Postharvest Biology and Technology 95 (2014) 28–35

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Postharvest Biology and Technology

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

Effects of electron-beam irradiation on blueberries inoculated with

Escherichia coli and their nutritional quality and shelf life
Qiulian Kong a , Aizhong Wu a , Wenyuan Qi a,∗∗ , Rongdi Qi a , John Mark Carter b ,
Reuven Rasooly b , Xiaohua He b,∗
a
Shanghai Shuneng Irradiation Technology Co., Ltd, Shanghai Academy of Agricultural Sciences, Shanghai 201400, China
b
Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, CA 94710, USA

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

Article history: Fresh blueberries have become a popular new functional food because of their remarkably high levels of
Received 8 January 2014 antioxidant phytonutrients and health benefits. However, the potential prevalence of human pathogens
Accepted 5 April 2014 on blueberries has become an increased concern because they are consumed fresh. Procedures effective in
decontamination and extending shelf life without affecting fruit quality are needed. Electron-beam irra-
Keywords: diation was applied to fresh blueberries at the doses ranging from 0.5 to 3.0 kGy and its effectiveness for
Antioxidant activity
inactivating Escherichia coli (E. coli) K-12 and extending shelf life were investigated. The decimal reduction
Blueberry
dose, D10 values, of E. coli in cultural medium and blueberries were 0.43 ± 0.01 kGy and 0.37 ± 0.015 kGy,
E. coli K12
Electron beam irradiation respectively. Irradiation reduced bacteria inoculated on blueberries from 7.7 × 108 CFU/g to 6 CFU/g at
l-Ascorbic acid 3.13 kGy and decreased the decaying of blueberries stored at 4 ◦ C up to 72% and at room temperature up
Total monomeric anthocyanins to 70% at this dose. No significant effect on the total monomeric anthocyanins, antioxidant activity, and l-
ascorbic acid content of blueberries was observed from irradiation at doses ≤3 kGy. However, significant
decreases in the antioxidant activity and l-ascorbic acid content were found in both control and irradi-
ated blueberries after storage at 4 ◦ C for 7 and 15 d. Information obtained in this study indicates that low
dose electron-beam irradiation is effective in reducing E. coli and extending shelf life while maintaining
the antioxidant properties of blueberries.
Published by Elsevier B.V.

1. Introduction
Blueberries, having the highest antioxidant capacity of all fresh
fruits (Wu et al., 2004) have become the second most popular
berries in the United States, following closely behind strawberries,
according to the U.S. Department of Agriculture. The production
of blueberries has increased dramatically in the last twenty years,
which led to an increased concern in food safety because blueber-
ries, like many other ready-to-eat fresh produce, are consumed raw.
Recently, outbreaks of foodborne illness associated with the con-
sumption of fresh produce have increased (Warriner et al., 2009)
(www.cdc.gov/ecoli). Fruits such as cantaloupe, papaya, and straw-
berries have been reported in several notable outbreaks (Olaimat
and Holley, 2012). An outbreak of Escherichia coli (E. coli) O26 asso-
ciated with consumption of raw blueberries was also reported. Six
∗ Corresponding author. Tel.: +1 510 559 5823; fax: +1 510 559 5768.
∗∗ Corresponding author. Tel.: +86 21 37195191x8030; fax: +86 21 37195190.
E-mail addresses: sunny0123@vip.163.com (W. Qi), xiaohua.he@ars.usda.gov
(X. He).
people were sickened as part of the outbreak, and one was hos-
pitalized (Goetz, 2011). Besides the threat posed to public health,
these outbreaks are also the source of tremendous economic losses
in relation to medical costs, loss of productivity, and trade. Despite
ongoing efforts, elimination or reduction of foodborne pathogens
from fresh produce is a vexing challenge. While thermal pasteur-
ization of liquid foods is well established, it does not suit solid foods.
Chemical sanitizing procedures have inherent problems concern-
ing residues and environmental pollution (Nieuwenhuijsen et al.,
2000). In light of these, food irradiation is attracting renewed atten-
tion as a potential non-thermal decontamination strategy to ensure
the safety of fresh fruits and vegetables. The Food and Drug Admin-
istration (FDA) in the USA has allowed the use of irradiation as a
means for improving food safety and extending the shelf life of
fruits, vegetables, shell eggs, seeds for sprouting, spices, raw poul-
try, shellfish, and red meats since the first approval of the use of
irradiation to kill pests in wheat and flour in 1963 (FDA, 2011).
Currently, food irradiation is approved for use in over 55 countries
worldwide. Chinese legislation has remained supportive since the
announcement of National Standards for Food Irradiation in 1997.
Foods cleared to date include potatoes, onions, garlic, sausages, rice,

http://dx.doi.org/10.1016/j.postharvbio.2014.04.004
0925-5214/Published by Elsevier B.V.
 Q. Kong et al. / Postharvest Biology and Technology 95 (2014) 28–35
apples, peanuts, mushrooms, chickens, pollens, preserved fruits,
almonds, tomatoes, pork, litchi, oranges, wines, and lean meats. The
volume of commercially irradiated foods reached 150,000 metric
tons in 2006 (Gao et al., 2007).
The type of radiation of primary interest in food preservation is
ultraviolet-C (UV-C), gamma rays, X-rays and electron-beam (E-
beam). UV-C kills bacteria by disrupting cross-linking between
neighboring pyrimidine bases in DNA and has been used as a
surface disinfectant for fruits and vegetables (Kang et al., 2013).
However, the poor penetrating capacity of UV-C restricts its use in
food applications. Gamma rays are produced from a nuclear source
(cobalt-60) and have powerful penetration capability, enabling
treatment of commercial packages. However, because their sources
in the processing of food are cobalt-60, a radioisotope that could
cause cancers, the use of gamma-rays has become a safety concern.
E-beam and X-rays are electrically generated radiation technolo-
gies. The choice of E-beams vs X-rays depends on the density of
the food and the anticipated mass throughput. For products having
greater areal densities (density multiplied by thickness > 20 g/cm2 ),
it is better to use more penetrating X-rays. In terms of through-
put, E-beams are superior to X-rays because E-beams provide
higher dose rate although both have the switch-on and switch-
off capability (Castell-Perez and Moreira, 2011). E-beams provide
multiple advantages (Sugranes, 2005) over other popular con-
trol methods, such as chemical washes, fumigants, thermal and
high pressure. These advantages include: (1) No load pretreatment
needed; (2) Shorter sterilization exposure time; (3) No chemi-
cal residues; (4) No degassing or aeration process needed after
sterilization; (5) Environmentally friendly; (6) Faster processing;
(7) Materials can be re-sterilized; (8) Reduction of undesirable
microorganisms without using liquids which reduces the amount
of wax/bloom on blueberry fruits. E-beam technology has been
broadly used to reduce the growth of pathogenic and spoilage
microorganisms (Cabeza et al., 2010; Espinosa et al., 2012; Grasso
et al., 2011; Neal et al., 2008). However, as with other tech-
nologies, there are limitations and areas of concern associated
with E-beam technology. One of the biggest challenges in using
E-beam on food as a decontamination technology is the oppo-
sition based on psychological perception due to lack of public
knowledge, on the wholesomeness of irradiated food (Resurreccion
et al., 1995). Results from previous studies demonstrated that
the effective irradiation dose varies significantly from product
to product and proper irradiation process parameters should
be established for each specific food product. The primary goal
of E-beam irradiation is to use the minimum dose and reach
the maximum effect of sterilization. Over-dosage is costly, but
under-dosage can result in huge safety concerns (Kim et al.,
2011).
E-beam irradiation has been applied to multiple types of fresh
produce and has been demonstrated to be effective in reduc-
ing pathogenic microorganisms (Grasso et al., 2011; Mintier and
Foley, 2006; Sanglay et al., 2011; Schmidt et al., 2006). However,
studies focusing on the bactericidal efficacy of E-beam irradi-
ation on blueberries have not been reported. In this study, a
bacterial surrogate was used as a tool to validate the effec-
tiveness of E-beam technology on the sterilization process for
eliminating bacteria on blueberries. The specific objectives of this
research were to: (1) Determine the effect of E-beam irradia-
tion on inactivating E. coli K12 in culture medium; (2) Validate
the effectiveness of E-beam irradiation for disinfecting fresh
blueberries; (3) Evaluate the effect of E-beam irradiation on spon-
taneous fungal growth and shelf life of blueberries; (4) Examine
the effect of E-beam irradiation on the total monomeric antho-
cyanins (TMA), l-ascorbic acid (AA) content, and antioxidant
activity.
29
2. Materials and methods
2.1. Blueberries
Blueberries (Vaccinium corymbosum, cvs. Collins, Bluecrop) were
hand-harvested on July 16, 2012 from a blueberry plantation
in Huangdao district, Qingdao, China by Wallen Agriculture Ltd.
(Qingdao, China). Berries were packaged by hand into polystyrene
clamshells (100 g each package) that were placed 10 each into com-
mercial master trays. The berries were then transported to the
Shanghai Shuneng Irradiation facility (Shanghai, China) and stored
at 4 ◦ C with 80–90% relative humidity. Blueberries visibly free of
mechanical damage were selected for treatment within 72 h after
harvesting.
2.2. Inoculum preparation
Non-pathogenic strain, E. coli K-12 (CGMCC1.750), was acquired
from Beina Biotechnology (Beijing, China). Before irradiation treat-
ment, the bacterial culture was inoculated in fresh Tryptic Soy Broth
(TSB) and incubated at 35 ◦ C for 24 h. The growth of E. coli was con-
firmed by streaking TSB cultures onto plates of Eosin-methylene
blue (EMB) agar and incubated at 35 ◦ C for 24 h. Bacterial cells from
overnight cultures were harvested by centrifugation at 1500 × g
and re-suspended in 0.1% peptone water. The prepared inoculum
containing 108 –109 colony forming unit (CFU)/mL was used within
2 h after preparation and kept at room temperature during the
experiment.
2.3. Inoculation of blueberries with bacteria
To disinfect existing microorganisms, blueberries were cov-
ered in 1% chlorine dioxide solution (Ouyan, Beijing) for 10 min
at room temperature, and then triple rinsed with sterilized water.
Ten grams of the clean berries were grouped in individual stom-
acher bags (300 × 190 mm, BagLight PolySilk, Interscience, France)
and 1 mL of bacterial cultures containing 108 –109 CFU was added
to each bag. The bag was then closed and shaken for 1 min to assist
in uniform distribution. The inoculated blueberries were left in the
bags to dry at room temperature before the bags were sealed for
treatment.
2.4. Irradiation
All E-beam irradiation treatments were conducted at Shanghai
Shuneng Irradiation Technology Co., Ltd. (Shanghai, China). A sys-
tem with a 10-MeV and 12-kW linear accelerator (ESS-010-33,
IHI, Japan) was used in this study using a single beam (top only).
Stomacher bags containing liquid bacterial cultures (10 mL) in TSB
medium or blueberries (average diameter 1.12 cm) arranged in a
single layer without any stacking between each other were immo-
bilized by cello tape to avoid displacement. Samples were treated
with target absorbed doses ranging from 0.5 to 3 kGy within 2 h
post-preparation. The accelerator was set to deliver a constant dose
of 0.5 kGy by running the conveyor belt at 0.5 m s−1 . The room tem-
perature was around 28–30 ◦ C. To deliver doses larger than 0.5 kGy,
multiple passes were performed. Irradiation at each dose level was
conducted in triplicate for each experiment and each experiment
was repeated at least three times.
2.5. Dose uniformity ratio (DUR)
A single layer of blueberries was packed in stomacher bags
described above and four dosimeters (Opti-chromic detectors,
FWT-70-83, Far West Technology, Goleta, CA) were placed inside
30 Q. Kong et al. / Postharvest Biology and Technology 95 (2014) 28–35
the bags in the areas where maximum (top of the blueber-
ries) and minimum (bottom of the blueberries) absorbed dose
were measured during extensive dose mapping. After irradiation,
the dosimeters were read at 600 nm with a FWT-200 reader (Far
West Technology) to determine the dose variation within each
bag. Blueberries were treated with target absorbed doses of 0, 0.5,
1.0, 2.0 and 3 kGy. Each radiation dose was replicated 3 times in
each experiment and each experiment was repeated three times.
2.6. Microbiological analysis
For bacterial samples in culture medium, counts were deter-
mined immediately after E-beam radiation by spread-plating in
triplicate onto tryptone soya agar (TSA) and EMB agar plates. For
inoculated blueberry samples, 90 mL of sterile 0.1% peptone water
was added to each of the stomacher bag containing blueberry sam-
ples and blended for 1 min. Serial dilutions were made and spread
onto EMB agar plates. Plates were incubated for 24 h at 35 ◦ C. Viable
colonies of bacteria (CFU/mL) were counted.
2.7. Shelf life assessment
Blueberry samples for decay studies were prepared the same as
the samples for bacterial reduction studies. Decay percentage was
determined visually based on the appearance of fungal mycelium.
Triplicate samples, each consisting of 100 fruit, were used for mon-
itoring decay under ambient and 4 ◦ C conditions in each treatment
including control. Decay percentage was calculated as:
 number of decayed fruit 
Decay percentage(%) = × 100
total number of fruit
2.8. Preparation of extracts for analytical procedures
One hundred intact blueberries without visible mechanical
damage (100–125 g) were placed in a stomacher bag and irradi-
ated with different doses (0, 0.5, 1.0, 1.5, 2.0 and 3.0 kGy). After
treatment, blueberries were stored at 4 ◦ C up to 15 d before being
used for analysis of antioxidant properties (Rotten berries were
excluded). Each experiment was repeated three times.
2.8.1. Extracts for TMA analyses
The extracts for TMA content were prepared as previously
described (Poiana et al., 2012) with minor modifications. Briefly,
blueberry pericarp (0.1–0.5 g) was ground in 100 mL of 95% ethanol
acidified with 0.1% of hydrochloric acid (HCl) for 2 min in a blender.
The mixture was stored at room temperature in the dark for 24 h
and then filtered.
2.8.2. Extracts for antioxidant activity analyses
The extracts for antioxidant activity determination were pre-
pared by grinding intact blueberries (5 g) in 20 mL of 95% ethanol
acidified with 0.1% HCl. After incubation at room temperature for
60 min, the solution was filtered. The residue was extracted one
more time. The two extracts were combined and diluted to a vol-
ume of 50 mL with HCl-acidified ethanol. The extracts were further
diluted 5-fold to analyze the antioxidant activity.
2.8.3. Extracts for l-ascorbic acid analyses
The extracts for analyses of l-ascorbic acid content were pre-
pared by grinding peeled-blueberries (10–15 g) in 2.5 mL of 2%
oxalic acid, followed by adding 1% oxalic acid up to 50 mL. The
extracts were filtered and 10 mL of the filtrate was used for deter-
mination of l-ascorbic acid content.
2.9. Analysis of TMA content
The TMA content was determined using a pH differential
method (Lee et al., 2005), which is a rapid spectrophotometric
method based on the anthocyanin structural transformation that
occurs with a change in pH (colored at pH 1 and colorless at pH
4.5). Aliquots of blueberry extracts were adjusted to pH 1.0 and pH
4.5, respectively, and diluted appropriately with 0.025 M potassium
chloride (pH 1.0) or 0.4 M sodium acetate (pH 4.5). The absorbance
of each dilution was measured at 520 nm and 700 nm using distilled
water as a blank. The absorbance value (A) calculated according
to the equation: A = (A520 − A700 )pH=1.0 − (A520 − A700 )pH=4.5 is pro-
portional to the TMA content. The TMA content was calculated by
using a molar extinction coefficient of 26,900 and the molecular
weight of 449.2 g mol−1 (the most prevalent anthocyanin, cyanidin-
3-glucoside) and expressed as g kg−1 pericarp. All experiments
were performed in triplicate.
2.10. Antioxidant activity
The antioxidant activity of fresh blueberries was measured using
the ferric reducing antioxidant power (FRAP) assay (Benzie and
Strain, 1996). This assay uses antioxidants as reductants to reduce
the ferric 2,4,6-tris (2-pyridyl)-1,3,5-triazine (TPTZ) complex to a
blue-colored ferrous form at 37 ◦ C in pH 3.6 sodium acetate buffer.
The FRAP values are obtained by comparing the absorbance change
at 593 nm in test reaction mixtures with those containing ferrous
ions in known concentration. An aliquot of 10 ␮L of extract was
added to 180 ␮L of diluted FRAP reagent (T-AOC Assay Kit, Beyotime
Institute of Biotechnology, Shanghai, China) and the samples were
incubated at 37 ◦ C for 30 min before measuring the absorbance at
593 nm. A standard curve was prepared using different concen-
trations (0.05–0.4 mmol Fe2+ L−1 ) of FeSO4 ·7H2 O. The antioxidant
activity based on the ability to reduce ferric ions of the extract was
expressed as molar concentration of Fe2+ per mass of fresh blue-
berries, mmol L−1 kg−1 . All solutions were prepared on the day of
the experiment and all were performed in triplicate.
2.11. Determination of l-ascorbic acid
l-Ascorbic acid content was determined via a redox titra-
tion using 2,6-dichlorophenolindophenol (DCPIP) (Sigma–Aldrich
Corp., St. Louis, MO) as an indicator (Tanner and Barnett, 1985).
EDTA was added as chelating agent to remove Fe and Cu interfer-
ences. DCPIP is a redox dye which develops a pink color in acid
conditions. In a titration, DCPIP was added to a10 mL blueberry
extract. DCPIP will be reduced to DCPIPH2 , which is colorless if
ascorbic acid, a reducing agent, is present in the solution. But when
all the ascorbic acid has been used up, there will not be any elec-
trons available to reduce the DCPIP to DCPIPH2 and the solution
will remain pink. The amount of dye used to turn the extract solu-
tion pink is used to determine the content of ascorbic acid. Results
were expressed in mass of l-ascorbic acid per mass of peeled fresh
blueberries, g kg−1 .
2.12. Data analysis
Colony counts (CFU/mL or CFU/g) were converted to log values
for data analysis. Estimated log reduction was determined by sub-
tracting the log count for the corresponding treatment from the
log count on control samples. Radiation decimal reduction, D10 ,
values for E. coli K-12 were determined from the reciprocal of the
slope of the regression line plotted from the log counts of surviving
bacteria against increasing doses of radiation. It was defined as the
dose in kGy required to reduce the bacterial population by a fac-
tor of ten. All results are presented as means ± standard deviation
 Q. Kong et al. / Postharvest Biology and Technology 95 (2014) 28–35
10
8 7
Log10CFU/mL
6
4 4
2 2
0 0
0 1 2 3
Dose (kGy)
Fig. 1. Inactivation of E. coli K12 cells in TSB medium as a function of electron beam
irradiation dose. Linear regression (r2 = 0.96) extrapolation produced a D10 value
of 0.43 kGy. Results represent the mean ± SD of three trials and triplicates were
performed for each trial. The 95% confidence limits are indicated by two dashed
lines.
(SD) of triplicate measurements and analyzed by one way ANOVA
followed with Tukey’s Multiple Comparison Test using GraphPad
Prism 5 (GraphPad Software Inc., San Diego, CA). The differences
were considered significant at P < 0.05.
3. Results and discussion
3.1. Elimination of E. coli on blueberries using E-beam irradiation
process
The aim of this study was to validate the effectiveness of E-beam
irradiation for decontamination of blueberries.
3.1.1. Selection of bacterial strain suitable for E-beam treatment
A non-pathogenic microorganism, E. coli strain K-12
(CGMCC1.750), was used as a surrogate. Use of a surrogate
microorganism in place of target pathogens for verification of a
given antimicrobial process or treatment for food is recommended
by U.S. regulatory agencies (FDA, 2000). The surrogate selected
must be a non-pathogenic strain and validated to be more resistant
to a particular treatment than target pathogens. A study designed
to identify a potential surrogate tested five non-pathogens: E.
coli K12 MG1655, Listeria innocua Seeliger 1983 (NRRL B-33003
and NRRL B-33014), Enterobacter aerogenes, and Salmonella LT2
for their sensitivity to E-beam irradiation, and found that the
non-pathogenic E. coli K-12 was most resistant to radiation in
comparison to the other strains (Rodriguez et al., 2006). Therefore,
E. coli K-12 was selected as an indicator pathogen in this study.
3.1.2. Effect of E-beam irradiation on bacteria in cultural medium
The effect of radiation on the E. coli K-12 in TSB cultural medium
was tested first. The initial bacterial population was 8.5 log CFU/mL.
Bacterial counts decreased significantly after E-beam radiation
and the counts were inversely proportional to the dose of energy
applied. A regression line (r2 = 0.96) was used to extrapolate a D10
value of 0.43 ± 0.01 kGy (Fig. 1), which is very similar to the D10
values reported for four food-borne bacteria: Enterococcus faecalis,
Listeria innocua, Salmonella enteritidis, and Pseudomonas fluorescens
grown in TSB cultural medium (Aguirre et al., 2011).
3.1.3. Effect of E-beam irradiation on inoculated blueberries
Irradiation of fresh produce can be very challenging because
they are generally very susceptible to over dosage that can result
in tissue damage and affect their quality. Unlike Gamma and X-
ray radiation, E-beam has high dose rate, but low penetration. It
requires higher doses to achieve bacterial reductions in blueber-
ries due to their high density (the mass per unit volume) compared
to thin leafy vegetables. When we applied E-beam to blueberries
31
9
8
Log10 CFU/g
6
5
3
1
0 0.9 1.47 1.73 2.3 3.13
Dose (kGy)
Fig. 2. Survival of E. coli K-12 on blueberries exposed to E-beam irradiation at differ-
ent doses using a 10 MeV linear accelerator at room temperature. Results represent
the mean plus one SD of three trials and triplicates were performed for each trial.
inoculated with E. coli K-12 at a level of 8.9 log CFU/g, the popula-
tion of surviving E. coli K-12 on the blueberries gradually decreased
with the increased dose. At 2.3 kGy, irradiation decreased bacte-
rial counts to 28 CFU/g, and at 3.13 kGy bacterial counts were only
6 CFU/g of blueberries. The average D10 value for E. coli K-12 on
blueberries was 0.37 ± 0.015 kGy (Fig. 2).
3.1.4. Selection of growth medium for bacteria recovery
It is known that E-beam treatment may induce sub-lethal injury
and selective medium may inhibit the recovery of sub-lethally
injured cells (these cells may recover during the storage of blue-
berries) and therefore increase the risk of over-estimating the
effectiveness of the E-beam treatment (Tesfai et al., 2011). We
compared the D10 value of E. coli K-12 in TSB using non-selective
medium TSA and selective medium EMB. No significant difference
was found between these two media (data not shown), suggesting
that the EMB is an appropriate medium to use in this study. It not
only has the capability to differentiate and enumerate target bac-
terium but also recovers sub-lethally injured cells equally well as
the non-selective medium.
3.2. Dose uniformity ratio (DUR)
Because of the round shape of blueberries, the DUR was mea-
sured using the method described previously (Wall and Khan,
2008) to ensure the uniformity ratio was within an acceptable
level. For target doses of 0.5-, 1.0-, 2.0-, and 3.0-kGy, the average
minimum and maximum absorbed doses obtained inside the bags
were 0.44–0.53, 1.07–1.21, 2.06–2.43 and 3.01–3.52 kGy with cor-
responding DUR of 1.20, 1.13, 1.18, and 1.17 respectively. It must be
pointed out that these results were obtained from a single layer of
blueberries. It is much more complicated when irradiating a box of
blueberries because of the large differences in densities inside of the
boxes. The random distribution of the berries around the air pockets
can result in a large dose uniformity ratio inside the box. Studies on
the dose distribution within a tray of blueberries exposed to E-beam
irradiation at levels of 1.0–3.2 kGy demonstrated that the dose was
not uniformly distributed. Dose levels at the bottom of the trays
were 18% higher than at the top (Moreno et al., 2008). Therefore,
when irradiating blueberries in bulk or commercial packages, a pre-
cise design of the irradiation process is needed to ensure uniform
dose distribution. To accurately determine the correlation between
radiation dose and inactivation of bacteria, it is critical that the
radiation dose be as uniform as possible throughout the blueber-
ries being treated. Therefore, only single layer of blueberries was
treated in this study.
32 Q. Kong et al. / Postharvest Biology and Technology 95 (2014) 28–35

Table 1
Spontaneous rot decay (%)a of blueberries stored at room temperature (28–30 ◦ C) and 4 ◦ C after E-beam treatment.

Treatment Fruit Storage time (days) at room temperature Storage time (days) at 4 ◦ C
no.
1 2 4 6 8 14 20 26

Control 100 0 40 (a) 70 (a) 70 (a) 100 (a) 39 (a) 49 (a) 88 (a)
0.5 kGy 100 0 10 (b) 50 (b) 60 (b) 100 (a) 39 (a) 38 (b) 86 (a)
1.0 kGy 100 0 10 (b) 40 (c) 60 (b) 70 (b) 24 (b) 38 (b) 86 (a)
2.0 kGy 100 0 0 (c) 10 (d) 50 (c) 60 (c) 8 (c) 37 (b) 73 (b)
3.0 kGy 100 0 0 (c) 0 (e) 30 (d) 60 (c) 3 (d) 11 (c) 16 (c)

Values are means, n = 3; numbers with different letters within each column were statistically different (P < 0.05), with same letter were statistically not different.
a
Decay percentage was determined visually based on the appearance of fungal mycelium.

3.3. Shelf life of irradiated blueberries

Irradiation has been shown to extend the shelf life of fruits by

delaying ripening, sprouting or by controlling microorganisms that
can cause spoilage (Dharkar et al., 2006; Hussain et al., 2010; Rezaee
et al., 2011; Yu et al., 1995). In this study, blueberries were stored
at 4 ◦ C before use after being obtained from the producer. After
irradiation, replicate sets of berries were stored at either 28–30 ◦ C
or 4 ◦ C. A significant effect of E-beam irradiation on suppressing
proliferation of fungi on blueberries was observed and consistent
among different experiments (the same blueberry stock was used
for control and different treatments in each experiment). Table 1
shows the decay percentage was 40% in control samples, 10% in
samples treated with 0.5 kGy dose, and 0% in samples treated with
2 kGy or higher dose after 2 d of storage at room temperature
(28–30 ◦ C). After 4 d of storage at room temperature, 70% of con-
trol samples decayed, but still no decay was found in samples
treated with a 3 kGy dose. Among samples stored at 4 ◦ C, no decays
were observed at 7 days (data not shown), 39% of control samples
decayed after 14 d, but only 8% decay were found in samples treated
with a 2 kGy dose and 3% decay in samples treated with 3 kGy
irradiation, suggesting that the shelf life of blueberries positively
correlates with the irradiation dose. Fig. 3 shows the appearance
of the fresh blueberries and of the blueberries after being stored
4 d after E-beam treatment. The appearance of blueberries treated
with 3 kGy dose was similar to those treated with 2 kGy dose (data
not shown). These results indicate that E-beam irradiation is useful
for extending shelf-life of blueberries. It is worth mentioning that
these experiments were performed in July in Shanghai. The rela-
tive humidity was very high (80–90%) and the room temperature
fluctuated from 28 to 30 ◦ C. There was no air conditioner in the
laboratory. Therefore, the decay percentage obtained in this study
could be higher than that obtained in other studies performed in
different seasons.

3.4. Effects of irradiation on chemical properties of blueberries

stored at 4 ◦ C

Although irradiation has not been found to induce products that

are harmful to human health, it could affect the quality of fruits
or degrade nutrients sensitive to irradiation treatment (Miller and
McDonald, 1995). It has been reported that the overall quality, tex-
ture and aroma of blueberries were found unacceptable by sensory
panelists when fruit were exposed to 3.2 kGy. However, this dose
did not affect the density, pH, moisture content, acidity and juici-
ness of the fruits (Moreno et al., 2007). Therefore, it is necessary
to study the effect of radiation on the quality of raw blueberries
stored at a low temperature. Because antioxidant compounds and
their health benefits are important factors influencing consumers’
interest in blueberries, three chemical parameters including TMA Fig. 3. (A) Appearance of blueberries before E-beam treatment. (B) Appearance of
content, antioxidant activity, and l-ascorbic acid content were blueberries stored at room temperature for 4 d after E-beam treatment. Decay was
investigated. determined visually based on the appearance of fungal mycelium.
 Q. Kong et al. / Postharvest Biology and Technology 95 (2014) 28–35 33

3..50

3..00

TMA content (g kg-1)

2..50

2..00 0 kGyy
0.5 kG
Gy
1..50 1 kGyy
2 kGyy
1.00
3 kGyy
0..50

0..00
0 7 15
Days aftter irradiattion

Fig. 4. The TMA content of blueberries stored at 4 ◦ C for 0, 7, and 15 d, respectively, following different irradiation doses. Results represent the mean plus one SD of three
trials and triplicates were performed for each trial.

90
0.00
Antioxidant activity (mmoL L-1 kg-1)

75
5.00

60
0.00
0 kGy
45
5.00 0
0.5 kGy
1 kGy
30
0.00 2 kGy
3 kGy
15
5.00

0
0.00
0 7 15
Days
D after irradiation

Fig. 5. The antioxidant activity of blueberries stored at 4 ◦ C for 0, 7, and 15 d, respectively, following different irradiation doses. Results represent the mean plus one SD of
three trials and triplicates were performed for each trial.

3.4.1. TMA content the TMA content among blueberries stored at 4 ◦ C for 7 d and
Fig. 4 shows that the TMA contents of blueberries treated 15 d. In contrast, a massive decrease of TMA content (81–84%)
with different doses are similar to that found in the control in blueberries was observed after thermal processing (80 ◦ C
blueberries and there was no significant effect of radiation on for 30 min) (Poiana et al., 2012), suggesting that anthocyanins

5
5.00
L-ascorbic acid (g kg-1 x 10-2)

4
4.00

3
3.00 Gy
0 kG
0.5 kGy

2
2.00 Gy
1 kG
Gy
2 kG
Gy
3 kG
1.00

0
0.00
0 7 15
Days affter irradia
ation

Fig. 6. The l-ascorbic acid content of blueberries stored at 4 ◦ C for 0, 7, and 15 d, respectively, following different irradiation doses. Results represent the mean plus one SD
of three trials and triplicates were performed for each trial.
34 Q. Kong et al. / Postharvest Biology and Technology 95 (2014) 28–35
are much less sensitive to irradiation at low doses than to
temperature.
3.4.2. Antioxidant activity
Besides TMA content, antioxidant activity is another impor-
tant property affecting the quality of blueberries. In this study,
the antioxidant activity of fresh blueberries was estimated from
their ability to reduce the ferric 2,4,6-tris (2-pyridyl)-1,3,5-triazine
(TPTZ) complex. Results shown in Fig. 5 indicate that there is no
significant difference in antioxidant activity among non-irradiated
blueberry (control) and blueberries irradiated with doses ≤3 kGy.
However, the antioxidant activity of blueberries in all groups
including treated and non-treated groups decreased significantly
(P < 0.05) after being stored at 4 ◦ C for 15 d. In terms of change in
antioxidant activity in response to storage, our result was consis-
tent with previous results obtained from Kale juice and blueberry
jam (Poiana et al., 2012; Song et al., 2006).
3.4.3. l-Ascorbic acid
It was reported that ascorbic acid is one of the major antioxi-
dants in fruit and it is also the most sensitive water-soluble vitamin
to irradiation (Kilcast, 1994). In this study, no loss of ascorbic acid
was observed after treatment with a dose of up to 3 kGy compared
to the control (Fig. 6). However, after storage there was a signif-
icant decrease of ascorbic acid content in control and irradiated
blueberries with a higher reduction (ranging from 32 to 54%) at
7 d after storage (P < 0.05). Similar results have been reported in
the literature (Harb et al., 2010; Moreno et al., 2008). However, no
change in ascorbic acid level in blueberries stored for 8 d at 0, 10,
20 and 30 ◦ C was also reported (Kalt et al., 1999). These discrepan-
cies could be caused by the quality of starting materials, conditions
during packaging, transportation, and storage, or cultivars used.
Reports about the effect of irradiation on vitamin C content in
different fruits and vegetables have been controversial. No major
effects on vitamin C content were observed in irradiated mango
fruit (≤1.5 kGy) (Reyes and Cisneros-Zevallos, 2007). Similarly, irra-
diation at 1 kGy did not affect the vitamin C content in grapefruits
(Girennavar et al., 2008). However, irradiation with doses ≤1 kGy
resulted in significantly lower vitamin C content in spinach (Gomes
et al., 2008). Another study found that irradiation affected the vita-
min C concentration in strawberries; however, the change was
small in comparison with the large variations observed between
varieties (Graham and Stevenson, 1997). These differences could
be due to the variations in fruit species, analytical methods, irradi-
ation dosage, or cultivars of the same varieties. It is difficult to make
general conclusions regarding the effect of irradiation on vitamin
C of fruits.
4. Conclusions
E-beam irradiation can be a useful tool for the treatment of blue-
berries in a single layer when used properly. Results from this study
showed that doses less than 3 kGy provided effective inhibition of
E. coli in blueberries and the shelf life of blueberries was extended
significantly as measured by the decay percentage, while important
chemical properties or nutritional qualities, such as antioxidant
activity, content of TMA and l-ascorbic acid were not changed.
Further studies are needed to optimize conditions of the treat-
ment for commercial uses. Results obtained in this study could be
used as a reference for other species of fresh produce, but not all
fruits are good candidates for irradiation treatment, proper irradi-
ation process parameters have to be established for each specific
product.
Acknowledgments
This research was supported by the projects of the Science and
Technology Bureau of Shanghai project numbers 12390700700,
11dz0502800 and USDA-ARS National Program NP108, CRIS project
5325-42000-048-00D. The U.S. Department of Agriculture is an
equal opportunity provider and employer.
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