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. -:-–-
https://doi.org/10.1016/j.japr.2020.04.001

1
Corresponding author: [email protected] or [email protected]
2 JAPR: Research Report

DESCRIPTION OF PROBLEM slightly AEW to reduce the population of

inoculated bacterial pathogens on chicken
The Centers for Disease Control and Pre- breasts (Rahman et al., 2012) and carcasses
vention (CDC, 2019) reported that Salmonella (Rasschaert et al., 2013). Oxidation–reduction
is the most common pathogen related to potential has been considered as one of the
broiler chicken outbreaks. Nowadays, sani- major factors affecting antimicrobial activity
tizing agents commonly used for fresh poultry (Aider et al., 2012). The safety of antimicro-
decontamination interventions involve chlo- bial interventions has been extensively dis-
rine compounds, trisodium phosphate, and cussed in the European Union owing to
organic acids. Neutral electrolyzed water lingering toxic residues at the consumption
(NEW) is gaining importance in the food in- stage (EFSA, 2005; Hugas and Tsigarida,
dustry mainly owing to its effective antimi- 2008; FAO/WHO, 2018). The use of NaClO
crobial activity, noncorrosiveness, in the presence of organic matter promotes
environment-friendly properties, in situ pro- THM formation by an oxidation reaction, and
duction at low cost, and safe handling (Aider thus, it has been considered a great disad-
et al., 2012; Jiménez-Pichardo et al., 2016). vantage. Chloroform is considered as an in-
Neutral electrolyzed water was obtained from dicator of these toxic compounds and has
an electrolytic process using sodium chloride been detected after NaClO treatments in
(NaCl) solution (0.1–1% w/v) that generates broiler chickens, which represents a chemical
highly oxidizing chlorine species in automated hazard because of its carcinogenic properties
dispensers, whose titer is usually measured as (Robinson et al., 1981; Vizzier-Thaxton et al.,
total available chlorine (TAC) (Rahman et al., 2010). No evidence has been found about
2010). Several compounds are produced, and THM formation resulting from the use of
the most abundant species are hypochlorous electrolyzed water. This work was aimed to
acid (HOCl), hypochlorite ions (ClO2), chlo- study the effectiveness of NEW antimicrobial
rine dioxide (ClO2), and ozone (O3) (Al-Haq intervention in broiler chickens, their physi-
et al., 2005). The mechanism of action of cochemical properties after treatments, and
NEW has been associated with membrane THM generation.
damage, enzyme inhibition, weakening of
membrane transport capacity, and high
oxidation–reduction potential (ORP) (Rahman MATERIALS AND METHODS
et al., 2016; Ye et al., 2017).
Chemicals
Neutral electrolyzed water is permitted in
the United States for poultry processing at Neutral electrolyzed water was obtained by
doses #50 mg/L (FSIS, 2019), but reports dissolving 1% NaCl in water and subjected to
have mainly focused on its antimicrobial ac- an electrolytic process in an experimental
tivity (Rasschaert et al., 2013; Al-Holy et al., prototype (Center for Research and Develop-
2015) without evaluation of trihalomethanes ment of Electrochemistry, CIDETEQ, Queré-
(THM) formation in treated broiler chickens. taro, Mexico). After production, NEW was
However, nowadays other countries do not adjusted to a pH of 6.5 and showed an ORP of
have legislation concerning its use. Acidic 1,123 mV (normalized to a standard hydrogen
electrolyzed water (AEW) has been previ- electrode). A commercial brand of NaClO
ously used to prevent the presence of food- (Cloralex, Nuevo León, Mexico) showed pH
borne pathogens in chicken carcasses of 10.8, and ORP of 695 mV. A chromato-
(Fabrizio et al., 2002) and chicken legs (Al- graphic standard mixture (Supelco, Bellafonte,
Holy et al., 2015). However, this oxidizing PA) of 4 THM (2,000 mg/L each): chloroform
acidic water is becoming less popular because (CHCl3), bromodichloromethane (CHBrCl2),
of potential corrosion of stainless steel sur- chlorodibromomethane (CHClBr2), and bro-
faces (Len et al., 2002). On the other hand, moform (CHBr3) diluted in methanol was
there are few reports of NEW antimicrobial used to set up calibration curves for THM
interventions on broiler chickens, using quantification.
HERNÁNDEZ-PIMENTEL ET AL: ANTIMICROBIALS ON CHICKEN 3

Bacterial Inoculum and Nalidixic Acid 15 mg/L) and NaClO (25, 50, 75, 100, and
Adaptation 125 mg/L). Exposure times for both treatments
were 1, 2, 3, 4, and 5 min, and a control with
Salmonella enterica subsp. enterica ser. deionized water was used. Chlorinated com-
Typhimurium ATCC 14028 and S. enterica pounds were inactivated by performing the first
subsp. enterica ser. Infantis strains (Department decimal dilution in neutralizing buffer, and
of Food Research and Postgraduate Studies of subsequent 10-fold serial dilutions were per-
the Autonomous University of Queretaro) were formed in SS. Surviving microorganisms from
kept frozen in glass beads at 270
C. The each treatment were determined by the spread
serotype Typhimurium is among the most plate method (detection limit $1 log cfu/mL)
common Salmonella serotypes with resistance using TSA containing NA (50 mg/L) and then
to antibiotics, while CDC (2019) reported a incubated at 37
C for 24 h.
multistate outbreak in which the serotype
Infantis was linked to raw chicken products.
Cells Visualization Using Fluorescence and
The strains were transferred to tryptic soy broth
Scanning Electron Microscopy
(TSB) (Bioxon, Cuautitlan, Mexico) and incu-
bated at 37
C in an orbital shaker (Lab-Line, IL) One milliliter of the bacterial mixture was
at 180 rpm during 24 h. Then, 0.1 mL of each treated separately with 9 mL of NaClO or NEW
culture was transferred to 250-mL flasks con- at 50 mg/L TAC, and after 1 min, 1 mL from
taining 90 mL TSB and incubated at 37
C for each treatment was filtered and fixed on a 0.22-
24 h. After 2 passage stages, an equal volume of mm pore size hydrophilic polyvinylidene fluo-
each culture was centrifuged (Eppendorf ride membrane (Millipore, MA) using 2% (v/v)
5804 R, Hamburg, Germany) at 4,000 3 g for glutaraldehyde (J.T. Baker, Xalostoc, México)
10 min at 4
C, washed twice with saline solu- for 24 h. Serial dehydration steps were followed
tion (SS, 0.85% w/v NaCl), and finally resus- using ethanol (J.T. Baker) [30, 50, 80, and
pended in 10 mL of SS to achieve 8 log cfu/mL 100% (v/v)] for 15 min each, and then, samples
of each culture. The culture population was were coated with gold-platinum (Denton Vac-
determined by spread plate method using uum Desk II, NJ) followed by observation under
0.1 mL aliquots on tryptic soy agar (TSA) plates a microscope (Zeiss Evo 50 Ultraplus, Ober-
(Bioxon). kochen, Germany).
S. Typhimurium ATCC 14028 and S. Infantis For fluorescence microscopy, 1 mL from the
cultures were adapted to 50 mg/L nalidixic acid bacterial mixture treated with NaClO or NEW
(NA) (Sigma-Aldrich, MO) as previously re- (50 mg/L of TAC) was filtered through a 0.45-
ported (Chintagari et al., 2015). Each strain was mm pore size black polycarbonate membrane
cultured separately in 10 mL TSB broth for (Whatman, Clifton, NJ). The Live/Dead Bac-
18 h at 37
C. Then, 200 mL aliquots of each Light bacterial viability kit (Molecular Probes,
culture was transferred to TSB supplemented OR) comprises a mixture of 2 fluorochromes:
with increasing concentrations of NA (0, 5, 10, SYTO 9 (6 mmol) and propidium iodide
20, 30, 40, and 50 mg/L) to promote cell (30 mmol). A mixture of 50 mL of each fluoro-
adaptation and incubated at 37
C in an orbital chrome was used on treated cells for 15 min in
shaker (Lab-Line) at 150 rpm for 24 h. Both the dark. When exposed to excitation wave-
adapted cultures were used as pure culture length of 480/500 nm, an emission showing
mixture. fluorescent green color is attributed to intact cell
membranes, whereas fluorescent red color is
Antimicrobial Activity on Pure Culture associated to damaged cell membranes (Liato
Mixture et al., 2017). Untreated cells were used as con-
trol, followed by observation under a fluores-
According to Rahman et al. (2010), 1 mL of cence microscope (Axioskop 40, FICT filter;
the bacterial mixture (8 log cfu/mL) was added Zeiss, Göttingen, Germany), fitted with an Axio
into tubes containing 9 mL of different aqueous CamMRc camera and ZEN pro 2,012 imaging
concentrations of NEW (5, 7, 9, 11, 13, and software (version 1.1.2.0.)
4 JAPR: Research Report

Neutral Electrolyzed Water Antimicrobial Means comparison was conducted using Stu-
Intervention on Inoculated Broiler Chicken dent t test (P , 0.05).
Carcasses
Neutral Electrolyzed Water Antimicrobial
The local slaughterhouse (Pilgrim’s Pride, Activity on Broiler Chicken Carcasses
TIF A-282; Carretera a los Cues 3, Querétaro,
Qro., 76,246, México) initiated processing by In this study, 180 broiler chicken carcasses
manual hang, and then, the birds were electri- were selected from the local slaughterhouse
cally stunned and killed by exsanguination. (Pilgrim’s Pride, TIF A-282) before immersion
Carcasses were scalded at 54
C 6 1
C before chilling, and transported within 30 min at 2
C
mechanical plucking and evisceration, followed to 4
C to the biotechnology laboratory of the
by washing inside and outside with cool water, University of Querétaro. Three different in-
and then chilled by immersion in water at dependent treatments on 60 broiler carcasses
2
C 6 1
C for 1.5 h. Ninety chicken carcasses were conducted using NEW or NaClO at
were randomly taken from the processing line 50 mg/L of TAC, and the control using
before immersion chilling. Carcasses were distilled water. Treatments were applied by
placed altogether in a disinfected 200-L immersion in separate 100-L containers at
container and transported at 5C 6 1C in a 3 6 1
C for 1.5 h, in agreement with the local
refrigerated truck and processed the same day slaughterhouse processing conditions. After
(about 0.5 h between sampling and laboratory treatments, carcasses were individually and
processing.) aseptically packaged in sterile low-density
The difference in specific growth rate of multilayer polyethylene bags (Food Saver
wild and NA-resistant S. Typhimurium was V3800, NY). This manufacturer claims that
only 0.01 h21 (1.79 h21 and 1.80 h21, bags show very low water vapor permeability
respectively), whereas that difference for S. and low permeability to oxygen and carbon
Infantis was 0.02 h21 (1.85 h21 for the wild dioxide. All treatments were maintained under
strain vs. 1.83 h21 for the NA-resistant strain). refrigeration (5
C 6 1
C), and 5 carcasses per
Thus, it was considered that the experiments treatment were sampled after 0, 4, 8, and 10 D
were not biased because of growth rate dif- of storage. Mean comparison was conducted
ferences of the 2 strains. One mL of the among treatments using Tukey’s test (P ,
resistant Salmonella pure cultures mixture 0.05); blocking by sampling day was used to
adjusted to 6 log cfu/mL was inoculated, discriminate natural changes with storage
allowing it to drain through the walls of the time.
gastrointestinal cavity of the previously evis-
cerated carcasses and then-air dried for 10 min
in a laminar flow cabinet. Three independent Total Viable Counts and Total Coliforms on
experiments were carried out involving 30 Broiler Chicken Carcasses
carcasses per treatment, which were immersed
in a solution (for 1 h at 3
C) containing On each sampling day, the natural microflora
50 mg/L TAC (for NEW and NaClO) and was determined. Each carcass was placed in a
distilled water as control. Each carcass was polyethylene bag containing 100 mL of peptone
placed in a polyethylene bag containing water and scrubbed during 2 min. Then, 1 mL
100 mL of neutralizing buffer (Difco, MD), aliquot was added to a tube containing 9 mL of
scrubbed during 2 min, and 1 mL aliquot was SS, followed by 10-fold serial dilutions. The
added to a tube containing 9 mL of SS, fol- appropriate dilution was placed on plate count
lowed by 10-fold serial dilutions. An appro- agar for total viable count (TVC) and on Violet
priate dilution was plated on TSA added with Red Bile agar for total coliforms (TC) and
50 mg/L NA, followed by incubation at 37
C incubated for 24 h at 37
C and 35
C, respec-
during 24 h. Salmonella population after each tively. Results were expressed as log cfu/mL of
treatment is reported as the mean 6 SD. TVC and TC.
HERNÁNDEZ-PIMENTEL ET AL: ANTIMICROBIALS ON CHICKEN 5

Color Determination of Broiler Chicken For pH measurements, filtered solutions from

Carcasses After NEW Treatments TVB-N tests were analyzed using a pH meter
(Thermo Fisher Scientific, MA).
In accordance with the CIELAB method
(Hunter and Harold, 1987), the parameters L* Trihalomethane Detection and Quantification
(lightness), a* (red/green), and b* (yellow/blue) on Chicken Carcasses
were evaluated in the breast (frontal and lateral)
and in the back. A Konica colorimeter (Minolta A total of 120 carcasses treated with 50 mg/L
CR400; Konica, Osaka, Japan), fitted with TAC of NEW or NaClO treatments were
illumination source D65, 10
angle, and auto- analyzed for THM detection, and a control with
matic white calibration, was used. Color distilled water was used. All treatments were
changes (DE) for each part of chicken carcasses applied by immersion in 100 L of antimicrobials
during 10 D of storage was determined using solutions, at 3
C 6 1
C during 90 min. Re-
equation (1): covery tests were conducted on 3 tissues (fat,
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi meat, and skin; 5 g) were cut from each carcass
  and placed in 10 mL methanol (J.T. Baker).
DE ¼ ðDLÞ2 1ðDaÞ2 1ðDbÞ2 (1)
Samples were manually stirred for 3 min, fol-
Total Volatile Basic Nitrogen and pH lowed by filtration through a 0.22-mm pore
diameter membrane (Millipore) and injected to a
Total volatile basic nitrogen (TVB-N) is a Gas Chromatography/Mass Spectra (GC/MS)
reference index that has been used to evaluate equipment (Agilent 6850/5973, Santa Clara,
a chicken’s freshness, and its level increases CA) fitted with an autosampler; the injection
with bacterial spoilage and/or enzymatic volume was 1 mL, and split was set at 1:60. The
degradation (Urmila et al., 2015). The Conway capillary column used (HP-MS5, Agilent) was
microdiffusion method was used (Hong et al., 30 m long x 0.25 mm internal diameter, coated
2015), involving samples of 5 g from each with 0.32-mm-thick film comprising 95%
carcass that were homogenized in 45 mL dimethyl–5% diphenylpolysiloxane. Chromato-
distilled water for 2 min and then centrifuged graphic conditions were as follows: Injection
at 21,000 3 g for 5 min. The supernatant was temperature 250
C; column temperature 48
C
adjusted to 50 mL with distilled water and for 1 min, increased 2
C/min up to 52
C, fol-
filtered (Whatman No. 1). One mL of the lowed by increasing 5
C/min up to 57
C, and
filtrate was placed into the outer space of the kept for 3 min. The electron capture detector
Conway dish, whereas 1 mL of 0.01 N boric temperature was 250
C, and injection volume
acid (H3BO3) and 2 drops of Conway reagent was 2 mL in splitless mode. A 20 mg/L stock
[1:1, methyl red 0.066% (w/v):bromocresol solution was prepared from the THM standard,
green 0.066% (w/v)] were placed in the inner and the method 8260C (EPA, 2018) was fol-
space. The dish was sealed immediately after lowed. Calibration curves were developed for
adding 1 mL of saturated K2CO3 solution in the stock solutions, using concentrations be-
the outer diffusion chamber, slightly shaken, tween 10 and 500 mg/L. Detection and quanti-
and incubated at 37
C for 120 min. The boric fication were conducted by the enhanced
acid in the inner space was titrated with ChemStation (v. d38, 2,001).
0.02 N H2SO4, and TVBN was quantified
using the following equation (Min et al., Trihalomethane Detection and Quantification
2007): on Chicken Legs

ða  bÞ3f 328:014 The following experiments were conducted to

TVBN0mg% ¼ 3100 (2) determine the effect of NEW at over-chlorination
s
condition. Thirty-six chicken legs were obtained
where S is the sample weight (g), b is the from a local market (“General Mariano Esco-
volume of H2SO4 added to the blank (mL), a is bedo,” Lucas Alamán 56, Centro, Querétaro,
the volume of H2SO4 added to the sample Qro., 76000, México) and refrigerated until
(mL), and f is the H2SO4 concentration. analysis on each of 3 experiments. Legs were
6 JAPR: Research Report

selected because of good skin coverage as well as time, whereas NaClO achieved this activity at
exposed meat tissue and fat (Vizzier-Thaxton 125 mg/L TAC. In addition, when exposure time
et al., 2010). A completely randomized 2 3 3 was 5 min, the total pathogens inhibition was
experimental design was used and 3 independent achieved with NEW at 5 mg/L TAC, whereas
experiments were conducted in duplicate, and the NaClO required 50 mg/L, indicating that the
experimental unit comprised 2 legs. One factor antimicrobial activity of NEW was about 10
was the antimicrobial agent at 2 levels (NEW and times higher than that of NaClO (Figures 1A
NaClO), and the other was antimicrobials con- and 1B). This effect was attributed to the com-
centration at 3 levels (50, 100, and 150 mg/L of bination of oxidizing compounds present in
TAC), and one control was carried out with NEW that show several antimicrobial mecha-
distilled water. The response variable was THM nisms of action, which together with a high
concentration. Legs were initially washed with redox potential make it more efficient than
distilled water to remove blood residues and NaClO.
remaining feathers. All treatments were applied Acidic electrolyzed water is obtained from an
by immersion into a 1-L container at 3
C 6 1
C, electrolysis chamber containing dilute sodium
pH 6.5, during 1 h with slight agitation using a chloride solution using a membrane separating
magnetic stirrer. After treatments, 3 samples (5 g) the electrodes. Two types of electrolyzed water
from each leg comprising skin, meat, and fat are generated simultaneously: AEW is produced
tissue were randomly cut, placed into vials with in the anode, whereas basic electrolyzed water is
10 mL of methanol, and stirred manually for generated in the cathode. A report using
3 min. Then, tissues were removed, and the 100 mg/L TAC of AEW achieved .5 log cfu/
remaining methanol solution was filtered through mL reduction of S. Typhimurium contacted for
a 0.22-mm pore diameter membrane (Millipore) 15 min (Fabrizio et al., 2003). There is however
and injected into the GC/MS equipment for a disadvantage of using AEW, which is its
analysis using the same chromatographic condi- corrosive potential on stainless steel surfaces
tions described in the previous section. Response (Len et al., 2002).
variable was the quantification of each type of Untreated Salmonella pure culture mixture
THM: chloroform (CHCl3), bromodichloro- was observed by scanning electron microscopy
methane (BrCHCl2), chlorodibromomethane (Figure 2A). Visualization of cells treated with
(ClCHBr2), and bromoform (CHBr3). Statistical 50 mg/L TAC of NaClO showed sublethal
analysis was conducted using Tukey’s test (P # damage (Figure 2B), whereas NEW treatment at
0.05) by comparing the effect of each factor and same TAC concentration produced cell lysis
its interaction. (Figure 2C). Treatment of Salmonella pure
culture mixture with 9 mg/L TAC of NEW
Statistical Analysis produced morphologic changes and cell
swelling (Figures 2D and 2E), showing that
Results are shown as the mean of 3 inde- membrane damage is a relevant NEW mecha-
pendent experiments 6 SD. Data in all experi- nism of action. Similar damage of S. Typhi-
ments were analyzed with JMP 8.0 software murium cells was reported after NaClO
(SAS Institute, NC), and difference between treatment using 200 mg/L TAC (Su and
treatments was considered significant when P , D’Souza, 2012). Comparable cell damage has
0.05. been reported for a mixture of 5 Salmonella
strains treated with high-pressure processing
(Sheen et al., 2015).
RESULTS AND DISCUSSION The effect of NEW and NaClO treatments on
Neutral Electrolyzed Water Antimicrobial membrane integrity of Salmonella cells was
Activity on Salmonella Pure Culture Mixture studied by fluorescence microscopy. Untreated
cells showed only green fluorescence due to
Neutral electrolyzed water at 14 mg/L TAC intact cells without surface damage (Figure 3A).
completely inhibited (.6 log cfu/mL) the Sal- Conversely, after 50 mg/L TAC of NaClO
monella pure culture mixture after 1 min contact (sublethal treatment), red-stained cells were
HERNÁNDEZ-PIMENTEL ET AL: ANTIMICROBIALS ON CHICKEN 7

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 used to demonstrate that electroactivated solu-
green fluorescence indicated surviving bacteria tions of HClO showed more efficient antimi-
(Figure 3B). On the other hand, micrographs of crobial activity against Salmonella cells than
NEW treatment (50 mg/L TAC) only showed weak organic acids (Liato et al., 2017).
red-stained cells. In addition, a red mist was
observed indicating cell lysis (Figure 3C). Neutral Electrolyzed Water Antimicrobial
Moreover, the bactericidal effect was also Activity Against Salmonella on Broiler
confirmed by plate counting achieving a Chicken Carcasses
reduction of .6 log cfu/mL using NEW and of
1 log cfu/mL with NaClO after 1 min treatment Neutral electrolyzed water antimicrobial
(as per Figure 1B). This technique has also been activity decreased its marked advantage over
8 JAPR: Research Report

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
Figure 2. Scanning electron micrographs (11,000X) of safe and convenient because it is highly avail-
Salmonella Typhimurium ATCC 14028, and S. able, does not represent a health hazard, and it is
Infantis–free cells after antimicrobial treatments. (A)
Control; (B) Sublethal treatment with sodium hypo- not required constant monitoring and occupa-
chlorite (50 mg/L TAC); (C) Treatment with neutral tional safety precautions when used (Wang
electrolyzed water (50 mg/L TAC); (D) Sublethal treat- et al., 2018).
ment with neutral electrolyzed water (9 mg/L); (E)
Same as (D) at 94,640X magnification. Abbreviation:
TAC = total available chlorine. pH, TVB-N, and Color Changes in Broiler
Chickens after Antimicrobial Treatments

NaClO when applied on broiler chicken car- Broiler chicken carcasses treated with NEW
casses (90 experimental units) with results showed significant difference (P , 0.05) in pH
showing no significant difference. Both agents compared with NaClO and control after 10 D of
HERNÁNDEZ-PIMENTEL ET AL: ANTIMICROBIALS ON CHICKEN 9

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
Figure 3. Fluorescence micrographs of Salmonella From GC/MS, the retention time of each
Typhimurium ATCC 14028 and S. Infantis mixed cul-
ture after NEW treatment. (A) Control; (B) NEW sub- chemical comprising the THM standard mixture
lethal treatment (9 mg/L TAC); (c) NEW lethal treatment was identified, followed by comparison with the
(50 mg/L TAC). Cells with intact membrane show NIST database. Standard detection and quanti-
fluorescent green color, whereas cells with damaged
membrane exhibit fluorescent red color. Abbreviation: fication were optimized by single ion moni-
NEW = neutral electrolyzed water; TAC = total avail- toring analysis mode, using m/z values: CHCl3:
able chlorine. 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
storage (Figure 5A). Broiler chickens treated with for CHCl3, CHBrCl2, CHClBr2, and CHBr3
NEW achieved the lowest pH value (6.2) after were 1.4, 1.9, 3.0, and 4.5 min, respectively. In
10 D of study, similar to a previous report addition, recoveries of CHCl3, CHBrCl2,
(Fabrizio et al., 2002). Carcasses treated with CHClBr2, and CHBr3 were 99.1, 99.3, 98.9, and
NEW showed significantly (P , 0.05) lower 99.3%, respectively. The determination coeffi-
TVB-N values than using NaClO after 10 D of cient (R2) of each standard curve was .0.98,
storage, whereas NaClO treatment showed and the detection thresholds were CHCl3
similar value as the control, in agreement with .55 mg/L, CHBrCl2 .42 mg/L, CHClBr2
Duan et al. (2017). To our knowledge, there is no .51 mg/L, and CHBr3 .18 mg/L.
10 JAPR: Research Report

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). Abbreviation:
NEW = neutral electrolyzed water; TAC = total available chlorine.

Trihalomethanes were not detected from NaClO at 15
C for 5 min, produced up to
broiler chicken carcasses treated with 50 mg/L 447 mg/kg of CHCl3 by using a petroleum ether
of NEW or NaClO. In addition, the distilled extraction technique, whereas different treat-
water controls did not show any THM. The ment temperature and exposure time (5
C for
most reported THM resulting from NaClO 20 min) led to 146 mg/kg (Robinson et al.,
treatments is CHCl3 (FAO/WHO, 2018). 1981). However, using a purge-and-trap sys-
Chicken carcasses treated with 50 mg/L of tem and thiosulphate extraction of CHCl3, a
HERNÁNDEZ-PIMENTEL ET AL: ANTIMICROBIALS ON CHICKEN 11

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. Abbreviation: NEW = neutral electrolyzed water; TAC =
total available chlorine.
12 JAPR: Research Report

Table 1. Trihalomethanes determination on chicken legs after neutral 1electrolyzed water and sodium
hypochlorite treatments.
Trihalomethanes (mg/kg)
Agent Concentration (mg/L) Chloroform Bromodichloromethane Bromoform
Neutral Electrolyzed Water 50 ,0.011 C
,0.008 C
,0.003B
100 0.037 6 0.001B ,0.008C ,0.003B
150 0.043 6 0.001A ,0.008C ,0.003B
Sodium Hypochlorite 50 ,0.011C ,0.008C ,0.003B
100 0.039 6 0.001B 0.026 6 0.001B 0.020 6 0.001A
150 0.044 6 0.001A 0.031 6 0.001A 0.023 6 0.001A
Recovery (%) 99.1 98.9 99.3
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 the main molecule (Jiménez-Pichardo et al.,
quantified from broiler chickens after NaClO 2016). From these 2 species mainly, ClO2 ox-
treatment .100 mg/L. Nevertheless, after idizes organic matter, and this may explain why
50 mg/L treatments, no THM could be detected lower amounts of ClO2 in NEW led to lower
(Vizzier-Thaxton et al., 2010). THM generation at equal TAC concentrations.
In the United States, THM mixtures for
Trihalomethane Detection and Quantification chlorinated drinking water are permitted at
on Chicken Legs 80 mg/kg, whereas the European Union legis-
lation permits 100 mg/kg (EFSA, 2005; EPA,
Trihalomethanes were not detected from 2018). No reports were found about THM pro-
chicken legs using 50 mg/L of NEW or NaClO duction by using NEW. Furthermore, chemical
treatments (Table 1). However, the non- hazards are a health concern for broiler chicken
corrosiveness, environment-friendly properties, consumers, and it is important to validate that
safe handling, and in situ production at low cost NEW treatments are safe by using whole
make NEW a better alternative than NaClO chicken carcasses under processing conditions
(Aider et al., 2012). In addition, we found 0.037 similar to those used in the poultry industry. In
and 0.043 mg/kg of CHCl3 after using NEW addition, in a previous report, an official method
treatments with 100 mg/L TAC and 150 mg/L of analysis after NaClO treatments was not used
TAC, respectively, much higher than the (Robinson et al., 1981), whereas another report
maximum antimicrobial intervention concen- used official methods for chicken parts only
tration allowed (#50 mg/L) for poultry pro- instead of whole carcasses (Vizzier-Thaxton
cessing in the United States (FSIS, 2018). et al., 2010).
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- CONCLUSIONS AND APPLICATIONS
bial agent concentration significantly (P , 0.05)
affected the CHCl3 concentration generated, 1. Neutral electrolyzed water achieved 10 times
whereas concentration and type of agent higher antimicrobial activity than NaClO on
significantly affected the concentrations of Salmonella pure culture mixture.
CHBrCl2 and CHBr3. No THM were detected 2. Morphologic changes and cell swelling
after NEW treatments except CHCl3, whereas indicated that membrane damage is a rele-
NaClO treatments generated CHBrCl2 and vant mechanism of action of NEW.
CHBr3 (Table 1) representing a health hazard. 3. Neutral electrolyzed water was more efficient
The equilibrium HClO/ClO2 is pH dependent in controlling TVC and TC populations than
and considering that for NaClO treatments at NaClO on chicken carcasses during 10 D of
pH $ 10, the ClO2 concentration is close to storage, without affecting color and pH; its
100%, whereas NEW at pH 6.5 shows HOCl as use produced lower TVB-N in chicken meat.
HERNÁNDEZ-PIMENTEL ET AL: ANTIMICROBIALS ON CHICKEN 13

4. The antimicrobial agent concentration suspensions of Salmonella Typhimurium and Listeria

significantly affected CHCl3 generation on monocytogenes. J. Food Protect. 66:1379–1384.
FAO/WHO. Benefits and risks of the use of chlorine-
chicken legs after antimicrobial intervention, containing disinfectants in food production and food pro-
but concentration and type significantly cessing. Accessed Jan. 2019. www.fao.org/docrep/012/
increased the generation of CHBrCl2 and i1357e/i1357e00.html.
CHBr3. Trihalomethanes were not detected FSIS, Food Safety and Inspection Service. Safe and
Suitable Ingredients Used in the Production of Meat,
in chicken legs or carcasses (skin, fat, or Poultry, and Egg Products. United States Department of
meat tissue) when using 50 mg/L TAC Agriculture. Accessed Jan. 2019. www.fsis.usda.gov/wps/
treatment of either NaClO or NEW. wcm/connect/bab10e09-aefa-483b-8be8-809a1f051d4
c/7120.1.pdf?MOD=AJPERES.
5. Neutral electrolyzed water could represent a
GB National Standards of People’s Republic of China.
safe alternative as antimicrobial intervention National Standards-Meat. Accessed Jan. 2018. www.mpi.
agent during poultry processing, owing to govt.nz/document-vault/14392.
high availability, safe handling, low corro- Hong, G. E., J. H. Kim, S. J. Ahn, and C. H. Lee. 2015.
Changes in meat quality characteristics of sous-vide cooked
sion capacity, and low toxicity because of no chicken breast during refrigerated storage. Korean J. Food
by-product generation. Sci. 35:757–764.
Hugas, M., and E. Tsigarida. 2008. Pros and cons of
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