Food Control 115 (2020) 107277

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Food Control
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The efficacy of X-ray doses on Vibrio vulnificus in pure culture and Vibrio T
parahaemolyticus in pure culture and inoculated farm raised live oysters
(Crassostrea virginica) with different acceleration voltages☆
Yuwei Wua, Sam K.C. Changa,b,∗
a
Experimental Seafood Processing Laboratory, Coastal and Research Extension Center, Mississippi State University, Pascagoula, MS, 39567, USA
b
Food Science, Nutrition, and Health Promotion Department, Mississippi State University, MS State, MS, 39759, USA

A R T I C LE I N FO A B S T R A C T

Keywords: Vibrio vulnificus and Vibrio parahaemolyticus are halophilic bacteria and can cause illness associated with the
Vibrio parahaemolyticus consumption of oysters. In this study, we tested the hypothesis to see if the adjustment of the acceleration
Vibrio vulnificus voltage can change the efficacy of X-ray doses on V. vulnificus and V. parahaemolyticus in pure culture and
X-ray inoculated V. parahaemolyticus within farm-raised live oysters (Crassostrea virginica). By using a tailor-made X-
Acceleration voltage
ray irradiator (Kimtron 350), the acceleration voltage was adjusted to 50, 200 or 350 kV and irradiated on pure
Live oysters
culture of V. vulnificus or V. parahaemolyticus and farm-raised live oysters accumulated with a three-strain
mixture of V. parahaemolyticus. Inactivation of V. vulnificus and V. parahaemolyticus pure cultures without filter
had a lower inactivation when compared with filtered X-ray. With a 1 mm aluminum filter, 250–300 Gy X-ray
reduced more than 6 log CFU ml−1 at 350 kV acceleration voltage. When 350 kV X-ray was applied on live
oysters with the aluminum filter, 6 log MPN g−1 reduction of V. parahaemolyticus was achieved with 1250 Gy.
The V. parahaemolyticus was significantly (p < 0.05) reduced in whole shell oysters from 8.11 ± 0.19 to
6.11 ± 0.19, 3.97 ± 0.47, 4.17 ± 0.17 and 2.77 ± 0.28 log MPN g−1 after processing with 250, 500, 750
and 1000 Gy, respectively. These results showed that the pathogen inactivation can be improved by the opti-
mization of energy level with the adjustment of irradiation filter and acceleration voltage.

1. Introduction was sufficient to produce a 6-log reduction of V. parahaemolyticus in

The presence of the pathogens in oysters has a serious impact on
public health and international trade because oysters can serve as ve-
hicles for foodborne pathogenic microorganisms by filtering of sea-
water (Le Guyader, Atmar, & Le Pendu, 2012; Mahmoud, 2009b). Vi-
brios exist naturally in the marine environment water, where oysters
are grown (Campos & Lees, 2014). Vibrio vulnificus is a cause of human
illness and death to individuals with underlying diseases especially liver
disease, occurring naturally in warm estuarine environments, such as
the Gulf Coast water (Mahmoud, 2009b). Vibrio parahaemolyticus can
cause illness with symptoms, including gastroenteritis, vomiting, diar-
rhea, headache, and nausea (Mahmoud, 2009a; Mahmoud & Burrage,
2009).
Irradiation is one of FDA-approved technologies for killing vibrios in
oysters. Jakabi et al. (2003) reported 1.0 kGy gamma radiation (60Co)
oysters. Irradiation dose at 3.0 kGy gamma ray did not kill the oysters
or affect their sensory attributes (Jakabi et al., 2003). Our laboratory
further reported the survivability of live oysters was not significantly
affected by 5.0 kGy X-ray for up to 10 days during storage at 5 °C (Wu
et al., 2017).
In the industrial application, live oysters and blanched shellfish
from the Gulf of Mexico are irradiated to reduce the risk of con-
tamination by virulent microorganisms, particularly Vibrio species
(Ehlermann, 2016). Oysters have been successfully processed to reduce
vibrios by Gateway America located in Gulfport, Mississippi. Gateway
America has a pool type gamma ray irradiator for food phytosanitation.
From our personal communication, Gateway America is planning to
build X-ray facilities for their new plants. A study submitted to National
Nuclear Security Administration (NNSA) by Fermi National Accelerator
Laboratory predicted an increasing trend of X-ray application for cold

☆
This paper is a journal article of the Mississippi Agriculture and Forestry Experiment Station.
∗
Corresponding author. Experimental Seafood Processing Laboratory, Coastal and Research Extension Center, Mississippi State University, Pascagoula, MS, 39567,
USA.
E-mail address: sc1690@msstate.edu (S.K.C. Chang).

https://doi.org/10.1016/j.foodcont.2020.107277
Received 10 January 2020; Received in revised form 24 March 2020; Accepted 25 March 2020
Available online 30 March 2020
0956-7135/ © 2020 Published by Elsevier Ltd.
Y. Wu and S.K.C. Chang
sterilization (Kroc, Kephart, Thangaraj, & Penning, 2017). X-ray sys-
tems, can be turned on and turned off, are even more economical when
compared with large size gamma-ray facilities because of the im-
provement of modern technology. X-ray meets the highest requirements
for treating food in pallets with very fast speed, excellent dose uni-
formity and adjustable power output.
Both gamma ray and X-ray without filter have been reported to have
4 logs of reductions of V. parahaemolyticus in the pure culture by 500 Gy
exposure (Mahmoud & Burrage, 2009; Thupila, Ratana-Arporn, &
Wilaipun, 2011). When applied to whole shell oysters, 1000 Gy gamma
ray can produce a 6-log reduction of inoculated V. parahaemolyticus
(Andrews, Jahncke, & Mallikarjunan, 2003; Jakabi et al., 2003; Thupila
et al., 2011) while 5000 Gy X-ray without filter is needed to have the
similar reduction (Mahmoud & Burrage, 2009). It is necessary to im-
prove the sterilization of vibrios within oysters by X-ray for industrial
application. Gamma ray production is due to radionuclide decay and
emitted at distinctive energy with mega-electron-volt (MeV) level,
while X-ray is polychromatic ionizing radiation of a continuous spec-
trum and has a sharp cutoff due to the limited energy of the incoming
electrons which is equal to acceleration voltage (Urbain, 2012; Zhang,
Ha, Seck, & Zhou, 2019). Ionizing radiations deposit energy on matter
through three kinds of mechanism: the photoelectric effect, Compton
scattering, and pair production (Urbain, 2012). Although the me-
chanism of depositing energy on matter is determined by the energy
level of the radiation, there is no report about the impact on pathogen
inactivation by different energy levels of ionizing irradiation.
To test the hypothesis if acceleration voltage can change the in-
activation efficacy of X-ray doses on vibrios, our laboratory acquired a
tailor-made X-ray irradiator (Kimtron 350) to optimize the best pa-
thogen inactivation and food preservation by using irradiation filter
and varying acceleration voltage. Our results on Escherichia coli
O157:H7 showed that the pathogen inactivation can be improved by
the optimization of energy level with the adjustment of irradiation filter
and acceleration voltage (Wu & Chang, 2019). The objective of this
study was to determine the effect of the X-ray irradiation of Vibrios
under various acceleration voltages (50, 200 and 350 kV) to produce
various dosages 0–300 Gy with 50 Gy interval for pure culture treat-
ment (with or without an 1 mm thickness aluminum filter) and filtrated
irradiation ranging from 0 to 1250 Gy with 250 Gy intervals for live
oyster treatment.
2. Materials and methods
2.1. Bacterial strains and growing conditions
A cocktail of three V. vulnificus (ATCC 27562, ATCC 33815 or ATCC
33816) or V. parahaemolyticus strains (ATCC17802, ATCC35117 and
ATCC35118) purchased from American Type Culture Collection (ATCC)
were used for this study. Each bacterial was grown in trypticase soy
broth with 1% NaCl (TSB-1% NaCl) and incubated at 35 °C for 18–24 h
with continuous agitation (200 rpm) on a Corning® LSE™ 71L Shaking
Incubator (Corning, NY, USA) as described by Mahmoud and Burrage
(2009). The cultures were streaked onto individual plates of trypticase
soy agar supplemented with 1% NaCl (TSA-1% NaCl) and incubated at
35 °C for 18–24 h. A single colony was selected from the TSA-1% NaCl
plate and enriched in TSB-1% NaCl at 35 °C for 18–24 h (approx.
108−9 CFU ml−1).
2.2. Atlantic oysters (Crassostrea virginica)
Live, freshly harvested Atlantic oysters (Crassostrea virginica),
graded the size to 3 inches by sorting machine were obtained from an
oyster breeder (Murder Point Oyster Co, Bayou La Batre, AL, USA) off
Alabama's coastline. The length of oysters was 73.33 ± 3.12 mm after
measuring 21 oysters. The oysters were transported immediately (ap-
proximately 30 min) in a cooler with ground ice to the laboratory. The
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Food Control 115 (2020) 107277
oysters were washed with tap water to remove mud from the shells and
kept at refrigerator temperature until use.
2.3. Preparation of pure culture and bioaccumulation in whole-shell oysters
2.3.1. Pure culture
Each bacterial strain culture was centrifuged at 3000 rpm (BD
420104 Dynac III Centrifuge; AriaMedical Equipment, San Antonio, TX,
USA) for 10 min. The supernatant was discarded and the cell pellet was
washed and resuspended in Phosphate Buffered Saline (PBS, pH 7.4).
The three strains of V. vulnificus or V. parahaemolyticus were then
combined at an equal volume to produce a culture cocktail as described
by Mahmoud and Burrage (2009).
2.3.2. Oysters
Live oysters were allowed to accumulate the V. parahaemolyticus
mixture according to our previously described method (Wu et al., 2017)
with a few modifications. For each batch, 18 live oysters were culti-
vated in polyethylene tank containing recirculating 60 L of artificial
seawater (1.5% Instant Ocean® Sea Salt, Instant Ocean Spectrum
Brands, Blacksburg, VA) at room temperature (20–22 °C). Oysters were
activated to start filtering water by using phyto-plankton (Reef Phyto-
plankton, Seachem, Madison, GA) according to the instruction of the
manufacturer. The oyster's vitality was checked by tapping with a long
stick to see if it quickly closed the oyster shells. Only the oysters quickly
closed after a tapping were left for the following experiment. The cul-
tures of V. parahaemolyticus were added to the tank to reach approxi-
mately 107–108 CFU ml−1. The water temperature of the tank was
maintained at room temperature (20–22 °C) for V. parahaemolyticus
accumulation for 12–18 h before X-ray treatment.
2.4. Description of radiator and generation of X-ray
Specific corresponding doses (50, 100, 150, 200, 250, 300, 500,
750, 1000, and 1250 Gy) as measured from the X-ray generator source
were generated at 50, 200 and 350 kV using Kimtron 350 (Kimtron Inc,
Oxford, CT, USA). Kimtron 350 is a tailor-made X-ray irradiator cus-
tomized for food irradiation research. The irradiation beam filter and
acceleration voltage can be optimized for the best pathogen inactiva-
tion and food preservation. An internal ion chamber (TM7862 Monitor
chamber, PTW, Freiburg, Germany) is mounted at the filter-installation
box and measures the output dose in real time. The integrated dose
control system of Kimtron 350 including mounting device, ion
chamber, dose meter and all associated hardware and software enables
the operator to enter a desired corresponding dose at sample shelf via
the touch-panel controller and terminates the exposure once the desired
dose has been achieved. The X-ray tube was mounted enabling it
downward-pointing attitude.
2.5. Treatment of pure cultures and inoculated oysters with X-ray
2.5.1. Pure culture
The suspension of V. vulnificus or V. parahaemolyticus was prepared
as mentioned above. The cells suspension with 9.5 ml volume were
placed in the 6-well flat bottom plate (9.5 cm2 culture area, no surface
treatment) and downward-pointing irradiated specific irradiation doses
(50, 100, 150, 200, 250 and 300 Gy) were generated at 50, 200 and
350 kV. Taking advantage of this downward-pointing orientation, X-ray
does not need to pass through the substance of the container, ensuring
that X-ray acts directly on the suspension. In order to compare the effect
of filter on the bactericidal effect, the pure culture experiments were
divided into two groups, one was unfiltered, and one with an aluminum
filter with a thickness of 1 mm. Irradiation was operated at 22 °C and
55–60% relative humidity as described by Wu et al. (2016).
Y. Wu and S.K.C. Chang
2.5.2. Oyster
For comparison, three non-inoculated and inoculated live oysters
were used to determine the initial numbers of inherent bacteria and
accumulated V. parahaemolyticus before X-ray processing. The oysters
were also placed in the container inside the exposure chamber and
processed with different doses of X-ray (250, 500, 750, 1000, and
1250 Gy). The container contained ground ice so that the lower half of
the oyster was buried in the ground ice while the upper half was ex-
posed to the downward-pointing X-ray source.
2.6. Microbial enumeration
2.6.1. Pure culture
For examining V. vulnificus in pure culture samples, serial 10-fold
dilutions were made with PBS. Bacterial populations were enumerated
using overlay plating method as described by Mahmoud (2009b) with
some modification. The diluted solutions (0.1 ml) were spread plated
onto TSA–1.0% NaCl agar plates and incubated at 35 °C for 4–6 h fol-
lowed by pouring a layer of selective agar (Cellobiose Polyxymin B
Colistin Modified Agar (mCPC Agar)) on the top of the resuscitated cells
and re-incubated for 18–20 h. Colonies were counted and results ex-
pressed as log CFU ml−1. For examining V. parahaemolyticus, similar
method was applied as described above except selective agar was
changed to thiosulfate-citrate-bile salts-sucrose agar (TCBS agar)) as
described by Mahmoud and Burrage (2009).
2.6.2. Oysters
V. parahaemolyticus was extracted from oysters as described by Wu
et al. (2017) with a few modifications. To shuck the oysters, a sterile
oyster knife was inserted to the point between the shells on the ventral
side after making a small opening with a part of sterile bone cutting
forceps. The adductor muscle was cut from the upper flat shell and
drained shell liquor into the blender. The whole animals were trans-
ferred to the sterile blender after severing the adductor muscle con-
nection to the lower shell. In each assay, two whole shell oysters were
shucked and pooled. The samples were homogenized by a sterilized
blender for 2 min after diluting 10 times with PBS. A three-tube MPN
method was used to determine the populations of V. parahaemolyticus as
described by Mahmoud and Burrage (2009). All sample dilutions were
individually inoculated into three tubes of alkaline peptone water
(APW) after serial 10-fold dilutions. Inoculated APW tubes were in-
cubated at 35 °C for 18 h and one loopful of samples from the top
3
Food Control 115 (2020) 107277
Fig. 1. Changes in the counts (log CFU ml−1) of
V. vulnificus pure culture by 0, 50, 100, 150, 200,
250 and 300 Gy X-ray at 50, 200 and 350 kV
without filter. Different lower-case letters in-
dicated differences (p < 0.05) among the levels
of within each kind of different acceleration
voltage (p < 0.05). Different capital letters in-
dicated differences (p < 0.05) among same ir-
radiation dosage.
1.0 cm of a turbid tube was streaked onto individual TCBS agar for V.
parahaemolyticus detection. All plates were incubated at 35 °C for 24 h.
Formation of colonies that were round (2–3 mm diameter) and green or
bluish on TCBS agar were counted as positive V. parahaemolyticus. The
results expressed as log MPN g−1. For determining inherent microflora
on oysters, serial dilutions were prepared by PBS and a 0.1 ml portion
of each dilution was spread on trypticase soy agar (TSA) plates and
incubated at 35 °C for 48 h. Colonies were counted and results ex-
pressed as log CFU g−1.
2.7. Statistical analysis
All experiments were replicated three times. The mean values and
standard deviations were determined using 2014 SAS (Version 9.4, SAS
Inst. Inc., Cary, NC, U.S.A.). Significant differences among means were
made with the Least Significant Difference (LSD) analysis using prob-
ability level of 0.05. For D10-value determination, a first-order kinetic
model (linear model) was used to analyze the data for log of surviving
organisms per treatment dose (Mahmoud, 2009c). The D10-values (X-
ray dose required for a 90% reduction) were determined using survival
data for X-ray dose (Gy) during treatment. D10-values analyses were
performed using Microsoft Excel (Microsoft, Redmond, WA, USA).
3. Results and discussion
3.1. Inactivation of V. vulnificus pure culture with or without 1 mm
aluminum filter
The effect of X-ray treatment on the population of V. vulnificus in
pure culture without the 1 mm aluminum filter is shown in Fig. 1. The
log CFU ml−1 was significantly (p < 0.05) reduced from 8.32 ± 0.19
to 7.90 ± 0.14 after treatment with 50 Gy at 50 kV. No significant
differences were observed between the samples treated with 50, 100,
150, 200, 250 and 300 Gy. More reduction of log CFU ml−1 was found
when the acceleration voltage was increased to 200 kV. The log CFU
ml−1 was significantly (p < 0.05) reduced from 8.32 ± 0.19 to
7.82 ± 0.08, 7.48 ± 0.39, 7.24 ± 0.19 and 7.00 ± 0.14 after
treatments with 150, 200, 250 and 300 Gy at 200 kV. Among the ir-
radiation voltage treatments on pure culture without filter, the 350 kV
X-ray had the highest V. vulnificus inactivation. The log CFU ml−1 was
significantly (p < 0.05) reduced from 8.32 ± 0.19 to 7.24 ± 0.12,
6.67 ± 0.28, 6.55 ± 0.23 and 6.09 ± 0.18 after treatments with
Y. Wu and S.K.C. Chang
150, 200, 250 and 300 Gy at 350 kV, respectively.
Inactivation of V. vulnificus pure culture without filter had a lower
inactivation when compared with previous reports which used X-ray as
a source for irradiation. As described previously reported by our la-
boratory (Mahmoud, 2009b), the V. vulnificus pure cultures had a 3-log
reduction by 100 Gy X-ray, irradiated using X-ray irradiator as RS 2400
(Rad Source Technologies, Inc.). We noticed that although even the
RS2400 had no installed filter, its stainless-steel cylinder might have
acted as a filter to produce filtrated radiation. Further research required
us to use of filters with different materials and thickness to examine the
effect on sterilization effects.
The effect of X-ray treatment on the population of V. vulnificus in
pure culture with the 1 mm aluminum filter is shown in Fig. 2. The log
CFU ml−1 was significantly (p < 0.05) reduced from 8.32 ± 0.19 to
7.67 ± 0.12, 7.33 ± 0.05, 6.83 ± 0.12, 5.89 ± 0.17, 4.84 ± 0.07,
and 3.72 ± 0.30 after treatments with 50, 100, 150, 200, 250 and
300 Gy, respectively, at 50 kV of acceleration voltage. The log CFU
ml−1 was significantly (p < 0.05) reduced from 8.32 ± 0.19 to
4
Food Control 115 (2020) 107277
Fig. 2. Changes in the counts (log CFU ml−1) of
V. vulnificus pure culture by 0, 50, 100, 150, 200,
250 and 300 Gy X-ray at 50, 200 and 350 kV
with 1 mm aluminum filter. Different lower-case
letters indicated differences (p < 0.05) among
the levels of within each kind of different ac-
celeration voltage (p < 0.05). Different capital
letters indicated differences (p < 0.05) among
same irradiation dosage.
Fig. 3. Changes in the counts (log CFU ml−1) of
V. parahaemolyticus pure culture by 0, 50, 100,
150, 200, 250 and 300 Gy X-ray at 50, 200 and
350 kV without filter. Different lower-case let-
ters indicated differences (p < 0.05) among the
levels of within each kind of different accelera-
tion voltage (p < 0.05). Different capital letters
indicated differences (p < 0.05) among same
irradiation dosage.
7.70 ± 0.11, 7.16 ± 0.04, 5.51 ± 0.08, 3.46 ± 0.17, 2.93 ± 0.32
and 3.11 ± 0.20 after treatments with 50, 100, 150, 200, 250 and
300 Gy, respectively, at 200 kV. At the acceleration voltage of 350 kV,
the log CFU ml−1 was reduced to non-detectable level at the dose of
300 Gy (more than 6 log CFU ml−1). The log CFU ml−1 was sig-
nificantly (p < 0.05) reduced from 8.32 ± 0.19 to 7.70 ± 0.11,
5.79 ± 0.17, 4.18 ± 0.27, 2.95 ± 0.13 and 1.76 ± 0.00 after
treatment with 50, 100, 150, 200 and 250 Gy, respectively. These re-
sults showed that the pathogen inactivation can be improved by the
optimization of energy level with the adjustment of irradiation filter
and acceleration voltage. Andrews et al. (2003) reported 500 Gy could
only reduce less than 5 log CFU ml−1 of V. vulnificus pure culture. A 6-
log reduction of V. vulnificus pure culture was achieved with 750 Gy X-
ray (Mahmoud, 2009b). Our experimental results showed that the V.
vulnificus inactivation was enhanced when an aluminum filter was in-
stalled. Under the experimental conditions in this study, the inactiva-
tion in pure culture (6 log CFU ml−1 reduction by 300 Gy) was much
more effective than that previously reported using gamma ray or X-ray.
Y. Wu and S.K.C. Chang
Because the low-energy wavelength X-rays were filtered out by the
presence of an aluminum filter, the dose rate was decreased at the same
time. It is not clear to what extent the simultaneous decrease in dose
rate contributed to the total inactivation of V. vulnificus and is worth
further study.
3.2. Inactivation of V. parahaemolyticus pure culture
The effect of X-ray treatment on the population of V. para-
haemolyticus in pure culture without filter is shown in Fig. 3. The log
CFU ml−1 was significantly (p < 0.05) reduced from 8.16 ± 0.03 to
7.73 ± 0.07, 7.78 ± 0.23, 7.86 ± 0.18, 7.00 ± 0.05 and
6.97 ± 0.05 after treatment with 100, 150, 200, 250 and 300 Gy at the
acceleration voltage of 50 kV, respectively. More reductions of log CFU
ml−1 were found at the same doses when the acceleration voltage was
increased to 200 kV acceleration voltage. The log CFU ml−1 was sig-
nificantly (p < 0.05) reduced from 8.16 ± 0.03 to 7.01 ± 0.09,
7.14 ± 0.01, 6.64 ± 0.13, 5.92 ± 0.09 and 5.86 ± 0.07 after
treatments with 100, 150, 200, 250 and 300 Gy at 200 kV, respectively.
Among the acceleration voltages without filter, the 350 kV acceleration
voltage had the highest V. parahaemolyticus inactivation. The log CFU
ml−1 was significantly (p < 0.05) reduced from 8.16 ± 0.03 to
7.76 ± 0.15, 7.27 ± 0.14, 6.92 ± 0.18, 6.45 ± 0.13, 5.57 ± 0.12
and 4.75 ± 0.04 after treatment with 50, 100, 150, 200, 250 and
300 Gy at 350 kV, respectively. The results above showed that 50 kV X-
ray without filter had a lower impact on the survival on V. para-
haemolyticus pure culture when compared with the literatures values
which reported a 4-log reduction by 500 Gy gamma ray or X-ray
(Mahmoud & Burrage, 2009; Thupila et al., 2011) which performed
their experiments with an initial culture inoculum of approximately
107–108 CFU ml−1. When 350 kV acceleration voltage was applied, the
impact on the survival on V. parahaemolyticus pure culture was not less
than gamma ray or X-ray described in previous reports.
The effect of X-ray treatment on the population of V. para-
haemolyticus in pure culture with filter is shown in Fig. 4. The log CFU
ml−1 was significantly (p < 0.05) reduced from 8.16 ± 0.03 to
7.07 ± 0.05, 5.29 ± 0.14, 4.05 ± 0.11, 4.01 ± 0.05 and
1.91 ± 0.31 after treatments with 50, 100, 150, 200 and 250 Gy at
50 kV, respectively. The log CFU ml−1 was significantly (p < 0.05)
reduced from 8.16 ± 0.03 to 7.38 ± 0.07, 5.42 ± 0.11,
3.85 ± 0.02, 3.40 ± 0.05 and 1.36 ± 0.08 after treatments with 50,
100, 150, 200 and 250 Gy at 200 kV, respectively. The log CFU ml−1
5
Food Control 115 (2020) 107277
was significantly (p < 0.05) reduced from 8.16 ± 0.03 to
7.20 ± 0.11, 5.32 ± 0.13, 3.89 ± 0.03 and 3.39 ± 0.01 after
treatments with 50, 100, 150 and 200 Gy at 350 kV, respectively. The
improvement of pathogen inactivation is much more when compared
with previous researchers’ results of a 4-log reduction by 500 Gy
(Mahmoud & Burrage, 2009; Thupila et al., 2011) using either gamma
ray or unfiltered X-ray.
The researches in our laboratory demonstrated a lower suscept-
ibility in the pure culture of Escherichia coli O157:H7 to X-ray when no
filter was applied (Wu & Chang, 2019). Treatments with 1250 Gy X-ray
without filter achieved the reductions of 5.5 log CFU ml−1 at 350 kV or
2.5 and 2.2 log CFU g−1 at the acceleration voltage of 200 and 50 kV,
respectively. When a 1 mm aluminum filter was added, E. coli O157:H7
was reduced to below detection limit (< 2.0 log CFU ml−1) when X-ray
doses were equal or higher than 750 Gy. Treatments with 500 Gy X-ray
achieved the reductions of 6.6 log CFU ml−1 at 50 kV or 5.9, and 4.8
log CFU ml−1 at 200 and 350 kV, respectively. Based on these ob-
servations, a higher the acceleration voltage not always has a better
inactivation of bacteria. Further research is necessary to optimize the
acceleration voltage for specific bacteria and the different operation
conditions. It is also needed to conduct more comprehensive researches
on the metal material of the filter and its thickness to determine and
optimize the effectiveness for various pathogens.
3.3. Inactivation of V. parahaemolyticus within whole shell live oysters with
or without 1 mm aluminum filter
The effect of X-ray treatment on the population of V. para-
haemolyticus in whole shell oysters is shown in Fig. 5. When 350 kV
accelerated X-ray was applied on live oysters, 6 log MPN g−1 was
achieved with 1250 Gy. The log MPN g−1 was significantly (p < 0.05)
reduced in whole shell oysters from 8.11 ± 0.19 to 6.11 ± 0.19,
3.97 ± 0.47, 4.17 ± 0.17, 2.77 ± 0.28, and 1.88 ± 0.35 after
treatment with 250, 500, 750, 1000 and 1250 Gy X-ray, respectively.
Less inactivation of V. parahaemolyticus was found when using a lower
range of acceleration voltage. Literature had reported that 1000 Gy
gamma ray could produce a 6-log reduction of inoculated V. para-
haemolyticus (Andrews et al., 2003; Jakabi et al., 2003; Thupila et al.,
2011) in oysters, while 5000 Gy X-ray without filter is needed to pro-
duce a similar reduction (Mahmoud & Burrage, 2009). However, in our
current study with filter at 350 kV, more than 6-logs of reduction in live
oysters could be achieved by using 1250 Gy, representing a dramatic
Fig. 4. Changes in the counts (log CFU ml−1) of
V. parahaemolyticus pure culture by 0, 50, 100,
150, 200, 250 and 300 Gy X-ray at 50, 200 and
350 kV with 1 mm aluminum filter. Different
lower-case letters indicated differences
(p < 0.05) among the levels of within each kind
of different acceleration voltage (p < 0.05).
Different capital letters indicated differences
(p < 0.05) among same irradiation dosage.
Y. Wu and S.K.C. Chang Food Control 115 (2020) 107277

Fig. 5. Changes in the counts (log MPN g−1) of

V. parahaemolyticus within live oysters by 0,
250, 500, 750, 1000 and 1250 Gy X-ray at 50,
200 and 350 kV with 1 mm aluminum filter.
Different lower-case letters indicated differences
(p < 0.05) among the levels of within each
kind of different acceleration voltage
(p < 0.05). Different capital letters indicated
differences (p < 0.05) among same irradiation
dosage.

Fig. 6. Changes in the mesophilic bacterial counts (log CFU g−1) of treated whole-shell oysters with 0, 250, 500, 750, 1000 and 1250 Gy X-ray at 50, 200 and 350 kV
with 1 mm aluminum filter. Different lower-case letters indicated differences (p < 0.05) among the levels of within each kind of different acceleration voltage
(p < 0.05). Different capital letters indicated differences (p < 0.05) among same irradiation dosage.

enhancement in efficiency to that achieved by gamma ray at 1000 Gy.
The log MPN g−1 was significantly (p < 0.05) reduced in whole
shell oysters from 8.11 ± 0.19 to 7.37 ± 0.43, 6.48 ± 0.22,
5.46 ± 0.12, 5.13 ± 0.35, and 3.93 ± 0.70 after treatment with
250, 500, 750, 1000 and 1250 Gy X-ray at 200 kV, respectively. When
50 kV accelerated X-ray was applied less than 2 log MPN g−1 was
achieved by 1250 Gy.
Relative to gamma ray, X-ray has been reported to require five times
the dose to achieve the same 6 log reduction of inoculated V.
parahaemolyticus in whole shell oysters (Andrews et al., 2003; Jakabi
et al., 2003; Mahmoud & Burrage, 2009; Thupila et al., 2011). In this
report, our research demonstrated that by adjusting the acceleration
voltage, X-ray would produce similar level V. parahaemolyticus in-
activation in live oysters when compared to gamma ray.
X-rays are emitted in the process of collision of electrons towards a
piece of metal called the "target" after acceleration in a vacuum of an X-
ray tube (Wagner, 2008; Wagner, Dillon, Blythe, & Ford, 2009). The X-
ray spectrum is a continuous spectrum with characteristic sharp peaks

6
Y. Wu and S.K.C. Chang
at specific energy level associated with the atoms in the target. The
distribution of X-ray spectrum is determined by to the limited energy of
the incoming electrons while the maximum kinetic energy of electron
acquired from acceleration is the acceleration voltage of the machine
(Reed, 2005). In this study, three acceleration voltages of 50, 200 and
350 kV were used. Photoelectric effect of ionizing radiation is the
dominant energy transfer mechanism for photons with energies below
50 keV which causes the ejection of that electron from the atom when
an ionizing irradiation photon interacts with and transfers its energy to
an atomic electron (Urbain, 2012). Compton scattering is the principal
absorption mechanism for gamma rays and X-ray in the intermediate
energy range 100 keV to 10 MeV, in which ionizing irradiation photons
lose enough energy to an atomic electron to cause its ejection, with the
remainder of the original photon's energy emitted as a new, lower en-
ergy photon. In the 50 kV experimental group, the radiation photon
deposits energy mainly through the photoelectric effect. In addition, in
the 200 kV and 350 kV experimental groups, Compton scattering is the
principal absorption mechanism. In the future, we plan to continue to
study and adjust the filter types and to identify the range of acceleration
voltage to optimize the sterilization effect of X-ray to inactivate specific
type of pathogens.
3.4. Inactivation of the mesophilic bacterial counts in life oysters with 1 mm
aluminum filter
Changes in the microflora in whole shell oysters after treatments at
50, 200 and 350 kV X-ray are shown in Fig. 6. Treatment with 250, 500,
750, 1000, and 1250 Gy at 50 kV acceleration voltage significantly
(p < 0.05) reduced the total microbial counts in whole shell oysters
from 4.79 ± 0.06 to 4.43 ± 0.13, 4.19 ± 0.23, 4.03 ± 0.00,
3.96 ± 0.08 and 3.72 ± 0.16, respectively. The log CFU g−1 was
significantly (p < 0.05) reduced from 4.79 ± 0.06 to 3.82 ± 0.31,
3.47 ± 0.14, 3.08 ± 0.27and 3.09 ± 0.17 after treatment with 500,
750, 1000 and 1250 Gy at 200 kV acceleration voltage. When 350 kV
acceleration voltage was applied on live oysters, microflora in whole
shell oysters was reduced to non-detectable level (< 2 log CFU g−1)
with 1250 Gy.
3.5. Determination of D10-Value
The D10-values (Gy) for V. vulnificus pure culture, V. para-
haemolyticus pure culture, V. parahaemolyticus within live oysters and
mesophilic bacterial counts (log CFU g−1) of treated whole-shell oysters
by 50, 200 and 350 kV X-ray with or without 1 mm aluminum filter are
shown in Table 1. In the same filter installation status (with or without),
the experimental group with higher voltages had lower D10-values
(Gy). The D10-value of V. vulnificus pure culture was significantly
(p < 0.05) reduced from about 660 to 235 and 125 Gy when the ac-
celeration voltage was increased from 50 kV to 200 and 350 kV without
filter installed (Table 1). Similarly, the D10-value of V. parahaemolyticus
pure culture was significantly (p < 0.05) reduced from about 240 to
Table 1
D10-values (Gy) for V. vulnificus pure culture, V. parahaemolyticus pure culture, V. parahaemolyticus within live oysters and mesophilic bacterial counts (log CFU g−1)
of treated whole-shell oysters by 50, 200 and 350 kV X-ray with or without 1 mm aluminum filter.
X-ray source V. vulnificus pure culture V. parahaemolyticus pure culture
D10-values R2 D10-values
50 kV without filter 661.2 ± 142.3 aA 0.62 241.5 ± 23.0 bA
200 kV without filter 235.5 ± 40.9 aB 0.97 118.7 ± 5.5 bB
350 kV without filter 125.5 ± 10.0 aC 0.95 90.9 ± 1.7 bC
50 kV with filter 67.1 ± 4.3 cC 0.96 42.0 ± 2.2 cD
200 kV with filter 46.3 ± 5.6 cC 0.93 36.8 ± 0.5 cD
350 kV with filter 36.0 ± 1.1 cC 0.99 38.9 ± 0.4 cD
Mean values with different lower case letters in same row or capital letters in same column are significantly different (p < 0.05).
7
Food Control 115 (2020) 107277
119 and 91 Gy when the acceleration voltage was increased from 50 kV
to 200 and 350 kV without filter installed. With the installation of 1 mm
aluminum filter, both V. vulnificus and V. parahaemolyticus pure culture
had similar magnitudes (p > 0.05) of D10 values at 350 kV, all well
below 50 Gy, and had much smaller D10-values (Gy) compared with
previous reports by gamma ray irradiation which ranged 350 and 75 Gy
for V. vulnificus and V. parahaemolyticus, respectively (Monk, Beuchat, &
Doyle, 1995). Our experimental results showed that in the absence of a
filter, the D10-values (Gy) of V. parahaemolyticus was significantly
(p < 0.05) smaller than vulnificus. Interestingly, V. parahaemolyticus
had been reported as more resistant to ionizing irradiation than V.
vulnificus (Hu, Mallikarjunan, Koo, Andrews, & Jahncke, 2005;
Mahmoud, 2009b; Mahmoud & Burrage, 2009).
Literatures of X-ray, electron beam and gamma ray research on the
sterilization effect of microorganisms, had not reported the general
principles that can explain the differences in bactericidal effect by these
technologies (Fan et al., 2017; Jeong, Shin, Chu, & Park, 2015; Lopez-
Gonzalez, Murano, Brennan, & Murano, 1999; Waje et al., 2009). The
energy level between gamma ray and X-ray are different but both of
them were often classified as ionizing radiation in a general sense.
Compared with the gamma ray of cobalt-60 emitted at 1.17 and
1.33 MeV (Bomford, Walter, Miller, Kunkler, & Sherriff, 1993), X-ray
technology can be adjusted manually to give various distribution of the
energy levels by changing acceleration voltage from low energy as
100 kV to 10 MeV. The X-ray features can be more versatile and ma-
nipulable than gamma ray because various filters can be used to pro-
duce different effects whereas, in gamma ray technology, filters are not
used.
For the inactivation of V. parahaemolyticus within oysters, 1 mm
aluminum filter was installed in this study. The D10-value of V. para-
haemolyticus within oysters was significantly (p < 0.05) reduced from
about 620 to 320 and 210 Gy when the acceleration voltage was in-
creased from 50 kV to 200 and 350 kV, respectively (Table 1). For the
inactivation of mesophilic bacterial counts (log CFU g−1) within oy-
sters, when 1 mm aluminum filter was used, the D10-value of meso-
philic bacterial counts (log CFU g−1) within oysters was significantly
(p < 0.05) reduced from about 1260 to 680 and 640 Gy when the
acceleration voltage was increased from 50 kV to 200 and 350 kV, re-
spectively. V. parahaemolyticus within live oysters seems to be more
sensitive to X-ray sterilization than mesophilic bacterial counts (log
CFU g−1). We have not tested V. vulnificus in live oysters since our
findings (Table 1) showed the resistance with filter was similar for the
two organisms in pure culture form.
4. Conclusion
Our study showed that changing the acceleration voltage of X-ray,
or placing a filter or not, greatly affected the inactivation efficiency in
the pure culture of two pathogenic vibrios species. We have shown that
X-ray's killing vibrio effect on the whole shell oysters can be increased
by increasing the acceleration voltage to 350 kV. Because our machine
V. parahaemolyticus within oysters mesophilic bacterial within oysters
R2 D10-values R2 D10-values R2
0.81
0.93
0.97
0.96 621.9 ± 94.0 bA 0.71 1263.2 ± 159.1 aA 0.95
0.98 316.0 ± 59.5 bB 0.99 682.5 ± 27.5 aB 0.95
0.97 212.8 ± 4.5 bB 0.93 638.9 ± 24.1 aB 0.91
Y. Wu and S.K.C. Chang
can only increase its voltage to 350 kV, it is likely that better ster-
ilization can be achieved by further increasing the voltage using a
higher voltage generating X-ray. Since there are many suitable filter
candidates that can be researched, we believe that the synergistic ef-
fects can be attained by optimize accelerating voltage at a higher ca-
pacity and using suitable filters simultaneously.
CRediT authorship contribution statement
Yuwei Wu: Conceptualization, Methodology, Investigation, Data
curation, Writing - original draft, Project administration. Sam K.C.
Chang: Conceptualization, Methodology, Resources, Data curation,
Writing - review & editing, Visualization, Supervision, Funding acqui-
sition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
Funding was provided by the USDA-ARS SCA agreement No.
5860667081 and the state CRIS project No MIS 081710 for MS Center
for Food Safety and Postharvest Technology of the Mississippi
Agriculture and Forestry Experiment Station of the Mississippi State
University. Technical assistance in Vibrio analysis from Dr. Jessica
Jones of the US Food and Drug Administration in Dolphin Island, AL is
appreciated.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodcont.2020.107277.
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