Food Control 74 (2017) 54e60

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Inactivation of Listeria monocytogenes and natural microbiota on raw

salmon llets using acidic electrolyzed water, ultraviolet light or/and
ultrasounds
Marta Miks-Krajnik a, 1, Lee Xuan James Feng a, Woo Suk Bang b, Hyun-Gyun Yuk a, *
a
Food Science and Technology Programme, C/O Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore
b
Department of Food and Nutrition, Yeungnam University, 214-1 Dae-dong, Gyeongsan-si, Gyeongsangbuk-do, 712-749, South Korea

a r t i c l e i n f o a b s t r a c t

Article history: The objective of this study was to investigate the effectiveness of various decontamination treatments of
Received 9 September 2016 raw salmon llets, namely acidic electrolyzed water (AEW), ultraviolet light (UV), ultrasound (US), and
Received in revised form their combinations against Listeria monocytogenes and natural microbiota including total viable count
21 November 2016
(TVC), total coliforms, Escherichia coli, and yeasts and molds. The changes in quality and sensory pa-
Accepted 22 November 2016
Available online 23 November 2016
rameters of treated salmon samples were also evaluated. The combined treatments: UV US and
UV US AEW showed signicantly (P  0.05) higher reduction in L. monocytogenes of 0.79 and 0.75 log
CFU/g, respectively, compared to control (0.17 log CFU/g) washed with sterile distilled water (dH2O). TVC
Keywords:
Raw salmon
was reduced by 0.59 and 0.64 log CFU/g after UV US and UV US AEW treatments, respectively. The
Combined treatment color and odor of salmon were signicantly affected after combined treatments, but the texture and
Acidic electrolyzed water rmness of tissue were not signicantly (P > 0.05) changed. These results indicate that UV US and
Ultraviolet UV US AEW were the most effective at reducing the populations of L. monocytogenes and natural
Ultrasound microbiota on raw salmon llets. The AEW treatment by itself was found to be ineffective for raw salmon
sanitation. However, these combined treatments should be improved by optimizing other factors such as
treatment temperature, time and the distance between UV and food sample to enhance their anti-
listerial or antimicrobial effects.
2016 Elsevier Ltd. All rights reserved.

1. Introduction (Tompkin, 2002). The handling steps include head-cutting, evis-

Listeria monocytogenes is a Gram-positive, psychrotrophic,
facultative anaerobic, foodborne pathogen responsible for listeri-
osis, an illness caused by the ingestion of food containing the in-
fectious dose of 103 CFU/g (FAO-WHO, 2004) of relatively high
mortality rate of about 24% (Farber & Peterkin, 1991). Due to its
severity, according to Codex Alimentarius Commission there was
set a zero tolerance for food products providing favorable condi-
tions for L. monocytogenes growth and the limit of 102 CFU/g for the
remaining foods (FAO, 2012).
Contamination of sh and seafood products with
L. monocytogenes is believed to occur during the processing phase
* Corresponding author.
1
E-mail address: chmyukhg@nus.edu.sg (H.-G. Yuk).
Permanent address. Chair of Industrial and Food Microbiology, Faculty of Food
Science, University of Warmia and Mazury in Olsztyn, Plac Cieszyn  ski 1, 10-726
Olsztyn, Poland.
ceration and lleting. In a cold-smoked salmon processing plant,
L. monocytogenes were frequently isolated from cutting boards,
deboning pins, conveyor belts and slicer, skinner equipment (Dass,
Abu-Ghannam, Antony-Babu, & Cummins, 2010). The contamina-
tion of L. monocytogenes was also observed in catsh llet samples
and sh contact surfaces at the various stages of production (Chen,
Pyla, Kim, Silva, & Jung, 2010).
Many physical intervention technologies have been investigated
for eliminating L. monocytogenes in seafood processing environ-
ment and sh commodities, including irradiation (Su, Duan, &
Morrissey, 2004), high pressure processing (HPP) (Ritz, Jugiau,
Federighi, Chapleau, & De Lamballerie, 2008) and pulsed ultravio-
let (UV) light treatment (Ozer & Demirci, 2006a). Apart from these
physical methods, the efcacy of chemical sanitizers, such as
chlorine and chlorine-based sanitizers (Kim, Huang, Marshall, &
Wei, 1999) and acidic electrolyzed water (AEW) (Mccarthy &
Burkhardt, 2012; Ozer & Demirci, 2006a; Rahman, Khan, & Oh,
2016) have been also assessed for disinfection of raw sh during

http://dx.doi.org/10.1016/j.foodcont.2016.11.033
0956-7135/ 2016 Elsevier Ltd. All rights reserved.
 M. Miks-Krajnik et al. / Food Control 74 (2017) 54e60
processing. Interestingly, AEW is broadly applied in Japan as a
washing step for raw sh in sushi industry (Rahman et al., 2016),
and advised as sanitizer in aquatic foods for shelf-life extension
(Rasco & Ovissipour, 2015). Furthermore, the concept of bio-
preservation, which involves the addition of antagonistic,
bacteriocin-producing lactic acid bacteria has been also introduced
to control the growth of Listeria spp. on smoked salmon (Vescovo,
Scolari, & Zacconi, 2006).
Since single antimicrobial treatments using UV and AEW were
found as not sufcient to inhibit or inactivate L. monocytogenes in
sh products (Ozer & Demirci, 2006a), the hurdle technology could
be implemented to control this invasive pathogen and retain
desirable quality attributes (Leistner, 2000; Rahman et al., 2016).
Dual treatment applying both physical and chemical methods
should work synergistically to control L. monocytogenes in sh
products. For example, the combination of HPP and lactoperoxidase
system (LPS) was shown to be effective against L. monocytogenes in
cold-smoked salmon (Montiel, Bravo, De Alba, Gaya, & Medina,
2012).
AEW and UV treatments were found to have limited, antilisterial
effect in raw salmon and ready-to-eat (RTE) salads when used
individually (Ozer & Demirci, 2006a). Ultrasound (US) waves also
have been shown to control L. monocytogenes populations in other
commodities (Baumann, Martin, & Feng, 2005; Sagong et al., 2011).
The hurdle approach of combined thermosonication (US with mild
heat 40e60  C) and AEW showed higher reduction in
L. monocytogenes counts on fresh-cut bell paper (Luo & Oh, 2015)
and kale (Mansur & Oh, 2015) compared to their single treatments.
However, little is known about the effects of AEW or/and UV
combined with US in inactivating L. monocytogenes on raw salmon.
In this study, individual technologies including AEW, US or UV
were applied to determine the most effective treatment time to
inactivate L. monocytogenes in raw salmon llets. Subsequently,
using the optimized parameters, the effectiveness of AEW or/and
UV in combination with US on the inactivation of L. monocytogenes
on raw salmon was examined. In addition, the effects of the various
treatments on the physical qualities such as rmness and color of
treated salmon were studied. A sensory evaluation was also con-
ducted based on the parameters odor, color and texture of the
treated salmon. These quality assessments were to ensure the
quality attributes of the salmon that would not be negatively
affected after treatment.
2. Materials and methods
2.1. Bacterial cultures
Three Listeria monocytogenes serovars 1/2a (ATCC BAA 679), 1/2b
(ATCC BAA 839) and 4b (ATCC 13932), from American Type Culture
Collection (Manassas, VA, USA), were activated in 10 mL of tryptone
soya broth (TSB, Oxoid, Basingstoke, Hampshire,UK) for 24 h at
37  C and adapted to 200 mg/mL nalidixic acid (Sigma-Aldrich, St
Louis, MO, USA) so that L. monocytogenes cells isolated from inoc-
ulated salmon were relatively free from other background bacterial
contaminants. Each serovar (0.1 mL) was cultured separately in
9.7 mL of TSB (Oxoid) containing 0.2 mL of 200 mg/mL nalidixic acid
at 37  C for 24 h.
2.2. Preparation of inoculum
All L. monocytogenes cultures in TSB (1.0 mL) containing nalidixic
acid were centrifuged (3500 g for 5 min at 4  C, Sorvall Legend
Micro 17R centrifuge, Thermo Scientic) and the pellets were
washed twice with 1.0 mL of 0.1% (w/v) peptone water (PW, Oxoid)
to remove impurities. The harvested pellets were resuspended in
55
1.0 mL of 0.1% (w/v) PW to obtain a nal cell density of approx.
108 CFU/mL. All three serovars were aseptically combined in 1:1:1
(v/v/v) ratio to prepare a L. monocytogenes cocktail, which was
serially diluted (1:10) to approximately 106 CFU/mL for subsequent
inoculation.
2.3. Inoculation of salmon llets
Raw salmon llets were purchased from a local supplier in
Singapore on the day of the experiment. They were always from the
same manufacturer, packaging and expiry dates, to avoid potential
microbial uctuations. The salmon llets were cut aseptically into
pieces of approximately 10 g each (4  2  1 cm) prior to inocu-
lation. The 0.1 mL of prepared 3-strain L. monocytogenes cocktail
was spotted (ten 10 ml spots) on the one side of salmon llet to
achieve inoculum level of approx. 105 log CFU/g. Inoculated samples
were kept in the refrigerator at 4  C overnight to enable the
attachment of the cells prior to the sanitation experiments.
2.4. Acidied electrolyzed water (AEW) treatment
The acidied electrolyzed water (AEW) was obtained using an
electrolyzed water generator (ROX-20TB2, Hoshizaki Electric
Company, Aichi, Japan) at a constant current of z10 A by the
electrolysis of (0.1%, w/v) sodium chloride solution. The pH
(2.6 0.2) and the oxidation/redaction potential (ORP,
1140 30 mV) of AEW were measured with pH/redox meter
(Mettler-Toledo, Greifensee, Switzerland), while available free
chlorine concentration (65 15 ppm) using a reectometer (Merck,
Darmstadt, Germany). The inoculated salmon llets (10 g) were
submerged in a 100 mL sterile, screw-cap bottle lled with AEW
without agitation for the suggested treatment time (1, 5 or 10 min)
at room temperature (Ozer & Demirci, 2006a). The untreated
salmon llets (10 g) were used as controls for enumeration of initial
microbial count.
2.5. Ultraviolet (UV) light treatment
Both sides of salmon llets (10 g) were exposed to short wave
ultraviolet (UVeC) light (254 nm) from the distance of approx. 8 cm
for 5 or 10 min. An ultraviolet uorescent lamp (XX-15G/FB, Fisher
Scientic Pte Ltd, Singapore) equipped with two 15 W clear
germicidal tubes (BLE-1T155, Fisher Scientic Pte Ltd) was used.
The lamp was placed over irradiated samples on the experimental
setup, covered with reecting aluminum foil inside Class II Bio-
logical safety cabinet (BSC) (Esco Micro Pte Ltd, Singapore). The
irradiance of UV-C light (10.3 0.3 W/cm2) was measured at the
surface of salmon samples using a UVX digital radiometer (UVP Inc.,
Ultra-violet Products, Cambridge, UK) equipped with UVX-25 probe
(UVP Inc.). The applied dosage of UV-C irradiation was calculated
using the following Eq. (1) (Maclean, Macgregor, Anderson, &
Woolsey, 2009):
EPt (1)
where E is a dose in J/cm2, P is irradiance in W/cm2, and t is time of
UV treatment in sec.
2.6. Ultrasound (US) treatment
A 10.8 L ultrasonicator (TI-H-10, Elma, Germany) was used at a
frequency of 45 kHz, an ultrasonic power of 200 W, and the heater
was set at room temperature. Inoculated salmon llets were soni-
cated in a stomacher bags (Gosellin, Hazebrouck Cedex, France)
lled with 100 mL of sterile distilled water (dH2O) or AEW, which
56 M. Miks-Krajnik et al. / Food Control 74 (2017) 54e60
were immersed in the ultrasound tank lled with 3 L of dH2O for a
1 min.
2.7. Combined treatments with AEW, UV and US
To achieve the combined effect, the treatments with AEW
(1 min), UV (5 min), and US (1 min) were carried out in the order
shown in Table 1.
2.8. Microbiological analysis
The treated salmon samples were transferred to sterile stom-
acher bags (Gosellin, Hazebrouck Cedex, France) containing 90 mL
of Dey/Engley (D/E) neutralizing broth (Acumedia, Michigan, USA)
to neutralize residual AEW, or 90 mL of 0.1% (w/v) PW (Oxoid) for
remaining treatments. The samples were homogenized using a
blender (IUL Instruments, Barcelona, Spain) for 2 min at room
temperature, and the homogenate was serially diluted using 0.1%
(w/v) PW (Oxoid). L. monocytogenes was enumerated on tryptic soy
agar (TSA; Oxoid) supplemented with 0.6% yeast extract (Oxoid)
and 200 mg/mL nalidixic acid (Sigma-Aldrich) and incubated at
37  C for 24e48 h. The total viable count (TVC) was determined
using TSA (Oxoid) containing 0.6% (w/v) yeast extract and incu-
bated at 37  C for 3 days. Yeasts and molds were counted on Rose
Bengal chloramphenicol agar (RBCA; Oxoid) incubated at 21  C for 5
days. Coliforms and Escherichia coli were analyzed using 3M
Petrilm E. coli/Coliform Count Plate (3M, St. Paul, Minnesota,
USA) incubated at 37  C for 24 h.
2.9. Physical quality analysis
The effect of applied treatments on salmon physical quality
parameters, namely, color and rmness, was investigated. The
colorimeter CM-3500d (Konica Minolta, Tokyo, Japan) working in
the reection mode was used to measure the color changes of the
treated salmon llets, using CIE LAB-values (L*a*b*). The whiteness
index (WI) was calculated according to Eq. (2) (Bermdez-Aguirre
& Barbosa-Canovas, 2013):
h 2 i1=2
WI 100  100  L* a*2 b*2
The rmness of salmon samples (10 g) was analyzed with TA-
XT2i texture analyzer (Stable Micro Systems, Godalming, Surrey,
UK) as described previously (Tan et al., 2015). Briey, the rmness
was given by the maximum peak force (kg) required for the P5S
spherical probe (5 mm diameter) of the texture analyzer to
compress the samples by a penetration distance of 8 mm, using
running speed of 1.0 mm/s.
2.10. Sensory evaluation
A sensory evaluation was conducted to assess the changes in
quality parameters, namely: odor (O), color (C) and texture (T) of
treated and untreated salmon samples, using a 3-point scoring
Table 1
The combined treatments with acidic electrolyzed water (AEW), ultraviolet light
(UV), and ultrasound (US) applied for raw salmon llets.
No. Treatments Time (min) and the order of treatments
1 US AEW US (1 min) AEW (1 min)
2 UV AEW UV (5 min) / AEW (1 min)
3 UV USa UV (5 min) / US (1 min)a
4 UV US AEW UV (5 min) / US (1 min) AEW (1 min)
a
Sterile distilled water (dH2O) was applied for US treatment.
system, where 3 very good, 2 good, and 1 unacceptable. An
untrained sensory panel of 36 subjects was instructed prior to
analysis on applying score system, and two independent experi-
ments were conducted (n 72). The salmon samples (10 g), coded
with set of three randomly chosen digit numbers, were placed in
separate Petri dishes on white background half an hour prior to the
evaluation. All observations were conducted under standardized
conditions as described before (Miks-Krajnik, Yoon, Ukuku, & Yuk,
2016).
2.11. Statistical analysis
The experiments were performed in triplicate with duplicate
sampling (n 6) for microbiological and physical quality analysis.
Bacterial counts were converted to log10 CFU/g and expressed as
mean standard deviation. To compare the effect of various
treatments, the mean values were analyzed by a single factor
ANOVA using Tukey HSD Test at the signicance level of P  0.05
(Statistica, ver. 10.0, StatSoft, Poland).
3. Results
3.1. Inactivation of L. monocytogenes and natural microbiota on
salmon llets
In the preliminary steps, the optimum times for individual
treatments with acidied electrolyzed water (AEW) and ultraviolet
light (UV) were established. The AEW treatment was conducted for
1, 5 and 10 min, and greater reduction of L. monocytogenes was
observed after 5 and 10 min compared to 1 min treatment (data not
shown). However, due to observable quality deterioration of the
tissue of salmon llets after longer treatments, shorter time (1 min)
was applied for further experiments. Preliminary decontamination
experiments with UV for 5 and 10 min, which corresponded to the
doses of 3083 87 and 6166 174 J/cm2, respectively, showed
comparable (P > 0.05) results (data not shown). Therefore, the
shorter treatment time (5 min) was chosen as more feasible to
utilize in industrial applications. While, ultrasound (US) treatment
was performed for 1 min at 45 MHz as previously described
(Alegria et al., 2009).
(2)
The effectiveness of various decontamination technologies,
including AEW, UV, US, and their combinations for the inactivation
of L. monocytogenes on the surface of raw salmon llets is sum-
marized in Fig. 1. The initial count of L. monocytogenes was 4.9e5.9
log CFU per gram of inoculated salmon samples (data not shown).
The individual AEW and US treatments have not resulted in a sig-
nicant (P > 0.05) reduction compared to washing with sterile
distilled water (dH2O). Even though, for US treatment alone, no
signicant reduction was observed, synergistic effect of AEW
combined with US gave a signicantly (P  0.05) higher
L. monocytogenes inactivation (0.4e0.5 log CFU/g) than AEW alone.
The results of raw salmon sanitation showed that UV alone and all
combined treatments were more effective compared to washing
with dH2O. Among all treatments applied, signicantly (P  0.05)
higher L. monocytogenes inactivation was observed for UV US and
UV US AEW, achieving nearly 0.8 log CFU/g reduction, compare
to the other treatments. Interestingly, all the treatments involving
UV showed higher reduction than the remaining ones, highlighting
the effectiveness of UV irradiation.
The decontamination of natural microbiota, including total
variable count (TVC), total coliforms, and yeast and molds, on the
surface of raw salmon llets was also investigated (Table 2). The
initial contamination of salmon was 4.2e5.9, 2.8e4.0 and 1.3e2.9
log CFU/g, for TVC, coliforms, and yeast and molds, respectively.
Escherichia coli were not detected in raw salmon samples. All
 M. Miks-Krajnik et al. / Food Control 74 (2017) 54e60 57

1.0
dH2O
AEW CD
US D

L. monocytogenes reduction
0.8
US + AEW
UV
BC
UV + AEW BC
[ log CFU/g ] 0.6 UV + US
UV + US + AEW B
AB
0.4

A
A
0.2

0.0

Treatments
Fig. 1. The reduction (log CFU/g) of Listeria monocytogenes inoculated on raw salmon llets after treatments with sterile distilled water (dH2O), acidied electrolyzed water (AEW),
ultrasounds (US), ultraviolet (UV) and combined treatments applying hurdle approach. Different letters (AeD) indicate signicant (P  0.05) differences between treatments (n 6).

applied treatments did not show signicant (P > 0.05) reduction of
total coliforms and yeast and molds counts when compared with
sterile dH2O washing. The combined treatments UV US and
UV US AEW showed signicantly higher inactivation of TVC
compared to dH2O. However, due to large variances in initial
microbiota counts and low contamination levels with background
microbiota, some inconsistencies in microbial reductions after
treatments were observed, resulting in high standard deviations of
the results.
3.2. Changes in physical quality of raw salmon llets after
treatments
The salmon llets exposed to the most effective treatments for
microbial inactivation, namely UV, UV US and UV US AEW
were subjected to further instrumental quality analysis including
texture, color and sensory evaluation. Regardless of treatment
condition, the rmness of treated and untreated salmon samples
was comparable (P > 0.05) and ranged 0.134e0.164 kg (Fig. 2). The
changes in color, based on CIE L*, a*, b* values and whiteness index
(WI) of the salmon samples after UV irradiation and the hurdle
treatments: UV US and UV AEW US, are shown in Fig. 3. The
observed a* (redness-greenness) and b* (yellowness-blueness)
values were signicantly (P  0.5) lower for the combined treat-
ments than for untreated control samples. While L* (lightness-
darkness) value and WI showed that salmon llets subjected to
UV US and UV AEW US were much (P  0.5) lighter compared
to controls and only UV-treated ones. The score results of sensory
evaluation, including odor, color and texture characteristics of
treated and control salmon llets are presented in Table 3. The odor
scores for hurdle treatments: UV US and UV AEW US were
signicantly lower (1.7) compared to the control salmon samples
(2.2e2.4). In terms of color, sensory results showed that the
treatments involving UV irradiation gave signicantly lower color

Table 2
The reduction (log10 CFU/g)a of natural microora, including: total viable count
(TVC), coliforms, and yeast and molds on raw salmon after treatments with acidic
electrolyzed water (AEW), ultraviolet light (UV), ultrasound (US) and their
combinations.

Treatment conditions The reduction (log CFU/g) of natural microora:

Total viable count Coliforms Yeast and molds

b
dH2O NRAB NRA NRA
AEW NRABC NRA NRA
US NRABCD 0.28 0.40A NRA
US AEW NRA NRA NRA
UV 0.39 0.29ABCD 0.24 0.49A 0.10 0.26A
UV AEW 0.43 0.24BCD 0.14 0.23A NRA
UV US 0.59 0.17CD 0.59 0.33A 0.38 0.73A
UV US AEW 0.64 0.36D 0.49 0.42A NRA
Fig. 2. The rmness of raw salmon llets after various treatment conditions, including
a
Across the different treatments, the means (n 6) with the same letter (A - D) control sample, sterile distilled water (dH2O), ultraviolet (UV) and ultrasound (US)
for the same microorganisms group are not signicantly different (P > 0.05). combined with acidied electrolyzed water (AEW), measured by the maximum peak
b
NR e no reduction observed (<0.10 log CFU/g). force (kg) required for the probe to compress the samples by 8 mm (n 6).
58 M. Miks-Krajnik et al. / Food Control 74 (2017) 54e60
Fig. 3. The color expressed as CIE Lab-values: (A) a*value, (B) b*value, (C) L*value and
(D) calculated whiteness index (WI) of raw salmon llets after various treatments,
including control sample, sterile distilled water (dH2O), ultraviolet (UV) and ultra-
sound (US) combined with acidied electrolyzed water (AEW). across the treatment
conditions, the means (n 6) with the different letters (AeC) are signicantly
(P  0.05) different. (For interpretation of the references to colour in this gure legend,
the reader is referred to the web version of this article.)
scores (2.0e2.1) than the untreated and dH2O washed samples
(2.4). The color score of salmon tissue treated with use of AEW
(UV AEW US) was signicantly lower than remaining treat-
ments. The scores for texture of all treated salmon samples
(1.9e2.0) were not signicantly different from controls (2.0e2.2),
which was consistent with the results obtained for the rmness
determination using the texture analyzer.
4. Discussion
Generalizing, the results of this study revealed that the hurdle
approach combining several decontamination technologies may
improve the microbiological quality of raw salmon compared with
the application of single treatment only. By merging multiple
treatments, the microbial cells are being exposed simultaneously or
continuously to several stress factors, which usually exhibit com-
plementary inactivation mechanisms, leading to the cell death in
considerably rapid manner.
In theory, acidic electrolyzed water (AEW) itself is a hurdle,
bactericidal concept, involving high positive oxidation-reduction
potential (ORP, > 1000 mV), high concentration of available
chlorine ions (20 ppm) and low pH (<3.0). Each of these factors
represents different antimicrobial mechanisms (Rahman et al.,
2016; Rahman, Park, Song, Al-Harbi, & Oh, 2012). High ORP might
lead to the disruption of metabolic uxes and ATP synthesis in
bacteria due to the change in electron ow (Liao, Chen, & Xiao,
2007). At low pH (<2.6), the outer membrane of bacterial cells is
Table 3
The mean scoresa for odor, color and texture of salmon samples after treatments
involving ultraviolet light (UV), ultrasound (US), acidic electrolyzed water (AEW)
and their combinations.
Control dH2O UV UV US UV US AEW
Odor 2.4 0.8A 2.2 0.6A 1.9 0.7AB 1.7 0.8B 1.7 0.6B
Color 2.4 0.7A 2.4 0.6A 2.1 0.7B 2.0 0.7B 1.6 0.6C
Texture 2.2 0.7A 2.0 0.7A 2.0 0.8A 2.0 0.8A 1.9 0.7A
a
The values are expressed as means SDs of (n 72) sensory scores. Across the
treatment conditions, means with the same letter (AeC) in the same row are not
signicantly (P > 0.05) different, Tukey HSD test.
sensitized, which enables the entry of free chlorine in form of hy-
pochlorous acid (HOCl) into the cells, where it can inhibit glucose
oxidation by chlorine-oxidizing sulfhydryl groups of the enzymes
involved in carbohydrate metabolism (Luo & Oh, 2015). However,
despite all of the advantages of AEW as disinfecting agent, in this
study AEW treatment alone was found to be ineffective for raw
salmon sanitation. Our observations are in the agreement with the
results obtained in other studies focused on seafood and sh
disinfection using AEW and reviewed recently by Rahman et al.
(2016). More precisely, AEW treatment (1e10 min) reduced
L. monocytogenes count on raw salmon only by 1.1e1.3 log CFU/g
(Al-Holy & Rasco, 2015) and has not inhibited its growth at 4  C on
raw salmon immersed in AEW for 5 min (Mccarthy & Burkhardt,
2012). Even 64 min long AEW treatment of salmon llets
decreased L. monocytogenes count by 0.8e1.1 log CFU/g only (Ozer
& Demirci, 2006a). Similarly, AEW reduced the microbial pop-
ulations by 2.0 and 2.8 log CFU/g for carp llet and skin, respec-
tively, when treated for 15 min (Mahmoud et al., 2004), and of
1.3e2.2 log CFU/cm2 for salmon skin soaked for 120 min (Phuvasate
& Su, 2010). It was speculated that AEW efciency could be affected
by presence of organic matter from the salmon, which might have
reacted with free chlorine to form combined available chlorine of
lower bactericidal activity than free chlorine at the same concen-
tration level (Al-Holy & Rasco, 2015; Oomori, Oka, Inuta, & Arata,
2000). Moreover, low bacterial reduction might be explained by
insufciently short time (1 min, in this study) of treatment and that
raw sh tissue is a difcult food matrix for decontamination, which
strongly attach bacterial cells, compared to skin surface (Mahmoud
et al., 2004). Hence, to overcome this drawback, combining effect of
two or more discontamination methods in lower quantities could
be applied (Rahman et al., 2016; Rasco & Ovissipour, 2015).
The application of ultrasounds (US) as an additional hurdle to
create the cavitation and enhance the cells detachment from the
food matrix, thereby making the microorganisms more susceptible
to chlorine, could be a potential solution. Combined US or ther-
mosonication (40e60  C) with AEW or near neutral pH electrolyzed
(NEO) water have been reported as effective for microbial decon-
tamination as well as the shelf life extension of fresh-cut kale
(Mansur & Oh, 2015), bell pepper (Luo & Oh, 2015), kashk
(Forghani, Eskandari, & Oh, 2015) and fresh produce such as lettuce
and tomatoes (Afari, Hung, King, & Hu, 2016). Similarly, to the re-
sults of our study, Mansur and Oh (2015) revealed that the com-
bination of US with AEW was more effective in reduction of
L. monocytogenes counts (3.0 log CFU/g) on fresh-cut kale compared
with dH2O, US dH2O and AEW treatments. It has been demon-
strated that US signicantly increases ORP of NEO from 847 to
950 mV, not affecting the pH and free chlorine content (pH 6.5,
155 mg/L), most probably due to the generation of free radicals in
liquid phase (Afari et al., 2016). With increased ORP, the antimi-
crobial efciency of AEW increases and thus indicates that
US AEW shows synergistic antimicrobial effect, probably by
increasing the mechanical disruption of cells (Luo & Oh, 2015).
However, in this study, US AEW treatment was not as effective as
expected, most likely due to the properties of salmon surface,
especially the presence of fats and proteins which promote bacte-
rial attachment (Mahmoud et al., 2004).
The UV radiation in the wave range of 250e260 nm is known to
be the most lethal and germicidal to microorganisms as it directly
alters microbial DNA through pyrimidine dimer formation, result-
ing in the loss of bacterias ability to proliferate and eventually cell
death (Bintsis, Litopoulou-Tzanetaki, & Robinson, 2000). In the
present study, 254 nm UV treatment applied as a single hurdle
demonstrated the most promising results of L. monocytogenes
inactivation. Similarly, Skowron, Bauza-Kaszewska, Dobrzanski,
Paluszak and Skowron, (2014) reported signicant reduction in
 M. Miks-Krajnik et al. / Food Control 74 (2017) 54e60
counts of Salmonella spp., E. coli and Enterococcus spp. (up to 4 log
units for a dose of 400 J/cm2) on salmon and cod meals after UV
treatment. However, Gram-positive L. monocytogenes was found to
be more resistant to UV treatment than Gram-negative E. coli, due
to the lower photoproducts generation during exposure to UV,
which lead to the structural DNA distortion and consequently cell
death (Kim, Kim, & Kang, 2016).
Ozer and Demirci (2006b) reported that pulsed UV-light (5.6 J/
cm2) inactivated approx. 1 log of L. monocytogenes on raw salmon
llets at 8 cm for 60 s treatment, which is comparable to this study.
On the contrary, photodynamic inactivation of natural aerobic
microbiota on raw salmon using pulsed light (PL) showed limited
effectiveness compared to raw pork roast or roast pork (Nicorescu,
Nguyen, Chevalier, & Orange, 2014). The usefulness of UV or PL is
restricted to the surface decontamination, due to its limited
penetration depth, and the presence of supercial lipids and pro-
teins of strong UV-absorbing properties, which form additional
barrier for UV photons availability to inactivate natural microbiota,
scattered throughout the rough food matrix (Bintsis et al., 2000;
Nicorescu et al., 2014). The enhanced survival of background
microbiota observed in this study after UV treatment, compared to
L. monocytogenes, could be explained by their natural biodiversity,
thus increased stress resistance, and stronger attachment to food
matrix than in vitro sub-cultured and inoculated bacterial strains
(Tan et al., 2015). Moreover, it was previously observed that the
adaptation of other foodborne pathogen (Escherichia coli O157:H7)
to nalidixic acid increases its sensitivity to ionizing radiation
(Niemira, 2005) and UV treatment (Chintagari, Hung, & Hamanaka,
2015), but not to the electrolyzed water (Jadeja and Hung, 2013).
Thus, the effects of various treatments on reduction of adapted
L. monocytogenes serotypes in this study should be taken with
caution.
The combined treatments, in this study, were superior in their
effectiveness compared to the corresponding individual methods,
evidencing the proof of concept. However, there was no signicant
difference observed between the combined treatments when using
dH2O versus AEW, which conrms that AEW might be not an
effective sanitizer for L. monocytogenes and microbiota naturally
associated with raw salmon. Recently, Shiroodi, Ovissipour, Ross
and Rasco, (2016) evaluated the effect of AEW-temperature
(20e40  C) combination on the reduction of L. monocytogenes
count on cold-smoked Atlantic salmon. It was observed that the
bactericidal effect of AEW increased with the increased tempera-
ture of treatment. Their results showed L. monocytogenes reduction
of 2.85 log CFU/g at 40  C, compared to 1.58 log CFU/g at 20  C on
salmon when treated for 10 min in AEW. Likewise, Ozer and
Demirci (2006a) reported higher inactivation of L. monocytogenes
Scott A (1.12 log CFU/g) at 35  C compared to 22  C (0.4 log CFU/g).
However, increased temperatures of AEW treatment were not
recommended for application in seafood industry due to possible
quality change of seafood (Rasco & Ovissipour, 2015).
In this study, the certain differences in quality parameters of raw
salmon after combined treatments (UV US and UV US AEW)
were observed, in particular a decrease in color (a*, b*) values, an
increase of brightness indicators (L* value and whiteness index, WI)
as well as lower sensory scores (odor and color) of treated samples.
The discoloration (bleaching) of raw salmon esh was previously
reported for chlorine washing (Kim et al., 1999), PL technology
(Nicorescu et al., 2014), pulsed UV-light (Ozer & Demirci, 2006b). It
was hypothesized that color alternation of sh or meat tissue could
be induced with chemical and thermal reactions initiated with low
pH, high ORP, active chlorine ions (Kim et al., 1999; Shimamura,
Shinke, Hiraishi, Tsuchiya, & Masuda, 2015), as well as the local
temperature increase due to the exposure to UV-light (Ozer &
Demirci, 2006b). These induced oxidative and thermal
59
deterioration can result in lipid oxidation, carotenoids degradation,
proteins denaturation (Rahman et al., 2012), as well as sensory al-
ternations, which were observed previously for PL treatment,
causing intense cooked off-avor (Nicorescu et al., 2014). Similarly,
the color and odor were the most affected quality attributes in this
study for treated salmon samples involving UV irradiation.
5. Conclusions
The present study shows that the AEW or US technologies
applied individually revealed no signicant effect on microbial
reduction. The combined treatments of UV and US (regardless of
AEW addition) showed the improved inactivation of TVC, coliforms
and L. monocytogenes on raw salmon surface. The further US
treatment in AEW water did not increased antimicrobial effect.
Thus, it might be concluded that the hurdle approach combining
these two physical decontamination methods (UV US) could
improve the microbiological quality of raw salmon compared to
their single treatments. However, these combined treatments
caused deterioration of sensory quality of salmon tissue affecting
color and odor, while the texture and rmness remained un-
changed. Therefore, it is necessary to optimize the treatment con-
dition to enhance the bactericidal effect of these combined
chemical and physical hurdles on foodborne pathogens and
spoilage microorganisms in perishable, RTE food commodities, like
raw salmon llets, minimizing quality change.
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