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Effect of gamma irradiation and heat treatment on the artificial

contamination of maize grains by Aspergillus flavus Link NRRL 5906

Article in Journal of Stored Products Research · February 2017

DOI: 10.1016/j.jspr.2017.01.003

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 Journal of Stored Products Research 71 (2017) 57e63

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Journal of Stored Products Research

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

Effect of gamma irradiation and heat treatment on the artificial

contamination of maize grains by Aspergillus flavus Link NRRL 5906
Mohamed Albuzaudi a, Tero Eerika €inen b, Ossi Turunen b, Mohamed Ghelawi a,
M. El Haj Assad c, *, M. Tawalbeh c, Dattatray Bedade d, Salem Shamekh b
a
Biotechnology Research Center, Food Analysis and Treatment Research Group, P. O. Box 30313, Tripoli, Libya
b
Aalto University, School of Chemical Technology, Department of Biotechnology and Chemical Technology, P. O. Box 16100 (Kemistintie 1), FIN-00076, Aalto,
Finland
c
University of Sharjah, SREE Department, P. O. Box 27272, United Arab Emirates
d
Department of Food Engineering and Technology, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga, Mumbai 400019, India

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

Article history: Maize (Zea mays L.) is one of the main crops, which is easily susceptable to Aspergillus flavus infection
Received 9 August 2016 resulting in huge losses worldwide. This study was carried out to investigate the effect of combining heat
Received in revised form and irradiation treatments in controlling the fungal growth in maize grains. Surface disinfected maize
21 January 2017
grains were artificially contaminated with spores of Aspergillus flavus Link NRRL 5906, and then exposed
Accepted 22 January 2017
to gamma radiation with doses of 3.0, 4.0 and 5.0 kGy. The samples were additionally heat treated at
60
C for 30 min. The heat and irradiation treatments showed a synergistic effect on controlling
Aspergillus flavus growth. The heat treatment reduced the required radiation dose of about 0.5e1.0 kGy
Chemical compounds:
Aflatoxin B1 (PubChem CID: 186907)
when 4.0 kGy or 5.0 kGy irradiation was used. The combined heat and irradiation treatment of moisture
Aflatoxin B2 (PubChem CID: 2724360) reduced the average CFU by 8 log cycles when 4 kGy or 5 kGy irradiation was used and by 7 log cycles
when 3 kGy irradiation was used. The heat treatment of moisture alone reduced the average CFU by only
Keywords: by 0.8 log cycles. Combining irradiation with heat treatment to reduce the required radiation dose is very
Gamma irradiation useful especially when there is a concern over biological side effects of irradiation.
Heat treatment
© 2017 Elsevier Ltd. All rights reserved.
Aspergillus flavus
Maize

1. Introduction
Molds are widely spread on agricultural commodities causing a
considerable economical damage and risk for human and animal
health (Bryden, 2007; De Lucca, 2007; Shephard, 2008). The mold
contamination can occur in the field, during handling, and/or
storage. Fungal mycotoxins occurring in food are of a great concern
worldwide, and especially in the developing countries (Kabak et al.,
2006; Bryden, 2007; Reddy et al., 2009; Rem za et al., 2014). There
are more than 100 countries who retain specific regulations or
detailed guidelines for mycotoxins in food nowadays (Van Egmond
et al., 2007). Despite of all of these regulations or guidelines, afla-
toxins were found worldwide on average in 26% of various samples
* Corresponding author.
E-mail addresses: mozidi125@yahoo.com (M. Albuzaudi), tero.eerikainen@aalto.
fi (T. Eerika€inen), ossi.turunen@aalto.fi (O. Turunen), maghelawi@gmail.com
(M. Ghelawi), massad@sharjah.ac.ae (M. El Haj Assad), ft13dk.bedade@pg.
ictmumbai.edu.in (D. Bedade), salem.shamekh@aalto.fi (S. Shamekh).
in 2009 (Rodrigues and Griessler, 2009). In fact, in that study 76% of
the samples contained at least one of the major studied mycotoxins.
Therefore, the impact of mycotoxins on the safety of agricultural
products is significant.
Maize (Zea mays L.) is susceptible worldwide to contamination
by different Fusarium and Aspergillus strains (Wood, 1992;
Munkvold, 2003; Reddy et al., 2009). In particular, toxigenic
strains of Aspergillus flavus grow on nuts, maize and other grains
and produce highly toxic carcinogenic compounds known as afla-
toxins. Aflatoxins represent a worldwide threat to public health
(Strosnider et al., 2006). They transmit transplacentally and to the
newborns through breast-feeding (Goldman and Shields, 2003).
Normally, Aspergillus flavus produces aflatoxin B1 (AFB1) and B2
(AFB2). AFB1 is the most potent natural carcinogen known and is
associated with human hepatocellular carcinomas (Bennett and
Klich, 2003; Ferguson and Philpott, 2008). AFM1 and AFM2,
which are the hydroxylated metabolites of AFB1 and AFB2, are
usually found in milk or milk products obtained from livestock that
have ingested contaminated feed (Dutton et al., 1985; Bennett and

http://dx.doi.org/10.1016/j.jspr.2017.01.003
0022-474X/© 2017 Elsevier Ltd. All rights reserved.
58 M. Albuzaudi et al. / Journal of Stored Products Research 71 (2017) 57e63
Klich, 2003). In all maize grains, the fungal population was above
104 CFU/mg after 30 days of incubation and the aflatoxin was over
23.7 mg/kg after 60 days of incubation (Franzolin et al., 1999). Large
number of stored maize samples (storage time from 1 year to 11
years) were collected in Lianoing Province, China, where all these
samples contained aflatoxins with an average content of 0.99 mg/kg
(Liu et al., 2006).
Pre- and post-harvest strategies are necessary to prevent mold
infestation and production of mycotoxins (Jouany, 2007). Adequate
working rules and handling procedures are crucial aspects in
reducing the risk of mycotoxin production in maize (Munkvold,
2003; Kabak et al., 2006; Strosnider et al., 2006). After harvest,
low moisture content, low storage temperature and aeration are
very critical factors espectially, when there is a possibilty of fungal
growth. In that case, the grains should be dried quickly at high
temperature (Munkvold, 2003). Usually, fungal growth is pre-
vented by using physical, chemical and/or biological treatments
(Davis et al., 1984). There are many factors that contribute to the
fungi production and aflatoxin growth. The most important ones
are humid climate, warm temperature and high moisture content.
After harvesting, a drying process is required to reduce the mois-
ture content to a safe level. In this study, heat is required to provide
80e90% relative humidity for at least one hour before gamma
irradiation treatment takes place.
There is a number of different specific methods to prevent the
mold contamination of crops (Suttajit, 1991; Kabak et al., 2006; Yin
et al., 2008). Inhibitors are also developed over the years for afla-
toxin production (Holmes et al., 2008). Many studies reported, 40
years ago, using irradiation to control A. flavus (Malla et al., 1967;
Jemmali and Guilbot, 1970; Applegate and Chipley, 1973, 1974).
Gamma irradiation has been in wide use in food preservation.
However, there are many concerns regarding its safety due to
reactive radiolytic products and other undesired effects (Sommer
and Fortlage, 1966; Grolichova et al., 2004; Smith and Pillai,
2004). The current view is that irradiation is the safest and one of
the most reliable methods for preservation of food and agricultural
commodities (Shah et al., 2014). Moreover, it is also shown that
doses below 10 kGy in controlling the contamination of food are
safe (Smith and Pillai, 2004). According to International Atomic
Energy Agency (IAEA, 1982), the lethal radiation dose for molds is in
the range of 2.5e6.0 kGy. In most studies, a dose of 1e3 kGy can
reduce significantly the amount of pathogens (Shah et al., 2014).
However, a dose 3e20 kGy was required to sufficiently reduce the
microbial populations in dry food ingredients (Farkas and Moh acsi-
Farkas, 2011). There are many factors affecting gamma radiation
process, such as initial mycotoxin concentration, absorbed dose and
the amount of moisture content (Calado et al., 2014). As a rule,
using a combination of two treatment methods, such as heat and
chemical treatment or fermentation and steamimg, reduces AFs
more than each individual respective method alone (Jalili, 2016).
The general efficiency of irradiation in reducing the growth of
pathogens is well-documented (Shah et al., 2014), however, there is
a concern that irradiation treatment of food commodities may
enhance the mycotoxin production by the remaining fungi (O'Neill
et al., 1996). Several studies found that gamma radiation in fact
increased the aflatoxin production (Jemmali and Guilbot, 1970;
Applegate and Chipley, 1973, 1974; Schindler et al., 1980;
Odamtten et al., 1986, 1987; Aziz et al., 2002). In addition, heat
and irradiation may eliminate the protecting natural strains (O'Neill
et al., 1996).
As an example, the irradiation has to be strong enough
(5e10 kGy) to prevent the mycotoxin production (Ferreira-Castro
et al., 2007). Furthermore, when low dose, 2.0 kGy, was used, the
production of mycotoxins was increased, and for Fusarium infection
of maize, 10 kGy was required to fully eliminate the mycotoxin
production (Ferreira-Castro et al., 2007). The dose of at least 10 kGy
is generally necessary for total prevention of aflatoxin production in
the sample (Van Dyck et al., 1982; Mutluer and Ergoc, 1987; Aziz
and Youssef, 2002; Aquino et al., 2005). When following the
fungal growth after irradiation for 100 storage days, it was observed
that while 4.0 kGy dose was required to significantly reduce the
fungal growth, the 6.0 kGy dose was required to completely inhibit
the fungal growth (Aziz et al., 2006). Several studies have identified
treatments or chemicals that sensitize the fungi to irradiation, thus,
lowering the required irradiation dose (Mohyuddin and Skoropad,
1977; Shahin and Aziz, 1997; Patel et al., 1989). Combined treat-
ment of heat and radiation has also been studied in the control of
fungal growth (Farkas, 1990). When treating fungi, the heat treat-
ment should precede the irradiation treatment, whereas with
bacterial spores the reverse order of the treatments is necessary
(Farkas, 1990). The double-strand breaks caused by ionizing radi-
ation are lethal if not repaired or if misrepaired. High temperature
can prevent DNA damage repair and therefore exposure to high
temperature enhances greatly the cell killing (El-Awady et al.,
2001).
The combination of heat and irradiation was more effective than
either treatment alone to reduce the amount of aflatoxins in
Aspergillus parasiticus grown on rice (Narvaiz et al., 1988). The
treatment of the spores by gamma radiation of 4.0 kGy in combi-
nation with moist heat of 60
C for 30 min stopped the A. flavus
growth and aflatoxin production in flask cultivations (Odamtten
et al., 1986, 1987). However, those authors did not study the ef-
fect in conditions mimicking the handling of maize crop.
Low radiation doses tend to increase the aflatoxin production,
since dilution of spores stimulates aflatoxin production (Odamtten
et al., 1986, 1987; Ferreira-Castro et al., 2007). Thus, the size of
inoculum affects the toxin production. Accordingly, the maize
grains inoculated with a high number of spores develop less afla-
toxin than the grains inoculated with a lower number of spores
(Picco et al., 1999). Aflatoxin production occurs during the period of
intense sporulation and dilution of the spores induces sporulation,
hence, the aflatoxin production. Therefore, an inadequate treat-
ment may lead to an increase in the aflatoxin production.
Aspergillus is one of the major seed-borne fungi contaminating
maize in Libya (Baraka et al., 1999). Maize grains are seasonally
cultivated for consumption as fresh or dried. Maize is important as
animal feed, and imported animal feeds containing maize grains
are usually stored for a long time (Baraka et al., 1999). Food irra-
diation research started in Libya at the beginning of 1980s. Ac-
cording to the IAEA Food Irradiation Clearances Database (FICDB),
the Libyan clearance irradiated food list contains potatoes, onions,
garlic, dates, dried spices and poultry since January 1989. The
objective of this study is to evaluate the effect of gamma radiation,
heat treatment and their combinations in controlling A. flavus
growth on maize grains produced in Tripoli, Libya.
2. Materials and methods
2.1. Sample materials
Samples of maize grains cultivated in Tripoli were collected from
the local market. A.flavus Link NRRL 5906 spores were obtained
from Tripoli University, Faculty of Agriculture, Libya, originally from
the International Mycological Institute (IMT).
2.2. Growing of A. flavus on maize agar and cracked maize
To prepare maize agar, two hundred grams of maize flour were
mixed well with about 400 ml of distilled water. Then the filtrate
was filled upto 1 liter with distilled water. Using a hot plate with
 M. Albuzaudi et al. / Journal of Stored Products Research 71 (2017) 57e63
magnetic stirrer, 20 g of agar were dissolved in the filtrate, and then
autoclaved at 121
C for 15 min. The medium was then cooled to
about 45
C, and 15 ml of it was aseptically poured into the steril-
ized Petri dishes. The maize extract media in Petri dishes was
inoculated with a pure A. flavus strain at 28
C for one week. Then,
the growing spores on the solid media were removed using 10 ml
sterilized 1% peptone solution and transferred into small glass
containers. For growing of A. flavus on cracked maize, 500 grams of
cracked maize were moistened with 5 ml distilled water and
autoclaved at 121
C for 15 min in order to sterilize the growing
medium for A. flavus. Several dilutions were made of the spor-
esuspension of A. flavus to get spore concentration of 3.1  107
spores/ml. After shaking, 2 ml of spore solution were used to
inoculate 500 g of cracked maize. Then the inoculated cracked
maize was incubated for one week at 28
C. More details about the
preparation of stock spore production can be found eleswhere
(Tedihou et al., 2012; El-Shanshoury et al., 2014; Okun et al., 2015).
2.3. Inoculation of maize grains
Whole maize grains samples of 1600 g were surface disinfected
(70% ethanol for 2 min) and then infected with A. flavus Link NRRL
5906 culture by mixing the whole maize grains and the cracked
maize (500 g) together. Then the cracked maize was removed by
sieving. About 50 grams of infected maize grains were placed in a
Petri dish and incubated at room temperature (25
C ± 2
C) in glass
cages for 24 h prior to the heat and irradiation treatments. A Petri
dish containing glycerol solution (35%) was then placed in each
cage to give 90% relative humidity (R.H.).
2.4. Heat and irradiation treatments
Heat treatment wascarried out at 60
C for 30 min before irra-
diation (1e2 h earlier). Hot water wasplaced inside an oven to
provide approximately 80e90% R.H. throughout the heat treatment
process. The maize grains were irradiated in polythene bags with
doses of 3.0, 4.0 and 5.0 kGy gamma radiationwith a dose rate of
7.46 kGy/hfrom aCobalt 60 unit (GB651) gamma irradiator at Tajura
Research Center (TRC), Tripoli.Each experiment was repeated twice.
No dose distribution chart was prepared, due to the small size of
the irradiated samples. To achieve a good dose distribution, the
sample waskept on a turning table during the irradiation treat-
ment. The Fricke dosimetry system (Fricke and Hart, 1966) was
used in this study to monitor the irradiation. The range at which the
Fricke dosimetry is used depends primary on the irradiation sour-
ces. In this study, the irradiationsused in most of the experiments
were 3, 4 and 5 kGy. Hence, Fricke dosimetry was utilized since it
can be used for doses in the range of 0.5 kGye10 kGy (Kabori et al.,
2010), with an acceptable accuracy in the range of 0.2e4.0 kGy
(IAEA, 2002) or 0.4e5.0 kGy (Henniger, 2014).
2.5. Assays
For measuring the water content, two replicates of ten-gram
samples of heat-treated and untreated maize grains were dehy-
drated over night at 100
C, and then the weight of the dried
samples was measured. Forcounting the number of colonies (i.e.
colony forming unit, CFU), ten grams of maize grains weremixed
with 100 ml of 1%peptone solution. One ml of the suspension was
used to prepare serial dilutions (101 to 107). Different dilutions
were mixed in Petri dishes with sufficient amount of OGYE media
(Mossel et al., 1970) which was used in order to stimulate the fungi
growth and to inhibit bacterial contamination. Then the Petri
dishes were incubated at 28
C for 3 weeks and CFUs were counted
on a weekly basis. Before aflatoxin assay, thetreated and untreated
59
maize grains were stored for 4 weeks at room temperature
(25
C ± 2
C) in glass jars at 90% R.H. The aflatoxin assay was then
carried out by using enzyme-linked immunosorbent assay (ELISA)
technique (Lui et al., 2013), RIDASCREEN Aflatoxin B1 kit, r-Bio-
pharm GmbH Company. Five grams of ground maize were vigor-
ously shaken with 25 ml of methanol (70%) for one minute, and
then the extract was filtered using filter paper. The obtained filtrate
was then used for aflatoxin assay.
2.6. Statistical analysis
Statistical analysis was performed to estimate the overall reli-
ability of all steps in the experimental series. Data was analyzed
using two-way ANOVA. Multilinear regression (MLR) method was
used to calculate interaction models for average CFUand logarith-
mic CFU from variables incubation (INC), radiation (RD) and heat
treatment (HT). Response surface models for multilevel variable
values were calculated with Modde 8.0 (Umetrics, Sweden).
Response plots describing the model behavior were created with
Modde. Temperature data transformation (1/T) was tested, how-
ever, it did not provide a noticeable enhancement to the model.
Therefore, no transformation to original variable values was used,
except for the response variable mentioned above. A mixed level
full factorial design with response surface method was used to
describe the interaction effect between radiation dose (kGy), tem-
perature (
C) and incubation time (days) on the Aspergillus flavus
count (log CFU$g1). Description and examples of response surface
methodology is given by Gabrielsson et al. (2002). The radiation
dose was varied from 0 to 5 kGy at four different levels (0, 3.0, 4.0
and 5.0 kGy). The incubation time was studied from the first day to
end of fourth week taking four equal intervals (1, 7, 14 and 21 days).
The heat treatment was done at 60
C for 30 min. A total of 32
(4  4  2) experimental runs were conducted in duplicate and the
Aspergillus count (log CFU$g1) was taken corresponding to each
run. The counts were taken in duplicate for each duplicate runs. The
second order polynomial equation (Equation (1)) was developed for
microbial count as a function of three different variables dose,
temperature and time, respectively.
Y ¼ b0 þ b1 x1 þ b2 x2 þ b3 x3 þ b4 x21 þ b5 x22 þ b6 x23 þ b7 x1 x2
þ b8 x2 x3 þ b9 x1 x3
(1)
where Y is the predicted value, b1, b2 and b3 are linear coefficients
while b0 is constant, x1, x2, x3 represents radiation dose, tempera-
ture and time respectively.Model adequacy was tested through
ANOVA data, p-value, adjusted R2 and F-value of the model. The
significance of each term was tested against its p-value. Response
surface model was developed to visualize the interaction effect
between two independent variables on the microbial count,
keeping the third variable fixed at a certain level.
3. Results
The results of this study showed that the infection of maize
grains by Aspergillus flavus was sensitive to radiation and heat
treatments as shown in Table 1, Figs. 1 and 2. The data was analyzed
following the two treatments (heating and radiation), in
completely randomized design, showed a significant increase in
average number of CFUs of Aspergillus flavus during the testing
period. The survival of the fungi after the inactivating treatments
was assayed by cultivating the fungi in peptone medium. The sta-
tistical analysis shown in Fig. 1A and B indicated that the three
weeks follow-up did not reveal all the surviving fungi, however, it
60 M. Albuzaudi et al. / Journal of Stored Products Research 71 (2017) 57e63

Table 1
Effect of gamma radiation and heat treatment and their combination in controlling of Aspergillus flavus growth on maize. The heat treatment was done at 60
C for 30 min.

Treatment Average CFU/g

1 day 7 days 14 days 21 days

4a 6a 7a
Control (untreated) 1.4 ± 0.015  10 1.1 ± 0.012  10 1.0 ± 0.020  10 1.1 ± 0.020  108a
3 kG 0.8 ± 0.015  101b 1.9 ± 0.009  101 b
0.1 ± 0.015  102b 0.1 ± 0.013  103b
4 kG 0.1 ± 0.02  101c 0.9 ± 0.015  101 c
0.1 ± 0.012  102 c 0.6 ± 0.015  102c
5 kG BD BD 0.1 ± 0.018  101d 0.6 ± 0.018  101d
Heat onl 1.0 ± 0.018  103d 1.8 ± 0.012  106d 1.8 ± 0.011  106e 1.4 ± 0.014  107e
3 kGy and heat 0.1 ± 0.015  101e 0.1 ± 0.018  101e 0.5 ± 0.017  101f 0.1 ± 0.009  102f
4 kGy and heat BD BD 0.1 ± 0.13  101g 0.1 ± 0.015  101g
5 kGy and heat BD BD BD 0.1 ± 0.013  101h

(BD ¼ below detection level, t-test was performed to determine comparison between each value of CFU. Data are result of triplicate analyses and all the standard deviations
were less than ±5%, different letters on each value indicates significantly different values).

Fig. 1. Response surface contour plots of logarithmic colony count (Ln CFU). LnCFUs are shown as functions of radiation dose (kGy) and incubation time (d) and heat treatment,
where heat is constant at 25
C (A) or 60
C (B) or incubation time is constant for 7 days (C).

Fig. 2. Residuals plot for the response surface interaction model. This normal probability plot of residuals helps to detect outliers and assess normality of the residuals.

allowed the measuring of the differences between the treatments.
The cultivation assays showed that the colonies grown decreased
significantly with the treatments. It was also evident from Table 1
that the irradiation doses from 3.0 to 5.0 kGy reduced the num-
ber of colonies in a dose-dependent manner. It was also clear from
Table 1 that combined treatment of heat and irradiation reduced
the colonies grown when compared to using either irradiation or
heat treatment.
It is evident that fungal growth was prevented completely
during the first week of the follow-up assay by both irradiation
 M. Albuzaudi et al. / Journal of Stored Products Research 71 (2017) 57e63
treatment with 5.0 kGy gamma radiation and the combined
treatment of heat (60
C for 30 min) and irradiation (4.0 kGy). On
the other hand, the combined treatment of heat and 5.0 kGy was
able to prevent the growth completely during the first two weeks of
incubation. However, heat treatment alone showed a higher colony
number after one week of incubation compared to the control
(untreated grains) case. The initial moisture content of heat-treated
and untreated grains was 19.3% and 18.3%, respectively. ANOVA
analysis revealed clear differences in interaction between the
treatments and the incubation periods. Significant differences were
noted between treatments (DF ¼ 7, 62, F ¼ 169.05, P < 0.01), period
(DF ¼ 3, 62, F ¼ 107.02, P < 0.01) and treatments x periods (DF ¼ 21,
62, F ¼ 323.1, P < 0.01).
The moist heat treatment reduced the average number of CFUs
by 0.8 log cycles (log means the numbers expressed in logarithmic
form) compared to the control (untreated grains). Whereas the
combined heat and irradiation (4.0 or 5.0 kGy) and 3.0 kG y
treatments of moist reduced the average CFUs by 8.0 and 7.0 log
cycles, respectively, as shown in Table 1. Irradiation alone using
doses of 3.0, 4.0 and 5.0 kG y reduced the average CFUs by 6.0, 6.2
and 7.2 log cycles, respectively (Table 1).
ELISA technique was carried out after four weeks of incubation
of contaminated maize to assay the presence of aflatoxin. ELISA
technique was used to detect aflatoxins in maize grains with a
detection limit of 0.5 mg/kg. The test indicated thatn one of the
maize grains (irradiated orunirradiated) contain detectable
amounts ofAFB1, AFB2, AFG1, and AFG2.Thisindicatedthat the
conditions, such as relative humidity, temperature, gamma radia-
tion dose and incubation time, in the four weeks incubation, did not
induce any larger amounts of aflatoxins in the fungus grown on
maize. Due to the short time of incubation, the produced aflatoxin
was below the detectable level. Response surface models were
calculated as shown in Table 2 and Fig. 1 to define accurately the
treatment effects and to see whether there is true interaction and
synergy with heat treatment and radiation.
Incubation time (INC), irradiation (RD) and heat treatment (HT)
were used as independent multilevel factors and the average CFU
and the natural logarithm of CFUs (Ln CFU) were defined as the
responses. Polynomial interaction response surface model for Ln
CFU is given by Equation (2).
Ln CFU ¼ 4:291 þ 2:252INC  4:629RD þ 0:411HT
 2:119INC  RD  1:586RD  HT
where Ln CFU is the logarithmic colony forming unit, INC is incu-
bation time (days), RD is radiation dose (kGy) and HT is heat
treatment temperature (
C). The coefficient of determination (R2)
and coefficient of prediction (Q2) were 0.895 and 0.819 respectively
(DF ¼ 21, F ¼ 35.78). The model predicted and the experimentally
measured values for logaritmic CFU along with the independent
variables are presented in Table 3.
Due to the high non-linearity of the growth processes, the
control samples (experiments 1e4) and one outlier (experiment
18) were ignorned in the linear interaction regression model.
Table 2
Regression coefficient model for logarithmic CFU (Ln CFU).
Ln CFU
Incubation time
Radiation dose
Heat treatment temperature
Incubation time  Radiation dose
Radiation dose  Heat treatment temperature
Legend: N ¼ 27, (number of experiments in the model), DF ¼ 21 (degree of freedom of residuals).
61
Normal probability plot of residuals showed that the model pre-
diction residuals were random and normally distributed, and none
of the samples chosen to model were outlier as shown in Fig. 2. Ln
CFU-model was statistically significant (Table 2). Fig. 1C revealed
that the effect of heating on moisture content might cause a
problem, where CFUs increased in low radiation levels.
The resulting model implies that radiation dose and incubation
time in heat treatment are highly significant factors. The radiation
and heat treatment interaction term (RD*HT) is significant, but it
seems that heating favors decreasing colony formationonly at
higher radiation levels. The comparison of the countours in Fig. 1
reveals that at 4.0e5.0 kGy radiation doses combined with the
heat treatment decreased CFUs to zero. The heat treatment effect
corresponds to about 0.5e1.0 kGy radiation dose above 4.0 kGy
radiation levels. This behavior might be seen in the response sur-
face plots in Fig. 1C.
4. Discussion
To reduce the risk of fungal growth in the food, it is necessary, to
have a monitoring Program of Good Manufacturing Produce (GMP)
and use of gamma-irrradiationto prevent the proliferation of toxi-
genic fungi and the associated production of mycotoxins (Ferreira-
Castro et al., 2007). Irradiation combined with good storage con-
ditions, proved a reliable process for conservation of many food
products, because it prevents the growth of fungi, particularly
aflatoxigenic Aspergillus. Since low irradiation doses that may be
effective in fungal growth control may still allow the production of
mycotoxins, this study endeavored to find out how the effect of
irradiation could be enhanced. At the same time, with the help of an
additional/combined treatment, the dose of irradiation could also
be reduced. Dried mycotoxins are extremely radioresistant,
whereas mycotoxins in solution are sensitive to irradiation. The
oxidative radicals that originate from water radiolysis are respon-
sible for their degradation (Calado et al., 2014).
It has been reported that, fungal load may be substantially
reduced with irradiation levels of 5 kGy and above. However, lower
radiation doses can also be effective if products are previously
treated with hot water (Calado et al., 2014). In the current study, the
effect of combined gamma radiation and heat treatment on artifi-
cial contamination of maize grains by Aspergillus flavus spores was
tested, thus, mimicking the contamination in practical grain
(2) handling. According to present results, the combination of irradi-
ation andheat treatment reduced the needed dose of ionizing
irradiation in the inhibition offungal growth, when compared to
irradiation treatment alone. Higher colony number was higher for
heat treatment after one week of incubation, this might be due to
the apparent increase in the moisture content. The statistical
modeling confirmed the reliability of this conclusion. In wheat, an
irradiation dose of 3 kGy was sufficient to reduce the presence of
Alternaria, Aspergillus, and Fusarium by 10-fold (Calado et al., 2014).
The fungal colonies were followed up for three weeks after the
treatments, and the CFUs were counted on 1, 7, 14 and 21 days. In
the three weeks follow-up, at least 4.0 kGy in combination with the
Regression model coefficient P
2.252 <0.05
4.629 <0.05
0.411 0.41
2.119 <0.05
1.586 <0.05
62 M. Albuzaudi et al. / Journal of Stored Products Research 71 (2017) 57e63

Table 3
Model predicted (PredictedLn CFU) and experimentally measured (Measured Ln CFU) values for logarithmic CFU.

Exp. No Incl/Excl INC (d) RD (kGy) HT (
C) Measured Ln CFU Predicted Ln CFU Exp. No Incl/Excl INC (d) RD (kGy) HT (
C) Measured Ln CFU Predicted Ln CFU

1a Excl 1 0.0 25 9.55 e 17 Incl 1 0.0 60 6.91 6.55

2a Excl 7 0.0 25 13.91 e 18a Excl 7 0.0 60 14.40 NA
3a Excl 14 0.0 25 16.12 e 19 Incl 14 0.0 60 14.40 12.23
4a Excl 21 0.0 25 18.52 e 20 Incl 21 0.0 60 16.46 15.29
5 Incl 1 3.0 25 2.20 1.44 21 Incl 1 3.0 60 0.69 1.63
6 Incl 7 3.0 25 3.00 2.54 22 Incl 7 3.0 60 0.69 2.73
7 Incl 14 3.0 25 2.40 3.82 23 Incl 14 3.0 60 1.79 4.01
8 Incl 21 3.0 25 4.62 5.10 24 Incl 21 3.0 60 2.40 5.29
9 Incl 1 4.0 25 0.69 1.07 25 Incl 1 4.0 60 0.00 0.01
10 Incl 7 4.0 25 2.30 1.66 26 Incl 7 4.0 60 0.00 0.58
11 Incl 14 4.0 25 2.40 2.35 27 Incl 14 4.0 60 0.69 1.27
12 Incl 21 4.0 25 4.11 3.03 28 Incl 21 4.0 60 0.69 1.95
13 Incl 1 5.0 25 0.00 0.70 29 Incl 1 5.0 60 0.00 1.65
14 Incl 7 5.0 25 0.00 0.78 30 Incl 7 5.0 60 0.00 1.57
15 Incl 14 5.0 25 0.69 0.88 31 Incl 14 5.0 60 0.00 1.47
16 Incl 21 5.0 25 1.95 0.97 32 Incl 21 5.0 60 0.69 1.38

Legend: Independent variables: INC ¼ incubation time (days), RD ¼ radiation dose (kGy), HT ¼ heat treatment temperature (
C), - ¼ not meausred.
a
Experiments 1e4 were excluded from the linear interaction model due to high non-linearity and experiment 18 as found to be an outlier (outside the normal variation
boundary, which was four times standard deviation).

heat treatment was necessary to keep the microbial growth low.
Irradiation doses above 5 kGy effectively inhibit growth of Fusarium
verticillioides in maize grains, although a complete elimination of
the fungal microflora requires 10 kGy (Ferreira-Castro et al., 2007).
Present study results indicated that the heat treatment allowed
maintaining the irradiationdoses below 5.0 kGy to stop the growth
of A. flavus. On the other hand, it was evident that 3.0 kGy, evenin
combination with the heat treatment, is a very low dose. The
testing of the surviving microbes with various assay (cultivation
after treatments) lengths showed that when more time was
elapsed the more microbes recovered togrow. This indicated that in
the practical usage probably stronger treatments would be useful
for longer storage periods. An unexpected finding in the modeling
was that the heat treatment in combination with irradiation might
increase the fungal growth below the radiation doses of 3.0 kGy.
This finding indicates that if the fungal growth is not stopped
completely, the stimulated growth is a risk in respect to fungal
mycotoxins. Earlier studies have reported an increase in mycotoxin
production by various genera of fungi after exposure to gamma
radiation alone. Inoculum size of microbes may also affect myco-
toxin production in A. flavus and A. parasiticus by suppressing
aflatoxin production, which occurs when the level of spores in
substrate exceeds certain levels (Braghini et al., 2015).
Investigation of the effect of using the combination treatment
method reduced aflatoxin production than each method alone
(Jalili, 2016) as it is evident in this study. In general, the method was
more effective than either treatment alone to reduce the amount of
aflatoxins in Aspergillus parasiticus grown on rice (Narvaiz et al.,
1988). It was proved experimentally that the treatment of the
grain maize by gamma radiation in combination with moist heat of
60
C at 4 kGy stopped the A. flavus growth and aflatoxin produc-
tion, which is consistent with previous investigation in flask cul-
tivations (Odamtten et al., 1986, 1987).
The heat and irradiation treatments showed effectiveness in
controlling Aspergillus flavus growth. The heat treatment reduced
the needed radiation dose about 0.5e1.0 kGy when at least ~4.0 kGy
irradiation was used. Too mild treatments may stimulate the fungal
growth. The combination of irradiation with heat to reduce the
radiation dose can be a useful strategy. It can be also concluded that
the combination of irradiation and heat decreases the colony
growth, heating of moisture content decreases colony formation
only at high radiation dose and the relative humidity of the mixture
should be kept at between 80 and 90%. Moreover, the combination
of heat and irradiation treatment of moisture reduces the average
CFU by 7 or 8 logs depending on the gamma radiation dose used.
Finally, the use of heat and irradiation treatment may constitute a
strategy to prevent and control the presence of toxigenic fungi. This
method could be useful for controlling A. flavus and its toxins in
other grains/food products.
Moreover, the combination of heat and irradiation treatments
might be applicable when water or steam is required for cleaning
during the preparation process of fruits and vegetables that are
sensitive to irradiation (e.g., tomato, citrus). It might also be
beneficial to use combined treatment to minimize the radiation
dose in treating foods that are rich in water or fat (meat and
poultry) to avoid fat oxidation and formation of free radicals.
Acknowledgements
The authors would like to thank Mr. Abubaker Edkymish,
Biotechnology Research Centre, Tripoli, Libya, for his assistance in
carrying out the statistical analysis part of this study. They would
likealsoto thank Dr. Abdullah Watban of the College of Languages
and Translation, Al-Imam University, for the review and editing of
this work. The authers would like also to acknowledge the
Biotechnology Research Centre and Aalto University for funding the
research.
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