Food Research International 107 (2018) 496–502

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Food Research International

journal homepage: www.elsevier.com/locate/foodres

Synergistic effect of X-ray irradiation and sodium hypochlorite against T

Salmonella enterica serovar Typhimurium biofilms on quail eggshells
⁎
Soo-Jin Junga, Shin Young Parkb, Sang-Do Haa,
a
School of Food Science and Technology, Advanced Food Safety Research Group, BrainKorea21 Plus, Chung-Ang University, 72-1 Nae-Ri, Daeduck-Myun, Ansung,
Kyunggido 456-756, Republic of Korea
b
Department of Seafood and Aquaculture Science, Institute of Marine Industry, Gyeongsang National University, Togyeong 53024, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Keywords: The present study investigated the synergistic bactericidal effects of combined X-ray irradiation (0.5, 1.0, 1.5,
Salmonella enterica serovar Typhimurium and 2.0 kGy) and sodium hypochlorite (NaOCl) (50, 100, 150, 200, and 300 ppm) treatment on the reduction of
Biofilms S. enterica serovar Typhimurium ATCC 14028 biofilms on quail eggshells. Additionally, the color change of the
X-ray irradiation quail eggshells was measured by hunter color “L” (lightness), “a” (red/green), “b” (yellow/blue), and “ΔE” (total
Sodium hypochlorite
color difference). Additionally, the puncture force was tested to evaluate eggshell thickness after the combined
Synergistic effect
treatments. The highest biofilm reduction values were observed as 4.6 log CFU/egg after X-ray (2.0 kGy) and
NaOCl (300 ppm) treatment. Moreover, the synergistic reduction in values after combined treatment was
evaluated, and the highest biofilm reduction value was 4.3 log CFU/egg by 2.0 kGy X-ray/50 ppm NaOCl (1.47
log higher than the sum of reduction values of the individual treatments). The color of the quail eggshell and
puncture force were not significantly changed by combined treatments (p > 0.05 at both cases). Consequently,
2.0 kGy X-ray/50 ppm NaOCl was considered optimal for combination treatment for eliminating S. enterica ser.
Typhimurium biofilms on eggshell without any color or thickness changes. Furthermore, combination treatment
could be useful for improving microbiological safety in the quail egg industry.

1. Introduction
Foodborne diseases are considered an international public health
concern. Salmonella infections have a considerable global impact on
human health, with an estimated 150 million cases resulting from
foodborne transmission in 2010 (Kirk et al., 2015). Moreover, Salmo-
nella infection accounts for $365 million in medical costs each year in
the USA (CDC, 2011). According to the food and drug statistical year-
book published by the Ministry of Food and Drug Safety (MFDS), sal-
monellosis occurred 24 times, involving more than 1400 patients in
2014 in Korea (MFDS, 2015). Approximately 95% of human salmo-
nellosis cases are associated with the consumption of various foods such
as meat, poultry, eggs, milk, seafood, and fresh produce (Acha &
Szyfres, 2003; Romich, 2008). Among them, contaminated poultry eggs
are an important source of Salmonella infections, especially when the
bacterium is able to penetrate the egg contents. Salmonella enterica
serovars Enteritidis and Typhimurium (herein referred to as S. En-
teritidis and S. Typhimurium) are the two most frequently identified
causes of human salmonellosis (Hendriksen et al., 2011). There are two
possible contamination routes by Salmonella: the typical pathway is to
reach the gastrointestinal tract of hens through consumption; the other
is via penetration into the eggshell from contaminated feces during or
after oviposition (Messens et al., 2005; De Reu et al., 2006; Gantois
et al., 2009).
Quail eggs are much smaller than chicken eggs, but have higher
levels of vitamin B1 while containing similar other general contents.
Quail eggs are mainly consumed in Korea, Japan, and the Philippines
(Azanza, 2004). Specifically, quail eggs are the frequently-used-food
material and menu items offered by school foodservice operations in
Korea (Han et al., 2016). According to a survey, quail egg was named
one of the world's top 20 food trends for mini desserts in 2014 (Ctpost,
2014). Therefore, it is expected that the demand for quail eggs and their
products will steadily increase. However, the quail eggshell is thinner
than that of chicken eggs (Yannakopoulos & Tserveni-Gousi, 1986). In a
recent study, heat resistant S. Enteritidis was found in quail eggs (Rustia
& Azanza, 2005). Harsha et al. (2011) highlighted that S. Typhimurium,
as well as serovars Worthington and Bareilly were detected in 14%,
28%, and 57% of quail eggs studied, respectively.
S. Typhimurium can penetrate the eggshell and reach the internal
contents within 3 days at low temperatures (4 °C) (Schoeni et al., 1995).

⁎
Corresponding author at: School of Food Science and Technology, Chung-Ang University, 72-1 Nae-ri, Daeduk-myun, Ansung, Gyunggido 456-756, Republic of Korea.
E-mail address: sangdoha@cau.ac.kr (S.-D. Ha).

https://doi.org/10.1016/j.foodres.2018.02.063
Received 19 September 2017; Received in revised form 22 February 2018; Accepted 25 February 2018
Available online 27 February 2018
0963-9969/ © 2018 Elsevier Ltd. All rights reserved.
S.-J. Jung et al.
Lee (2015) noted that S. Typhimurium could adhere to hen eggshells
and form a biofilm on the eggshell as well as various food contact
surfaces such as stainless steel, plastic, and rubber. Biofilms are archi-
tecturally complex assemblies of microorganisms on surfaces and in-
ferred to be a critical source of food contamination (Jahid & Ha, 2012;
Srey et al., 2013). Many outbreaks of pathogens have been attributed to
biofilms, and it is estimated that biofilm formation accounts for up to
80% of microbial infections (Epstein et al., 2011). The bacteria are
embedded into a biofilm matrix wherein they are resistant to physical
and chemical disinfectants (Milanov et al., 2009). Previously published
research has explained that the high tolerance and resistance of bac-
terial biofilms are due to limited diffusion of disinfectant and sanitizers
through the biofilms' extracellular carbohydrate complexes, as well as
physiological heterogeneity of the populations within biofilms (Jahid &
Ha, 2012). The resistance of sessile cells to chlorine could be partially
described by the limitation of disinfectant diffusion into the interior of
the biofilms, and due to the reaction of available agent particles with
biofilm organic substances in the extracellular polymeric substance
(Chen & Stewart, 1996; Stewart & Franklin, 2008). Food irradiation has
the ability to damage microbial DNA in order to prolong shelf-life and
enhance food safety without having any negative effects on the sen-
sorial or nutritional quality when applied at an appropriate irradiation
dose, which has led to a globally increased application of ionizing ra-
diation (McNulty, 1998; World Health Organization 1999; Molins 2001;
Diehl, 2002). However, the application of radiation in food has been
mostly performed using gamma rays (Byun & Lee, 2003; Wang & Yang,
2005; Deschreider, 1960; Byun, 1994). Nevertheless, because gamma
rays are created from radionuclides like cobalt-60 or cesium-137, they
are not suitable for commercial throughput. A relatively recent tech-
nique, X-ray irradiation, generated from machine sources at energies
not exceeding 5 million electron volts (MeV), is one of the physical
methods for processing food using ionizing radiation (CFR, 2016). X-ray
generated by electrons decelerated in high atomic number material
(Tantalum, gold etc.) So it called bremsstrahlung (braking or decel-
eration radiation). X-ray has low efficiency of conversion from electron
beam (maximum 8%). Also X-ray requires high power with high energy
accelerator. However, X-ray irradiation, which has potentially deeper
penetration depth than gamma rays, has a considerable advantage,
specifically an on and off system using a switch. Accordingly, interest in
X-ray irradiation has increased as a useful alternative method to the use
of gamma rays. Studies evaluating the application of X-ray irradiation
in the inactivation of organisms causing foodborne illness have been
reported for various foods such as spinach leaves, mangos, and live
oysters (Mahmoud et al., 2016; Mahmoud et al., 2010; Mahmoud,
2009). However, there has been no study regarding biofilm control
using X-ray irradiation, or the combined treatment of X-rays and che-
mical disinfectants.
Sodium hypochlorite (NaOCl) is an antimicrobial agent frequently
and widely used in the food industry as an effective biocide for the
treatment of microbial contamination (Kim et al., 2016). Many re-
searchers have conducted studies regarding the use of NaOCl treatment
to eliminate biofilms (Corcoran et al., 2013; Wang et al., 2014; Wong
et al., 2010).
The objectives of this study were to examine the reduction in S.
Typhimurium biofilms formed on quail eggshells after treatment with
X-ray irradiation (0.5, 1.0, 1.5 and 2.0 kGy) and NaOCl (50, 100, 150,
200 and 300 ppm). Moreover, the synergistic effect of combination
treatment was evaluated in the reduction of S. Typhimurium biofilms
and eggshell quality (color and thickness) following this combined
treatment.
2. Materials and methods
2.1. Bacterial strain
S. Typhimurium (ATCC 14028) was used for this experiment. One
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Food Research International 107 (2018) 496–502
hundred microliters of a stock culture (107–108 CFU/mL), stored at
−70 °C in 20% (w/v) glycerol, was inoculated into 10 mL tryptic soy
broth (TSB; Difco, Detroit, D, USA) and incubated at 37 °C for 24 h.
After incubation, 100 μL of the incubated culture was pipetted into
10 mL of fresh TSB for and incubated for an additional 24 h. The TSB
broth cultures were then centrifuged at 10,000 ×g for 10 min at 4 °C,
and the pellet was re-suspended and washed with Dulbecco's phosphate
buffer saline (DPBS; Sigma Aldrich, USA). This process was repeated
twice. The subculture cells were diluted and the cellular contration was
determined by plating on Xylose lysine deoxycholate agar (XLD agar)
(Difco Laboratories, Detroit, MI, USA). The initial dose used for each
inoculation was 106–107 CFU/mL.
2.2. Preparation of sample, inoculation, and biofilm formation
Quail eggs were purchased from a local grocery store in Anseong,
Korea (eggs were selected with a remaining shelf life at least 40 days,
and stored at 4 °C until use). Each egg was gently broken using a ster-
ilized knife, the liquid egg was discarded, and the eggshell was retained.
Immediately after, the eggshell was soaked into 70% alcohol for
10 min, washed three times with sterilized deionized water (DW), and
then dried for 15 min on a clean bench to remove background flora,
prior to inoculation.
Eggshell sample was put into 50-mL conical tube (Geenpia,
Gyeonggi-do, Korea) containing 10 mL of sterile TSB. The samples were
then inoculated by dipping the shell into the S. Typhimurium solution
(106–107 CFU/mL) and incubated at 37 °C for 24 h. The eggshell sam-
ples were incubated at 20 °C for 24 h for biofilm formation on the
eggshell surface (average value of biofilm on egg: 7.7 ± 0.1 log CFU/
egg).
2.3. Single treatment with X-ray irradiation or NaOCl
After the formation of biofilms, each sample was washed twice by
dipping in DW to remove loosely attached cells. Immediately after, the
samples were placed in sterilized 50-mL conical tubes. X-ray irradiation
was carried out at 0.5, 1.0, 1.5, and 2.0 kGy (5 MeV) with an electron
accelerator (ELV-4, 10 MeV, Fujifilm, Tokyo, Japan) at EB-Tech,
Daejeon, Korea.
The NaOCl solution was prepared using 12% NaOCl (6% free
chlorine equivalent, pH 12.5; Yakuri Pure Chemicals Co., Ltd., Kyoto,
Japan). The NaOCl solution was diluted in sterile DW to final con-
centrations of 50, 100, 150, 200, and 300 ppm. The disinfectant solu-
tions were freshly prepared immediately prior to the bactericidal ex-
periments. Each sample was then treated by soaking it in a respective
tube with 10 mL of each solution for 3 min. After treatment, each
sample was dipped into 10 mL of D/E Neutralizer broth (Becton,
Dickinson and company, Sparks, MD, USA) for 3 min.
2.4. Combined treatment and synergistic effect
The combined treatments (X-ray/NaOCl) were conducted with first
physical (X-ray irradiation) and then chemical disinfectant (NaOCl)
treatment. The efficacies of combined treatment were compared to the
individual reduction values after single treatment to calculate the sy-
nergistic effect. The synergistic values of the combined treatments were
calculated using the following equation:
Synergistic value = A − (B + C)
where A is the total reduction value of combined X-ray/NaOCl treat-
ment; B and C are the reduction values from single physical and single
chemical treatments, respectively.
2.5. Detachment of biofilm cell and enumeration
Analysis of biofilm detachment was performed as previously
S.-J. Jung et al. Food Research International 107 (2018) 496–502

Table 1
The bactericidal reduction values of S. Typhimurium biofilms on quail eggshells after combination treatment with X-ray/NaOCl.

Sample Treatment Mean ( ± SD) reduction value (log CFU/egg)

X-ray (kGy)

0 0.5 1.0 1.5 2.0

Egg shell NaOCl (ppm) 0 – 0.9 ± 0.09d,C

2.0 ± 0.20c,B
2.2 ± 0.07e,B
2.7 ± 0.06b,A
50 0.1 ± 0.06e,C 2.0 ± 0.12c,B 2.6 ± 0.12b,AB 3.2 ± 0.05d,A 4.3 ± 0.12a,A
100 0.5 ± 0.08d,E 2.1 ± 0.06bc,D 3.3 ± 0.12a,C 4.0 ± 0.08c,B 4.2 ± 0.10a,A
150 0.6 ± 0.08c,D 2.3 ± 0.06b,C 3.3 ± 0.11a,B 4.2 ± 0.08b,A 4.3 ± 0.07a,A
200 1.9 ± 0.06b,D 2.6 ± 0.08a,C 3.4 ± 0.28a,B 4.3 ± 0.08b,A 4.5 ± 0.07a,A
300 2.2 ± 0.11a,D 2.6 ± 0.26a,C 3.6 ± 0.08a,B 4.4 ± 0.05a,A 4.6 ± 0.10a,A

a,b,c,d
and e means are significantly different of same column by Duncan's multiple range test. (p < 0.05).
A,B,C,D
and E means are significantly different of same row by Duncan's multiple range test. (p < 0.05).

described (Jahid et al., 2014) with some modifications. To recover the
biofilms detached from the eggshells, each egg was placed into 50-mL
conical tubes that contained 10 mL of 0.1% peptone water (PW; Oxoid,
ants, U.K.) and 10 of glass beads (Glastechnique, ∮ 2 mm). The tubes
were physically vortexed for 1 min to disperse the biofilms by colliding
the egg with beads. The biofilms were serially diluted in PW to de-
termined biofilm cell numbers by plating onto XLD agar and incubated
at 37 °C for 24 h.
2.6. Field emission scanning electron microscopy (FE-SEM)
Biofilm formation of S. Typhimurium was observed using Sigma
field emission scanning electron microscopy (FE-SEM, Carl Zeiss) fol-
lowing incubation for 48 h on eggshells in TSB. The eggshell was rinsed
with phosphate buffered saline (PBS) (pH 7.2) once by soaking in a
beaker containing 50 mL of PBS solution to remove loosely attached
cell. And the egg shell was subjected to single treatment (X-ray 2.0 kGy
or NaOCl 50 ppm) or combined treatments (2.0 kGy X-ray/50 ppm
NaOCl). After treatment, the eggshell was rinsed three times with PBS.
The adhered cells were fixed in 4% glutaraldehyde (Sigma-Aldrich, St.
Louis, MO) in PBS for 24 h. The fixed cells were washed three times
with PBS for 15 min. Fixed cells were serially treated with 50% ethanol
for 15 min, 60% for 15 min, 80% for 15 min, 90% for 15 min, and 100%
for 15 min twice. Next, they were successively dehydrated by 33, 66,
and 100% hexamethyldisilazane (Sigma-Aldrich, St. Louis, MO) in
ethanol for 15 min each. The dehydrated samples were coated with
platinum and observed using FE-SEM, which was operated at an ac-
celerating voltage of 5 kV with a 5-mm working distance.
2.7. Color evaluation
The color measurement was conducted as previously described by
Kim et al. (2016). The shell color was evaluated immediately after
single and/or combined treatments (0.5, 2.0 kGy X-ray and/or 50,
300 ppm NaOCl) to estimate the quality of the eggshells. The treated
samples were placed in Whirl-Pak filter bags (Nasco, Fort Atkinson,
WI). The color measurement was conducted using a color difference
meter (UltraScan PRO; Hunterlab Co., USA) five times for each treated
sample and was presented as the Hunter colors of lightness (L⁎), red-
ness/greenness (a⁎), and yellowness/blueness (b⁎). The results were
compared with those of the untreated control sample. The standard
white plate used had values of 100 for L⁎, 0 for a⁎, and 0 for b⁎. The total
color difference values (ΔE⁎) of each eggshell after single and/or
combined treatment was calculated as (ΔL⁎2 + Δa⁎2 + Δb⁎2)1/2.
2.8. Puncture force evaluation
Eggshell thickness was determined using a texture analyzer
(TAHDi/500, TAHD) with an adaptor No. 5 (2 mm diameter). Each egg
sample was mounted on a platform and eggshells were punctured at the
top (small end) and bottom (large end). The force required to compress
the eggshell by 3% of its diameter was recorded at a strain rate of
60 mm/min. Samples were measured right after single and/or com-
bined treatments (0.5, 2.0 kGy X-ray and/or 50, 300 ppm NaOCl) and
puncture test evaluation was recorded as the Newtons (N) required to
puncture the whole egg. The mean of 5 measurements was recorded for
each sample.
2.9. Statistical analysis
All experiments were performed in triplicate. The average reduc-
tions in microbes were represented as log CFU/egg. One-way ANOVA
was used to determine any significant differences in the population of
biofilm cells under the different conditions, which were then compared
using Duncan's multiple range test. Color and texture data were also
analyzed via SAS (Version 9.2, SAS Institute Inc., Cary, NC, USA). The
significant (p < 0.05) means were grouped using Duncan's multiple-
range test.
3. Results and discussion
3.1. Bactericidal and synergistic effect of X-ray irradiation and NaOCl
treatment against S. Typhimurium biofilm on formation on quail eggshells
Many studies have been conducted to evaluate the inactivation ef-
ficiency or synergy of combined chemical treatments and physical sa-
nitization techniques compared with that of individual treatments.
Studies of combined disinfection treatments are common in the litera-
ture (Chawla et al., 2006; Kanatt et al., 2006; Park et al., 2012; Park
et al., 2014; Park et al., 2016).
The bactericidal effects of individual and combined treatments X-
ray/NaOCl treatment on S. Typhimurium biofilms is shown in Table 1.
To investigate the bactericidal effects of X-ray and NaOCl against S.
Typhimurium biofilms on eggshells, we performed combination treat-
ment with different X-ray irradiation doses and varying concentrations
of NaOCl.
After individual irradiation treatment with 0.5, 1.0, 1.5, or 2.0 kGy
X-ray, the reduction value of S. Typhimurium in quail eggshell biofilms
was 0.9, 2.0, 2.2, and 2.7 log CFU/egg, respectively. In X-ray single
treatment, significantly increased reductions of S. Typhimurium bio-
films were observed as the X-ray irradiation dose increased (p < 0.05).
However, no significant decrease (p > 0.05) was observed between
irradiations with 1.0 to 1.5 kGy. Mahmoud et al. (2015) noted that a 3-
strain mixture of Salmonella enterica (S. Enteritidis E190-88, S. Typhi-
murium ATCC 14028 and S. Montevideo ATCC 8387), which was
8.1 ± 0.1 log CFU/egg, was significantly reduced by 3.0 log on egg-
shells by treatment with 0.5 kGy X-ray irradiation. They also high-
lighted that irradiation with 1.0 kGy X-ray resulted in a > 6 log CFU/
egg reduction in Salmonella strains on eggshell. The present study
showed that the biofilm reduction value (0.9 and 2.0 log CFU/egg) was

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S.-J. Jung et al.
lesser than that in the previous study after 0.5 and 1.0 kGy X-ray irra-
diation, respectively. This reduction may be a result of differences in
resistance character between biofilms and attached cells; in the present
study allowed 24 h for the formation of biofilms, whereas Mahmoud
et al. (2015) allowed 30 min for the bacteria to attach on the eggshell.
Additionally, many studies have demonstrated that biofilms are more
resistant to various antimicrobial treatments compared with that of
planktonic cells (Niemira and Solomon, 2005; Joseph et al., 2001).
In the case of NaOCl single treatment, significantly (p < 0.05) in-
creasing reductions of S. Typhimurium biofilms on quail eggshell were
observed as the concentration of NaOCl increased. After treatment with
50, 100, 150, 200, and 300 ppm NaOCl, the reduction value of S.
Typhimurium in quail eggshell biofilms was 0.1, 0.5, 0.6, 1.9, and
2.2 log CFU/egg, respectively. NaOCl treatment was observed to cause a
significant gradually reduction in biofilm cells under all treatment
conditions. Corcoran et al. (2013) demonstrated that NaOCl single
treatment (500 ppm from 10 to 90 min) was not effective against the
mixture of Salmonella in established biofilms (during 48 h). In a similar
study, Kim et al. (2016) reported that after single treatment with 50,
100, 150, or 200 ppm of NaOCl, the reduction values for L. mono-
cytogenes biofilms on eggshells were 0.24, 1.07, 1.14, and 1.26 log CFU/
cm2, respectively. The sterilization mechanism of NaOCl is based on its
physicochemical properties. The antimicrobial efficacy of NaOCl is at-
tributed through hydrolysis (NaOCl + H2O → HOCl + NaOH). H
(Estrela et al., 2003). NaOCl translates into hypochlorous acid (HOCl).
HOCl is a weak acid and dissociates into the hypochlorite ion (−OCl)
and proton (H+) depending on the solution pH (Fukuzaki, 2006).
After combined treatments with 0.5–2.0 kGy X-ray and 50–300 ppm
NaOCl, the reduction values in S. Typhimurium biofilms on the quail
eggshells were 2.0 to 4.6 log CFU/egg. The reduction of S.
Typhimurium biofilms was significantly (p < 0.05) greater than that of
X-ray or NaOCl treatment alone. Specifically, when combined with
50 ppm NaOCl and various X-ray irradiation doses (0.5, 1.0, 1.5 and
2.0 kGy), the reduction values of S. Typhimurium biofilms were 2.0,
2.6, 3.2 and 4.3 log CFU/egg, respectively.
Table 2 shows the synergistic effects of the combination of X-ray
and NaOCl treatments against S. Typhimurium biofilms on the surface
of quail eggshells. The synergistic values of combined treatment against
S. Typhimurium biofilms on egg shell were 0.90, 0.45, 0.92 and
1.47 log CFU/egg after 0.5, 1.0, 1.5 and 2.0 kGy X-ray with 50 ppm
NaOCl. The least synergistic effect for the treatment of S. Typhimurium
biofilms on eggshells was a reduction of 0.06 log CFU/egg observed
using a combination of 1.5 kGy X-ray and 300 ppm NaOCl. Most sy-
nergistic reductions were lesser than 1 log (90%) except for those
achieved with 1.5 kGy X-ray and 100 or 150 ppm NaOCl, and 2.0 kGy X-
ray and 50 or 100 ppm NaOCl, which were 1.40, 1.34, 1.47, and
1.10 log CFU/egg, respectively. Especially, the highest synergistic re-
duction value against S. Typhimurium biofilms on eggshells was
1.47 log CFU/egg (total reduction value: 4.3 log CFU/egg), observed
with 2.0 kGy X-ray and 50 ppm NaOCl. After combined treatment with
Table 2
The synergistic effect values of S. Typhimurium biofilms on quail eggshells after combination treatment with X-ray/NaOCl.
Sample Treatment Mean ( ± SD) synergistic and antagonistic value of reduction (log CFU/egg)
X-ray (kGy)
0.5
Egg shell NaOCl (ppm) 50 0.90 ± 0.17
100 0.74 ± 0.15
150 0.75 ± 0.16
200 −0.27 ± 0.15
300 −0.48 ± 0.23
Synergistic effect value (+) = total reduction value of combined x-ray/NaOCl treatment − (reduction values from x-ray treatment + reduction values from NaOCl treatment).
Antagonistic effect value (−) = total reduction value of combined x-ray/NaOCl treatment − (reduction values from x-ray treatment + reduction values from NaOCl treatment).
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Food Research International 107 (2018) 496–502
2.0 kGy X-ray and 50 ppm NaOCl, a 32.92-fold decrease in S. Typhi-
murium biofilms was observed compared to that with 50 ppm NaOCl
treatment alone. Additionally, a 1.60-fold decrease in S. Typhimurium
biofilms was observed compared to that with X-ray irradiation
(2.0 kGy) alone. The synergistic effect against biofilms of S. Typhi-
murium was not dependent on the dosage of X-ray or concentration of
sodium chloride.
Lillard (1993) observed that the combined treatment of chlorine
(0.5 ppm) and 20-kHz ultrasound yielded a 2.4–4-log reduction of
Salmonella spp. on chicken skin. In addition, Seymour et al. (2002)
reported that S. Typhimurium was further reduced by 1 log unit on
iceberg lettuce by cleaning with a combined treatment of chlorine
(25 ppm) and ultrasound (32–40 kHz, 10–15 W/L) for 10 min compared
to that with ultrasound or chlorine alone. Kim et al. (2016) reported
that reduction values of L. monocytogenes biofilms on eggshells after
combined treatment of UV-C and NaOCl 300–50 to 1800–300 (mWs/
cm2/ppm) were 1.25 to 2.70 log CFU/cm2. Furthermore, they calcu-
lated the synergistic reduction effect value, which showed maximum
1.02 log CFU/cm2 reduction. According to Park et al. (2016), combined
treatment with ultrasound for 100 min and 200 ppm NaOCl reduced
Cronobacter sakazakii biofilms on lettuce with a maximum
4.44 log CFU/g reduction. After this combined treatment, the greatest
synergistic value obtained was < 1.0 log CFU/g.
Table 2 describes the combined treatment results; unfortunately, an
antagonistic effect on S. Typhimurium biofilm reduction on eggshells
was observed with combined treatment over 200 ppm NaOCl, except for
combinations of 1.5 kGy X-ray/200 ppm NaOCl and 1.5 kGy X-ray/
300 pm NaOCl. Similarly, some previous studies have shown antag-
onistic effects. Park, Song, & Ha (2014) reported that combined treat-
ment with 60–100 ultrasonication and 50 ppm chlorine resulted in a
0.4–0.2 log antagonistic reduction effect on E. coli in seaweed produc-
tion. Moreover, Park et al. (2016) observed that the combination of
ultrasonication and chlorine (80 min-150 ppm) resulted in a 0.13 log
antagonistic reduction effect of C. sakazakii on lettuce. In addition, Kim
et al. (2016) demonstrated that on eggshells, combined treatment with
UV and NaOCl resulted in antagonistic reduction effects (0.08 and
0.22 log CFU/cm2) of L. monocytogenes, using 1800 mWs/cm2 and
100 ppm, and 1800 mWs/cm2 and 150 ppm, respectively.
3.2. FE-SEM images of S. Typhimurium biofilm formation on egg shell
The efficacy of X-ray irradiation and NaOCl treatment in eliminating
biofilms was clearly demonstrated when evaluated by FE-SEM.
Representative FE-SEM images of S. Typhimurium biofilms on quail
eggshells are illustrated in Fig. 1. A clear reduction in biofilms was
observed via FE-SEM on eggshells after both single chemical treatment
and combination treatment.
Control samples treated with DW formed extensive and strong
biofilms on eggshells, and individual rod-shaped biofilm cells as well as
three-dimensional structures in the shape of a mushroom are shown
1.0 1.5 2.0
0.45 ± 0.26 0.92 ± 0.12 1.47 ± 0.10
0.83 ± 0.24 1.40 ± 0.14 1.10 ± 0.08
0.67 ± 0.25 1.34 ± 0.12 0.97 ± 0.14
−0.56 ± 0.24 0.18 ± 0.13 −0.06 ± 0.12
−0.65 ± 0.24 0.06 ± 0.06 −0.25 ± 0.10
S.-J. Jung et al.
Fig. 1. Field emission scanning electron microscopy images of S. Typhimurium biofilms on quail eggshells treated with: (a) sterile distilled water (5000× magnification); (b) NaOCl at
50 ppm (5000× magnification); (c) X-ray irradiation at 2.0 kGy (5000× magnification); and (d) combined 2.0 kGy X-ray and 50 ppm NaOCl (5000× magnification).
(Fig. 1A). Treatment with NaOCl alone affected the formation of S.
Typhimurium biofilms by altering the biofilm architecture on the sur-
face of the eggshells. After exposure to 50 ppm NaOCl, bacteria were
still compactly attached to the eggshell (Fig. 1B). After X-ray treatment
alone (2.0 kGy), cell aggregates were still formed on the surface of the
eggshell, and a monolayer of cell attachment was observed at 2.0 kGy
(Fig. 1C).
Notably, after combination treatment, biofilms on eggshells did not
exhibit a three-dimensional structure; instead, a monolayer was ob-
served (Fig. 1D). We also observed that background bacterial cells on
quail eggshells were clearly removed after combined treatment. In
previous study, they found that eggshells contained more compacted
biofilms with higher resistance than other food contact surface mate-
rials such as stainless steel, plastic, or rubber (Jung et al., 2017). Si-
milarly, in the present study, FE-SEM images show strong biofilm for-
mation on eggshells (Fig. 1). According to BAKTERIJ (2014), the
topography of a material surface can affect bacterial attachment and
biofilm formation. Surfaces with low shear forces that inhibit bacterial
attachment are significantly reduced on rough surfaces. Also food sur-
faces tend to provide more room for cells to attach due to the presence
of abundant nutrients environment. The efficacy of a disinfectant de-
pends on the ability of microorganisms to form biofilms on the surface,
stomata, trichomes, and cutting edges of materials (Jahid & Ha, 2012;
Olaimat & Holley, 2012).
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Food Research International 107 (2018) 496–502
3.3. Color and puncture force changes of eggshells after combination
treatment (X-ray/NaOCl)
Various combinations of chemical and physical food disinfectants
have been studied to reduce pathogenic bacteria populations, without
causing any simultaneous loss in quality (Chawla et al., 2006; Kanatt
et al., 2006). Likewise, the color and texture changes of eggshells were
analyzed after combination treatments with X-ray irradiation and
NaOCl. The color measurement values for eggshells subjected to com-
bination treatment are shown in Table 3. The results of the after no
treatment eggshell color after combined treatment were 80.7, −1.4 and
5.5 for lightness, greenness and yellowness, respectively. Color results
for the eggshells were not significantly different (p > 0.05) after
treatment with X-ray/NaOCl.
In a previous study (Mahmoud, 2012), no significant differences
(p > 0.05) were observed for color values (L⁎, a⁎, and b⁎) of whole
cantaloupes after X-ray irradiation with 2.0 kGy. Additionally, Jung
et al. (2015) reported no significant difference in the hunter color value
(p > 0.05) of red pepper powder after X-ray irradiation, even at 10 kGy
(2, 4, 6, 8, and 10 kGy). Kim et al. (2016) highlighted that there were no
significant changes (p > 0.05) in the colors represented by L⁎, a⁎, and
b⁎ for any of the eggshells treated with UV–C and/or NaOCl.
Total color difference (ΔE⁎) was observed as 0.6–1.2 unit after
combined treatments with X-ray irradiation and NaOCl. In the present
study, ΔE⁎ was investigated to confirm the overall visual color change.
S.-J. Jung et al. Food Research International 107 (2018) 496–502

Table 3
Change in Hunter color and texture values of eggshells after single and combination treatment with X-ray/NaOCl.

Treatment Hunter color Puncture force (N)

L⁎ a⁎ b⁎ ΔE

0 80.7 ± 0.85 −1.4 ± 0.08 5.5 ± 0.09 13.6 ± 2.2

X-ray 0.5 79.8 ± 0.55 −1.2 ± 0.20 5.5 ± 0.20 0.98 13.0 ± 2.2
2.0 80.2 ± 1.37 −1.3 ± 0.23 5.8 ± 0.25 0.64 13.7 ± 2.8
NaOCl 50 80.7 ± 1.16 −1.4 ± 0.08 5.3 ± 0.06 0.27 12.4 ± 0.5
300 81.3 ± 0.39 −1.5 ± 0.03 5.5 ± 0.06 0.54 11.6 ± 1.0
X-ray/NaOCl (kGy-ppm) 0.5–50 81.1 ± 0.86 −1.3 ± 0.13 5.1 ± 0.14 0.58 14.6 ± 0.7
0.5–300 81.7 ± 0.57 −1.5 ± 0.15 5.2 ± 0.17 1.00 14.5 ± 0.6
2.0–50 82.0 ± 0.55 −1.3 ± 0.16 5.3 ± 0.42 1.29 14.5 ± 0.7
2.0–300 81.8 ± 0.29 −1.3 ± 0.03 5.4 ± 0.14 1.11 13.7 ± 0.9

Values are mean ± standard deviation (n = 5).

L⁎ values = lightness.
a⁎ values = redness/greenness (+ = red,− = green).
b⁎ values = yellowness/blueness (+ = yellow, − = blue).
ΔE values = total color difference.

All of the ΔE⁎ values were observed to be < 1.0 unit after each single
treatment; this might be considered as no visible differences after X-ray
or NaOCl treatment alone. However, results with > 1.0 unit were ob-
served under several combined conditions. According to Francis &
Clydesdale (1975), if the ΔE⁎ value is > 2.0, the color difference could
be visually noticeable.
Changes in eggshell thickness as a result of combined treatments
were measured by puncture force, and the results are shown in Table 3.
Measurement of puncture force was monitored immediately after
treatment. A similar trend was observed for change in puncture force
after combination treatments, where no significant difference was ob-
served after treatment with up to 2.0 kGy X-ray and 300 ppm NaOCl.
The maximum force to puncture an eggshell of the control group was
13.6 N. No significant difference in puncture force was observed after
single or combined treatments compared with that of the control
samples (p > 0.05). Rababah et al. (2005) found that trained sensory
panelists evaluating chicken with no additives that had been irradiated
at 2.5 kGy could not detect any texture difference compared to that in
samples of non-irradiated chicken.
Carnarius et al. (1996) reported that puncture force and shell
thickness were significantly related (p < 0.01), and cracked and leaky
eggs were observed to have significantly lower puncture force
(p < 0.05). Based on the Carnarius et al. (1996) study, the present
puncture force results (Table 3) indicate that single and combination
treatments did not affect the thickness of the quail eggshell.
4. Conclusions
S. Typhimurium biofilms are strongly related to the contamination
of raw eggs and their products. In this study, combined treatment with
X-ray irradiation and NaOCl resulted in a synergistic anti-biofilm effect
on S. Typhimurium. Combined X-ray/NaOCl treatment showed the
highest synergistic reduction value on biofilms of 1.47 log CFU/egg at
X-ray 2.0 kGy and NaOCl 50 ppm, and the total reduction value on
biofilms was 4.3 log CFU/egg. Furthermore, these combined treatments
did not lead to any change in egg shell quality in either color or
thickness. Consequently, the combination strategies of X-ray irradiation
and chemical disinfectants can be used to enhance safety in the egg
industry.
Acknowledgments
This research was supported by the Chung-Ang University Graduate
Research Scholarship in 2017.
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