by
dissolving 1% NaCl in water and subjected to
an electrolytic process in an experimental
prototype (Center for Research and Develop-
ment of Electrochemistry, CIDETEQ, Queré-
taro, Mexico). After production, NEW was
adjusted to a pH of 6.5 and showed an ORP of
1,123 mV (normalized to a standard hydrogen
electrode). A commercial brand of NaClO
(Cloralex, Nuevo León, Mexico) showed pH
of 10.8, and ORP of 695 mV. A chromato-
graphic standard mixture (Supelco, Bellafonte,
PA) of 4 THM (2,000 mg/L each): chloroform
(CHCl3), bromodichloromethane (CHBrCl2),
chlorodibromomethane (CHClBr2), and bro-
moform (CHBr3) diluted in methanol was
used to set up calibration curves for THM
quantification.
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624
Bacterial Inoculum and Nalidixic Acid
Adaptation
Salmonella enterica subsp. enterica ser.
Typhimurium ATCC 14028 and S. enterica
subsp. enterica ser. Infantis strains (Department
of Food Research and Postgraduate Studies of
the Autonomous University of Queretaro) were
kept frozen in glass beads at 270
C. The
serotype Typhimurium is among the most
common Salmonella serotypes with resistance
to antibiotics, while CDC (2019) reported a
multistate outbreak in which the serotype
Infantis was linked to raw chicken products.
The strains were transferred to tryptic soy broth
(TSB) (Bioxon, Cuautitlan, Mexico) and incu-
bated at 37
C in an orbital shaker (Lab-Line, IL)
at 180 rpm during 24 h. Then, 0.1 mL of each
culture was transferred to 250-mL flasks con-
taining 90 mL TSB and incubated at 37
C for
24 h. After 2 passage stages, an equal volume of
each culture was centrifuged (Eppendorf
5804 R, Hamburg, Germany) at 4,000 3 g for
10 min at 4
C, washed twice with saline solu-
tion (SS, 0.85% w/v NaCl), and finally resus-
pended in 10 mL of SS to achieve 8 log cfu/mL
of each culture. The culture population was
determined by spread plate method using
0.1 mL aliquots on tryptic soy agar (TSA) plates
(Bioxon).
S. Typhimurium ATCC 14028 and S. Infantis
cultures were adapted to 50 mg/L nalidixic acid
(NA) (Sigma-Aldrich, MO) as previously re-
ported (Chintagari et al., 2015). Each strain was
cultured separately in 10 mL TSB broth for
18 h at 37
C. Then, 200 mL aliquots of each
culture was transferred to TSB supplemented
with increasing concentrations of NA (0, 5, 10,
20, 30, 40, and 50 mg/L) to promote cell
adaptation and incubated at 37
C in an orbital
shaker (Lab-Line) at 150 rpm for 24 h. Both
adapted cultures were used as pure culture
mixture.
Antimicrobial Activity on Pure Culture
Mixture
According to Rahman et al. (2010), 1 mL of
the bacterial mixture (8 log cfu/mL) was added
into tubes containing 9 mL of different aqueous
concentrations of NEW (5, 7, 9, 11, 13, and
JAPR: Research Report
15 mg/L) and NaClO (25, 50, 75, 100, and
125 mg/L). Exposure times for both treatments
were 1, 2, 3, 4, and 5 min, and a control with
deionized water was used. Chlorinated com-
pounds were inactivated by performing the first
decimal dilution in neutralizing buffer, and
subsequent 10-fold serial dilutions were per-
formed in SS. Surviving microorganisms from
each treatment were determined by the spread
plate method (detection limit $1 log cfu/mL)
using TSA containing NA (50 mg/L) and then
incubated at 37
C for 24 h.
Cells Visualization Using Fluorescence and
Scanning Electron Microscopy
One milliliter of the bacterial mixture was
treated separately with 9 mL of NaClO or NEW
at 50 mg/L TAC, and after 1 min, 1 mL from
each treatment was filtered and fixed on a 0.22-
mm pore size hydrophilic polyvinylidene fluo-
ride membrane (Millipore, MA) using 2% (v/v)
glutaraldehyde (J.T. Baker, Xalostoc, México)
for 24 h. Serial dehydration steps were followed
using ethanol (J.T. Baker) [30, 50, 80, and
100% (v/v)] for 15 min each, and then, samples
were coated with gold-platinum (Denton Vac-
uum Desk II, NJ) followed by observation under
a microscope (Zeiss Evo 50 Ultraplus, Ober-
kochen, Germany).
For fluorescence microscopy, 1 mL from the
bacterial mixture treated with NaClO or NEW
(50 mg/L of TAC) was filtered through a 0.45-
mm pore size black polycarbonate membrane
(Whatman, Clifton, NJ). The Live/Dead Bac-
Light bacterial viability kit (Molecular Probes,
OR) comprises a mixture of 2 fluorochromes:
SYTO 9 (6 mmol) and propidium iodide
(30 mmol). A mixture of 50 mL of each fluoro-
chrome was used on treated cells for 15 min in
the dark. When exposed to excitation wave-
length of 480/500 nm, an emission showing
fluorescent green color is attributed to intact cell
membranes, whereas fluorescent red color is
associated to damaged cell membranes (Liato
et al., 2017). Untreated cells were used as con-
trol, followed by observation under a fluores-
cence microscope (Axioskop 40, FICT filter;
Zeiss, Göttingen, Germany), fitted with an Axio
CamMRc camera and ZEN pro 2,012 imaging
software (version 1.1.2.0.)
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HERNÁNDEZ-PIMENTEL ET AL: ANTIMICROBIALS ON CHICKEN
Neutral Electrolyzed Water Antimicrobial
Intervention on Inoculated Broiler Chicken
Carcasses
The local slaughterhouse (Pilgrim’s Pride,
TIF A-282; Carretera a los Cues 3, Querétaro,
Qro., 76,246, México) initiated processing by
manual hang, and then, the birds were electri-
cally stunned and killed by exsanguination.
Carcasses were scalded at 54
C 6 1
C before
mechanical plucking and evisceration, followed
by washing inside and outside with cool water,
and then chilled by immersion in water at
2
C 6 1
C for 1.5 h. Ninety chicken carcasses
were randomly taken from the processing line
before immersion chilling. Carcasses were
placed altogether in a disinfected 200-L
container and transported at 5
C 6 1
C in a
refrigerated truck and processed the same day
(about 0.5 h between sampling and laboratory
processing.)
The difference in specific growth rate of
wild and NA-resistant S. Typhimurium was
only 0.01 h21 (1.79 h21 and 1.80 h21,
respectively), whereas that difference for S.
Infantis was 0.02 h21 (1.85 h21 for the wild
strain vs. 1.83 h21 for the NA-resistant strain).
Thus, it was considered that the experiments
were not biased because of growth rate dif-
ferences of the 2 strains. One mL of the
resistant Salmonella pure cultures mixture
adjusted to 6 log cfu/mL was inoculated,
allowing it to drain through the walls of the
gastrointestinal cavity of the previously evis-
cerated carcasses and then-air dried for 10 min
in a laminar flow cabinet. Three independent
experiments were carried out involving 30
carcasses per treatment, which were immersed
in a solution (for 1 h at 3
C) containing
50 mg/L TAC (for NEW and NaClO) and
distilled water as control. Each carcass was
placed in a polyethylene bag containing
100 mL of neutralizing buffer (Difco, MD),
scrubbed during 2 min, and 1 mL aliquot was
added to a tube containing 9 mL of SS, fol-
lowed by 10-fold serial dilutions. An appro-
priate dilution was plated on TSA added with
50 mg/L NA, followed by incubation at 37
C
during 24 h. Salmonella population after each
treatment is reported as the mean 6 SD.
625
Means comparison was conducted using Stu-
dent t test (P , 0.05).
Neutral Electrolyzed Water Antimicrobial
Activity on Broiler Chicken Carcasses
In this study, 180 broiler chicken carcasses
were selected from the local slaughterhouse
(Pilgrim’s Pride, TIF A-282) before immersion
chilling, and transported within 30 min at 2
C
to 4
C to the biotechnology laboratory of the
University of Querétaro. Three different in-
dependent treatments on 60 broiler carcasses
were conducted using NEW or NaClO at
50 mg/L of TAC, and the control using
distilled water. Treatments were applied by
immersion in separate 100-L containers at
3
C 6 1
C for 1.5 h, in agreement with the
local slaughterhouse processing conditions.
After treatments, carcasses were individually
and aseptically packaged in sterile low-density
multilayer polyethylene bags (Food Saver
V3800, NY). This manufacturer claims that
bags show very low water vapor permeability
and low permeability to oxygen and carbon
dioxide. All treatments were maintained under
refrigeration (5
C 6 1
C), and 5 carcasses per
treatment were sampled after 0, 4, 8, and 10 D
of storage. Mean comparison was conducted
among treatments using Tukey’s test (P ,
0.05); blocking by sampling day was used to
discriminate natural changes with storage
time.
Total Viable Counts and Total Coliforms on
Broiler Chicken Carcasses
On each sampling day, the natural microflora
was determined. Each carcass was placed in a
polyethylene bag containing 100 mL of peptone
water and scrubbed during 2 min. Then, 1 mL
aliquot was added to a tube containing 9 mL of
SS, followed by 10-fold serial dilutions. The
appropriate dilution was placed on plate count
agar for total viable count (TVC) and on Violet
Red Bile agar for total coliforms (TC) and
incubated for 24 h at 37
C and 35
C, respec-
tively. Results were expressed as log cfu/mL of
TVC and TC.
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626
Color Determination of Broiler Chicken
Carcasses After NEW Treatments
In accordance with the CIELAB method
(Hunter and Harold, 1987), the parameters L*
(lightness), a* (red/green), and b* (yellow/blue)
were evaluated in the breast (frontal and lateral)
and in the back. A Konica colorimeter (Minolta
CR400; Konica, Osaka, Japan), fitted with
illumination source D65, 10
angle, and auto-
matic white calibration, was used. Color
changes (DE) for each part of chicken carcasses
during 10 D of storage was determined using
equation (1):
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
 
DE ¼ ðDLÞ2 1ðDaÞ2 1ðDbÞ2
Total Volatile Basic Nitrogen and pH
Total volatile basic nitrogen (TVB-N) is a
reference index that has been used to evaluate
a chicken’s freshness, and its level increases
with bacterial spoilage and/or enzymatic
degradation (Urmila et al., 2015). The Conway
microdiffusion method was used (Hong et al.,
2015), involving samples of 5 g from each
carcass that were homogenized in 45 mL
distilled water for 2 min and then centrifuged
at 21,000 3 g for 5 min. The supernatant was
adjusted to 50 mL with distilled water and
filtered (Whatman No. 1). One mL of the
filtrate was placed into the outer space of the
Conway dish, whereas 1 mL of 0.01 N boric
acid (H3BO3) and 2 drops of Conway reagent
[1:1, methyl red 0.066% (w/v):bromocresol
green 0.066% (w/v)] were placed in the inner
space. The dish was sealed immediately after
adding 1 mL of saturated K2CO3 solution in
the outer diffusion chamber, slightly shaken,
and incubated at 37
C for 120 min. The boric
acid in the inner space was titrated with
0.02 N H2SO4, and TVBN was quantified
using the following equation (Min et al.,
2007):
ða  bÞ3f 328:014
TVBN mg% ¼ 3100 (2)
s
where S is the sample weight (g), b is the
volume of H2SO4 added to the blank (mL), a is
the volume of H2SO4 added to the sample
(mL), and f is the H2SO4 concentration.
JAPR: Research Report
For pH measurements, filtered solutions from
TVB-N tests were analyzed using a pH meter
(Thermo Fisher Scientific, MA).
Trihalomethane Detection and Quantification
on Chicken Carcasses
A total of 120 carcasses treated with 50 mg/L
TAC of NEW or NaClO treatments were
analyzed for THM detection, and a control with
distilled water was used. All treatments were
applied by immersion in 100 L of antimicrobials
solutions, at 3
C 6 1
C during 90 min. Re-
covery tests were conducted on 3 tissues (fat,
meat, and skin; 5 g) were cut from each carcass
and placed in 10 mL methanol (J.T. Baker).
(1)
Samples were manually stirred for 3 min, fol-
lowed by filtration through a 0.22-mm pore
diameter membrane (Millipore) and injected to a
Gas Chromatography/Mass Spectra (GC/MS)
equipment (Agilent 6850/5973, Santa Clara,
CA) fitted with an autosampler; the injection
volume was 1 mL, and split was set at 1:60. The
capillary column used (HP-MS5, Agilent) was
30 m long x 0.25 mm internal diameter, coated
with 0.32-mm-thick film comprising 95%
dimethyl–5% diphenylpolysiloxane. Chromato-
graphic conditions were as follows: Injection
temperature 250
C; column temperature 48
C
for 1 min, increased 2
C/min up to 52
C, fol-
lowed by increasing 5
C/min up to 57
C, and
kept for 3 min. The electron capture detector
temperature was 250
C, and injection volume
was 2 mL in splitless mode. A 20 mg/L stock
solution was prepared from the THM standard,
and the method 8260C (EPA, 2018) was fol-
lowed. Calibration curves were developed for
the stock solutions, using concentrations be-
tween 10 and 500 mg/L. Detection and quanti-
fication were conducted by the enhanced
ChemStation (v. d38, 2,001).
Trihalomethane Detection and Quantification
on Chicken Legs
The following experiments were conducted to
determine the effect of NEW at over-chlorination
condition. Thirty-six chicken legs were obtained
from a local market (“General Mariano Esco-
bedo,” Lucas Alamán 56, Centro, Querétaro,
Qro., 76000, México) and refrigerated until
analysis on each of 3 experiments. Legs were
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HERNÁNDEZ-PIMENTEL ET AL: ANTIMICROBIALS ON CHICKEN
selected because of good skin coverage as well as
exposed meat tissue and fat (Vizzier-Thaxton
et al., 2010). A completely randomized 2 3 3
experimental design was used and 3 independent
experiments were conducted in duplicate, and the
experimental unit comprised 2 legs. One factor
was the antimicrobial agent at 2 levels (NEW and
NaClO), and the other was antimicrobials con-
centration at 3 levels (50, 100, and 150 mg/L of
TAC), and one control was carried out with
distilled water. The response variable was THM
concentration. Legs were initially washed with
distilled water to remove blood residues and
remaining feathers. All treatments were applied
by immersion into a 1-L container at 3
C 6 1
C,
pH 6.5, during 1 h with slight agitation using a
magnetic stirrer. After treatments, 3 samples (5 g)
from each leg comprising skin, meat, and fat
tissue were randomly cut, placed into vials with
10 mL of methanol, and stirred manually for
3 min. Then, tissues were removed, and the
remaining methanol solution was filtered through
a 0.22-mm pore diameter membrane (Millipore)
and injected into the GC/MS equipment for
analysis using the same chromatographic condi-
tions described in the previous section. Response
variable was the quantification of each type of
THM: chloroform (CHCl3), bromodichloro-
methane (BrCHCl2), chlorodibromomethane
(ClCHBr2), and bromoform (CHBr3). Statistical
analysis was conducted using Tukey’s test (P #
0.05) by comparing the effect of each factor and
its interaction.
Statistical Analysis
Results are shown as the mean of 3 inde-
pendent experiments 6 SD. Data in all experi-
ments were analyzed with JMP 8.0 software
(SAS Institute, NC), and difference between
treatments was considered significant when P ,
0.05.
RESULTS AND DISCUSSION
Neutral Electrolyzed Water Antimicrobial
Activity on Salmonella Pure Culture Mixture
Neutral electrolyzed water at 14 mg/L TAC
completely inhibited (.6 log cfu/mL) the Sal-
monella pure culture mixture after 1 min contact
627
time, whereas NaClO achieved this activity at
125 mg/L TAC. In addition, when exposure time
was 5 min, the total pathogens inhibition was
achieved with NEW at 5 mg/L TAC, whereas
NaClO required 50 mg/L, indicating that the
antimicrobial activity of NEW was about 10
times higher than that of NaClO (Figures 1A
and 1B). This effect was attributed to the com-
bination of oxidizing compounds present in
NEW that show several antimicrobial mecha-
nisms of action, which together with a high
redox potential make it more efficient than
NaClO.
Acidic electrolyzed water is obtained from an
electrolysis chamber containing dilute sodium
chloride solution using a membrane separating
the electrodes. Two types of electrolyzed water
are generated simultaneously: AEW is produced
in the anode, whereas basic electrolyzed water is
generated in the cathode. A report using
100 mg/L TAC of AEW achieved .5 log cfu/
mL reduction of S. Typhimurium contacted for
15 min (Fabrizio et al., 2003). There is however
a disadvantage of using AEW, which is its
corrosive potential on stainless steel surfaces
(Len et al., 2002).
Untreated Salmonella pure culture mixture
was observed by scanning electron microscopy
(Figure 2A). Visualization of cells treated with
50 mg/L TAC of NaClO showed sublethal
damage (Figure 2B), whereas NEW treatment at
same TAC concentration produced cell lysis
(Figure 2C). Treatment of Salmonella pure
culture mixture with 9 mg/L TAC of NEW
produced morphologic changes and cell
swelling (Figures 2D and 2E), showing that
membrane damage is a relevant NEW mecha-
nism of action. Similar damage of S. Typhi-
murium cells was reported after NaClO
treatment using 200 mg/L TAC (Su and
D’Souza, 2012). Comparable cell damage has
been reported for a mixture of 5 Salmonella
strains treated with high-pressure processing
(Sheen et al., 2015).
The effect of NEW and NaClO treatments on
membrane integrity of Salmonella cells was
studied by fluorescence microscopy. Untreated
cells showed only green fluorescence due to
intact cells without surface damage (Figure 3A).
Conversely, after 50 mg/L TAC of NaClO
(sublethal treatment), red-stained cells were
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628 JAPR: Research Report

Figure 1. Surviving cells from pure cultures mixture (Salmonella Typhimurium ATCC 14028 and S. Infantis) after
antimicrobial treatments. (A) Effect of neutral electrolyzed water. (B) Effect of sodium hypochlorite.

observed suggesting cell damage, but scattered
green fluorescence indicated surviving bacteria
(Figure 3B). On the other hand, micrographs of
NEW treatment (50 mg/L TAC) only showed
red-stained cells. In addition, a red mist was
observed indicating cell lysis (Figure 3C).
Moreover, the bactericidal effect was also
confirmed by plate counting achieving a
reduction of .6 log cfu/mL using NEW and of
1 log cfu/mL with NaClO after 1 min treatment
(as per Figure 1B). This technique has also been
used to demonstrate that electroactivated solu-
tions of HClO showed more efficient antimi-
crobial activity against Salmonella cells than
weak organic acids (Liato et al., 2017).
Neutral Electrolyzed Water Antimicrobial
Activity Against Salmonella on Broiler
Chicken Carcasses
Neutral electrolyzed water antimicrobial
activity decreased its marked advantage over
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HERNÁNDEZ-PIMENTEL ET AL: ANTIMICROBIALS ON CHICKEN
Figure 2. Scanning electron micrographs (11,000X) of
Salmonella Typhimurium ATCC 14028, and S.
Infantis–free cells after antimicrobial treatments. (A)
Control. (B) Sublethal treatment with sodium hypo-
chlorite (50 mg/L TAC). (C) Treatment with neutral
electrolyzed water (50 mg/L TAC). (D) Sublethal treat-
ment with neutral electrolyzed water (9 mg/L). (E)
Same as (D) at 94,640X magnification. Abbreviation:
TAC, total available chlorine.
NaClO when applied on broiler chicken car-
casses (90 experimental units) with results
showing no significant difference. Both agents
629
exerted partial microbial inactivation, which
was attributed to the presence of organic matter
that reduced their efficacy, achieving a reduction
of 1.1 log cfu/mL. Despite NEW achieving the
same antimicrobial effect as NaClO on broiler
chicken carcasses, other features such as
competitive production costs, safe handling, and
low corrosion capacity and toxicity need
consideration. A reduction of 1.9 log cfu/mL of
S. Typhimurium in the chicken breast using
10 mg/L TAC of slightly AEW, for 10 min has
been reported (Rahman et al., 2012), which
represents a higher population inhibition than
this work. However, another report (Rasschaert
et al., 2013) showed a similar effect (1.31 log
cfu/mL) against Campylobacter in broiler
chickens carcasses with 50 mg/L TAC of NEW
exposure for 3 min.
Neutral Electrolyzed water Antimicrobial
Activity on Broiler Chickens
For TVC and TC after 10 D of refrigerated
storage, NEW achieved higher reduction (1.2
log cfu/mL) than NaClO treatments (0.4 log cfu/
mL), both applied on uninoculated carcasses
(Figures 4A and 4B). After 10 D, there was no
significant difference (P . 0.05) between
NaClO activity and the control (water) as pre-
viously reported (Northcutt et al., 2005),
whereas no reports on the effect of NEW against
TVC and TC in broiler chicken carcasses were
found.
The selection of chemical processing aids
should be performed carefully because some of
the most commonly used such as sodium chlo-
rite, trisodium phosphate, and peroxyacetic acid
are all toxic to humans and cause irritation on
the skin, eyes, throat, and lungs when exposed
(NIOSH, 2019). Therefore, the use of NEW is
safe and convenient because it is highly avail-
able, does not represent a health hazard, and it is
not required constant monitoring and occupa-
tional safety precautions when used (Wang
et al., 2018).
pH, TVB-N, and Color Changes in Broiler
Chickens after Antimicrobial Treatments
Broiler chicken carcasses treated with NEW
showed significant difference (P , 0.05) in pH
compared with NaClO and control after 10 D of
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630
Figure 3. Fluorescence micrographs of Salmonella
Typhimurium ATCC 14028 and S. Infantis mixed cul-
ture after NEW treatment. (A) Control. (B) NEW sub-
lethal treatment (9 mg/L TAC). (C) NEW lethal
treatment (50 mg/L TAC). Cells with intact membrane
show fluorescent green color, whereas cells with
damaged membrane exhibit fluorescent red color. Ab-
breviations: NEW, neutral electrolyzed water;
TAC, total available chlorine.
storage (Figure 5A). Broiler chickens treated with
NEW achieved the lowest pH value (6.2) after
10 D of study, similar to a previous report
(Fabrizio et al., 2002). Carcasses treated with
NEW showed significantly (P , 0.05) lower
TVB-N values than using NaClO after 10 D of
storage, whereas NaClO treatment showed
similar value as the control, in agreement with
Duan et al. (2017). To our knowledge, there is no
JAPR: Research Report
legislation relative to TVB-N in broiler chickens
besides China and Korea. Total volatile basic ni-
trogen compounds in chicken increase with
spoilage by either bacteria or enzymes, and their
levels in chicken meat are one of the important
chemical reference indices to evaluate freshness.
Neutral electrolyzed water was the only
treatment achieving the Korean acceptable limit
(#20 mg % TVB-N) after 8 D of storage
(Figure 5B) (Hong et al., 2015). In accordance
with the Chinese legislation (GB National
Standards of People’ s Republic of China,
2018), all treatments were lower than the
acceptable limit (#15 mg % TVB-N) at 4 D of
storage (Min et al., 2007). From these figures,
apparently, there is a relationship between
TVBN and pH because the higher the pH, the
lower microorganisms are inactivated, leading
to more meat protein hydrolysis resulting in
higher TVBN concentration (Urmila et al.,
2015).
Neutral electrolyzed water and control treat-
ments did not show significant color difference
(DE) after 10 D of storage in frontal and lateral
breast tissues (Figure 5C). NaClO treatment
showed significant color difference compared
with NEW and control treatments. However, the
back tissue showed significantly lower DE than
control and NaClO treatments.
Trihalomethane Detection and Quantification
on Chicken Carcasses
From GC/MS, the retention time of each
chemical comprising the THM standard mixture
was identified, followed by comparison with the
NIST database. Standard detection and quanti-
fication were optimized by single ion moni-
toring analysis mode, using m/z values: CHCl3:
119, 118, 85, and 83; CHBrCl2: 127, 123, 85,
and 83; CHClBr2: 208, 206, 207, and 129; and
CHBr3: 254, 252, 175, and 173. Retention times
for CHCl3, CHBrCl2, CHClBr2, and CHBr3
were 1.4, 1.9, 3.0, and 4.5 min, respectively. In
addition, recoveries of CHCl3, CHBrCl2,
CHClBr2, and CHBr3 were 99.1, 99.3, 98.9, and
99.3%, respectively. The determination coeffi-
cient (R2) of each standard curve was .0.98,
and the detection thresholds were CHCl3
.55 mg/L, CHBrCl2 .42 mg/L, CHClBr2
.51 mg/L, and CHBr3 .18 mg/L.
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HERNÁNDEZ-PIMENTEL ET AL: ANTIMICROBIALS ON CHICKEN 631

Figure 4. Recovery of bacterial population from chicken broilers treated with 50 mg/L TAC of NEW and NaClO at
2
C 6 2
C during 90 min and further storage of 10 D at 3
C 6 2
C. (A) Total viable counts. (B) Total coliforms. On
each figure, treatments with different capital letter indicate significant difference, Tukey test (P , 0.05). Abbrevia-
tions: NEW, neutral electrolyzed water; TAC, total available chlorine.

Trihalomethanes were not detected from
broiler chicken carcasses treated with 50 mg/L
of NEW or NaClO. In addition, the distilled
water controls did not show any THM. The
most reported THM resulting from NaClO
treatments is CHCl3 (FAO/WHO, 2018).
Chicken carcasses treated with 50 mg/L of
NaClO at 15
C for 5 min, produced up to
447 mg/kg of CHCl3 by using a petroleum ether
extraction technique, whereas different treat-
ment temperature and exposure time (5
C for
20 min) led to 146 mg/kg (Robinson et al.,
1981). However, using a purge-and-trap sys-
tem and thiosulphate extraction of CHCl3, a
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632 JAPR: Research Report

Figure 5. Physicochemical properties of broiler chicken meat after treatments of 50 mg/L TAC of NEW and NaClO at
2
C 6 2
C during 90 min and further storage during 10 D at 3
C 6 2
C. (A) pH; (B) total basic volatile nitrogen; and
(C) color changes measured in 3 different tissues after 10 D. Abbreviations: NEW, neutral electrolyzed water;
TAC, total available chlorine.
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HERNÁNDEZ-PIMENTEL ET AL: ANTIMICROBIALS ON CHICKEN
Table 1. Trihalomethanes determination on chicken legs after neutral 1electrolyzed water and sodium
hypochlorite treatments.
Agent Concentration (mg/L)
Neutral Electrolyzed Water 50
100
150
Sodium Hypochlorite 50
100
150
Recovery (%)
Different superscript capital letters in each trihalomethane column, indicate significant difference (Tukey test, P , 0.05).
A-C
Mean values 6 SD, n = 36.
significantly lower amount of 16.1 mg/kg was
quantified from broiler chickens after NaClO
treatment .100 mg/L. Nevertheless, after
50 mg/L treatments, no THM could be detected
(Vizzier-Thaxton et al., 2010).
Trihalomethane Detection and Quantification
on Chicken Legs
Trihalomethanes were not detected from
chicken legs using 50 mg/L of NEW or NaClO
treatments (Table 1). However, the non-
corrosiveness, environment-friendly properties,
safe handling, and in situ production at low cost
make NEW a better alternative than NaClO
(Aider et al., 2012). In addition, we found 0.037
and 0.043 mg/kg of CHCl3 after using NEW
treatments with 100 mg/L TAC and 150 mg/L
TAC, respectively, much higher than the
maximum antimicrobial intervention concen-
tration allowed (#50 mg/L) for poultry pro-
cessing in the United States (FSIS, 2019).
Moreover, CHCl3 concentrations were higher
(although not significant) after NaClO treat-
ments at the same TAC concentration (Table 1).
Statistical analysis showed that the antimicro-
bial agent concentration significantly (P , 0.05)
affected the CHCl3 concentration generated,
whereas concentration and type of agent
significantly affected the concentrations of
CHBrCl2 and CHBr3. No THM were detected
after NEW treatments except CHCl3, whereas
NaClO treatments generated CHBrCl2 and
CHBr3 (Table 1) representing a health hazard.
The equilibrium HClO/ClO2 is pH dependent
and considering that for NaClO treatments at
pH $ 10, the ClO2 concentration is close to
100%, whereas NEW at pH 6.5 shows HOCl as
633
Trihalomethanes (mg/kg)
Chloroform Bromodichloromethane Bromoform
,0.011 C
,0.008 C
,0.003B
0.037 6 0.001B ,0.008C ,0.003B
0.043 6 0.001A ,0.008C ,0.003B
,0.011C ,0.008C ,0.003B
0.039 6 0.001B 0.026 6 0.001B 0.020 6 0.001A
0.044 6 0.001A 0.031 6 0.001A 0.023 6 0.001A
99.1 98.9 99.3
the main molecule (Jiménez-Pichardo et al.,
2016). From these 2 species mainly, ClO2 ox-
idizes organic matter, and this may explain why
lower amounts of ClO2 in NEW led to lower
THM generation at equal TAC concentrations.
In the United States, THM mixtures for
chlorinated drinking water are permitted at
80 mg/kg, whereas the European Union legis-
lation permits 100 mg/kg (EFSA, 2005; EPA,
2018). No reports were found about THM pro-
duction by using NEW. Furthermore, chemical
hazards are a health concern for broiler chicken
consumers, and it is important to validate that
NEW treatments are safe by using whole
chicken carcasses under processing conditions
similar to those used in the poultry industry. In
addition, in a previous report, an official method
of analysis after NaClO treatments was not used
(Robinson et al., 1981), whereas another report
used official methods for chicken parts only
instead of whole carcasses (Vizzier-Thaxton
et al., 2010).
CONCLUSIONS AND APPLICATIONS
1. Neutral electrolyzed water achieved 10 times
higher antimicrobial activity than NaClO on
Salmonella pure culture mixture.
2. Morphologic changes and cell swelling
indicated that membrane damage is a rele-
vant mechanism of action of NEW.
3. Neutral electrolyzed water was more efficient
in controlling TVC and TC populations than
NaClO on chicken carcasses during 10 D of
storage, without affecting color and pH; its
use produced lower TVB-N in chicken meat.
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634
4. The antimicrobial agent concentration
significantly affected CHCl3 generation on
chicken legs after antimicrobial intervention,
but concentration and type significantly
increased the generation of CHBrCl2 and
CHBr3. Trihalomethanes were not detected
in chicken legs or carcasses (skin, fat, or
meat tissue) when using 50 mg/L TAC
treatment of either NaClO or NEW.
5. Neutral electrolyzed water could represent a
safe alternative as antimicrobial intervention
agent during poultry processing, owing to
high availability, safe handling, low corro-
sion capacity, and low toxicity because of no
by-product generation.
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Acknowledgments
Thank are given to CONACYT for PhD grant to VMHP.
We thank Ana Luisa Tovar Álvarez for technical support on
SEM micrographs.
Conflict of Interest Statement: The authors declare no
conflict of interest.