Effect of Co-60 gamma irradiation on Aspergillus flavus, Aflatoxin B1 and qualitative characteristics of pistachio nuts (Pistacia vera L.)

Abstract

In this study the effect of different doses of gamma irradiation (0, 0.5, 1, 1.5, 2, 4 and 6 kGy) on viable spore population of Aspergillus flavus, Aflatoxin B1 (AFB1) concentration and qualitative characteristics of pistachio samples were investigated. The results indicated that gamma irradiation at doses of 4 and 6 kGy reduced the viable spore population of A. flavus about 5 log. The maximum degradation of AFB1 in pistachio samples was 73.26% and 83.36% that was observed at doses of 4 and 6 kGy, respectively. The total phenol content (TPC) increased at dose of 2 kGy, but it was reduced at higher doses of irradiation. The increase of irradiation doses up to 4 kGy significantly increased the antioxidant activity, while irradiation dose of 6 kGy led to a reduction in antioxidant activity. All irradiation doses increased the amount of malondialdehyde in pistachio samples and the highest amount of malondialdehyde was observed at dose of 6 kGy. The chlorophyll and carotenoid content was decreased at all absorbed doses of gamma irradiation and affected the color features and resulted to a darker color of pistachio samples. Gamma irradiation slightly decreased the solubility of proteins and altered the pattern and intensity of the protein bands. The obtained results showed that gamma irradiation at doses higher than 2 kGy is capable of controlling and reducing the contamination of pistachio samples, while lower doses (kGy ≤ 2) of gamma irradiation caused minimum changes in pistachio samples quality.

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Vol.:(0123456789)
1 3
Journal of Food Measurement and Characterization
https://doi.org/10.1007/s11694-021-01060-z
ORIGINAL PAPER
Effect ofCo‑60 gamma irradiation onAspergillus flavus, Aflatoxin B1
andqualitative characteristics ofpistachio nuts (Pistacia vera L.)
MinaMakari1· MohammadHojjati1 · SamiraShahbazi2· HamedAskari2
Received: 19 April 2021 / Accepted: 5 July 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
In this study the effect of different doses of gamma irradiation (0, 0.5, 1, 1.5, 2, 4 and 6kGy) on viable spore population of
Aspergillus flavus, Aflatoxin B1 (AFB1) concentration and qualitative characteristics of pistachio samples were investigated.
The results indicated that gamma irradiation at doses of 4 and 6kGy reduced the viable spore population of A. flavus about
5 log. The maximum degradation of AFB1 in pistachio samples was 73.26% and 83.36% that was observed at doses of 4 and
6kGy, respectively. The total phenol content (TPC) increased at dose of 2kGy, but it was reduced at higher doses of irradia-
tion. The increase of irradiation doses up to 4kGy significantly increased the antioxidant activity, while irradiation dose of
6kGy led to a reduction in antioxidant activity. All irradiation doses increased the amount of malondialdehyde in pistachio
samples and the highest amount of malondialdehyde was observed at dose of 6kGy. The chlorophyll and carotenoid content
was decreased at all absorbed doses of gamma irradiation and affected the color features and resulted to a darker color of
pistachio samples. Gamma irradiation slightly decreased the solubility of proteins and altered the pattern and intensity of
the protein bands. The obtained results showed that gamma irradiation at doses higher than 2kGy is capable of controlling
and reducing the contamination of pistachio samples, while lower doses (kGy ≤ 2) of gamma irradiation caused minimum
changes in pistachio samples quality.
Keywords Pistachio· Aspergillus flavus· AFB1· SDS-PAGE· Color· HPLC
Introduction
Pistachio nut (Pistachia vera L.) is a member of anacardi-
aceae family, that has been consumed or exported in differ-
ent forms of raw, roasted and salted, and/or processed in
confections, deserts and ice creams. Pistachio kernel with
having about 20% of protein and 45% of oil is considered
as a rich source of protein and oil especially unsaturated
fatty acids (about 87%) [1]. Also this nut is one of the rich
sources of beneficial nutrients for human health like phe-
nolic compounds, anthocyanin, antioxidants, vitamins and
minerals [2]. Pistachio nut is one of the most susceptible
commodities to contamination by aflatoxin producing fungi
such as Aspergillus flavus and Aspergillus parasiticus, so
that pistachio contamination with these fungi during pre-
harvest, post-harvest and storage can reduce the quality of
the product and make it unsuitable for consumption and lead
to critical health issues and economic losses [1].
In accordance with the Food and Agriculture Organiza-
tion of United Nations (FAO) about 25% of produced nuts,
especially pistachios, is lost annually due to mycotoxin con-
tamination [3]. Recently, more than 400 compounds have
been identified as mycotoxins in the world that the most
important group among these mycotoxins are aflatoxins,
including AFB1, AFG1, AFB2 and AFG2, and considered
as a serious threat to human and animal health because of
their carcinogenic, teratogenic and mutagenic nature [4].
Among aflatoxins, AFB1, one of the four toxic secondary
metabolites produced by A. flavus and A. parasiticus, has
been ranked in group 1 definite carcinogen to humans by the
International Agency for Research on Cancer (IARC) [5].
According to European Commission regulations the permit-
ted maximum levels of total aflatoxins (TFAs) and AFB1 in
* Mohammad Hojjati
hojjati@asnrukh.ac.ir
1 Department ofFood Science andTechnology, Agricultural
Sciences andNatural Resources University ofKhuzestan,
Ahvaz, Iran
2 Nuclear Agriculture School, Nuclear Science andTechnology
Research Institute (NSTRI), Atomic Energy Organization
ofIran (AEOI), Karaj, Iran
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M.Makari et al.
1 3
pistachio nuts is 10µg/kg and 8µg/kg, respectively [6]. As a
result, it is necessary to use proper post-harvest preservation
methods to prevent fungi growth and production of myco-
toxins in this valuable product.
Irradiation is one of the most effective methods to prevent
the growth of aflatoxin producing fungi during food storage.
In food irradiation, the product is exposed to high energy
photons, including gamma rays, electron beam, and X-rays
[1, 7]. Application of gamma irradiation is preferred over
other radiation methods due to its high penetration capacity,
and the maximum allowable dose for commercial food irra-
diation in many countries is 10kGy [7, 8]. Common applica-
tions of gamma irradiation include pest control, prevention
of sprouting, delay ripening, increase storage time, decrease
storage losses, and improve the food safety by means of pas-
teurization and sterilization [3, 9]. Research have shown that
gamma irradiation can be effectively used as a decontamina-
tion method to reduce fungal and mycotoxin contamination
in various food products including nuts [4, 5, 10, 11].
The aim of this study was to investigate the effect of dif-
ferent doses of gamma irradiation on viable spore popula-
tion of A. flavus, reduction of AFB1 concentration and some
qualitative characteristics of pistachio nut samples.
Materials andmethods
Sample preparation
Pistachio nuts (Pistachia vera L.), cv. Akbari, were sup-
plied from the Iranian Pistachio Research Centre located in
Kerman Province in October of 2019. Unshelled pistachios
kernels were ground and packed in sterile petri dishes, then
stored appropriately in dry condition at 4 ± 1°C and pre-
vented from exposure to direct sunlight, until later analysis.
All chemicals and solutions were purchased mainly from
Merck (Darmstadt, Germany) and Sigma-Aldrich (St. Louis,
MO, US). Aflatoxin B1 from A. flavus (CAS No. 1162-65-
8; MW: 312.227g/mol) was obtained from Sigma-Aldrich
(Darmstadt, Germany).
Decontamination ofpistachio nuts withA. avus
andAflatoxin B1
The aflatoxigenic strain of A. flavus R5 [12] was cultured
on potato dextrose agar (PDA) medium and incubated at
28°C for 7days. Pistachio kernels inoculation with fungal
spores was done by rolling on the culture and ground with
other pistachio kernels. A suspension of 5g of contaminated
pistachio powder in 4.5ml sterile saline solution including
0.5% Tween 80 was prepared and the spore count was per-
formed using a hemocytometer. The population of spores in
pistachio powder was adjusted to (6.18 ± 1.18) × 105 spore/g
pistachio power. Some of the ground pistachios were spiked
with AFB1 (a final concertation of 400ppb). The contami-
nated pistachio powders were stored at petri dishes in proper
condition (at 4 ± 1°C) [11].
Gamma irradiation
Pistachio powder samples packed in sterile petri dishes
were exposed to different doses (0.5, 1, 1.5, 2, 4 and 6kGy)
of gamma ray in a Co60 gamma resource, in triplicates,
using a Gammacell 220 irradiator (MDS Nordion, Ottawa,
Canada) located at the Radiation Applications Research
School, Nuclear Science and Technology Research Institude,
AEOI, Tehran, Iran. The dose rate of gamma irradiation was
5.4kGy/h. The temperature and relative humidity during
irradiation process were 30 ± 1°C and 45% to 55%, respec-
tively. A Red-Perspex dosimeter (Hrwell Dosimeters, UK)
was used to assess the absorbed dose.
Evaluation ofA. avus total count
The spore count was done by preparing a suspension of each
irradiated pistachio powder samples in sterile saline (8.5g
NaCl in 1000ml distilled water) and serially diluted a cer-
tain amount (100µl) of each dilution was cultured in two
plates containing PDA medium (Merck, Germany) (includ-
ing 50ppm chloramphenicol and 0.033g/l of Rose-Bengal)
by surface plating method in triplicates. Fungal colonies
were counted after incubation at 28°C for 3–5days. The
results were expressed as log colony forming units per gram
(log CFU/g) [13].
Extraction ofAflatoxin B1 andHPLC determination
AFB1 was extracted from each 1g pistachio powder sam-
ples by mixing with methanol 80% v/v and shaking properly
(at 150rpm for 24h). The obtained extract was centrifuged
(10,000×g for 5min) and collected supernatant was filtered
and then purified by means of an ASPEC 401 immunoaffinity
column. In order to purify the extract, first 10ml of PBS was
passed through the immunoaffinity column, then loaded with
sample and washed with water (10ml), and the extract was
diluted with acetonitrile (1.5ml). 0.5ml of eluate was col-
lected in a glass vial then diluted with water (2ml). 400ml of
the diluted eluate was injected to the HPLC system equipped
with a Spherisorb Excel ODS1 (250 × 4.6mm; 5µm) column
with a guard column (25 × 4.6mm i.d.) and a Perkin Elmer
LC420 fluorescence detector (364nm excitation and 440nm
emission). The mobile phase was composed of water: metha-
nol: acetonitrile (56:14:30, v/v/v) with the flow rate of 0.86ml/
min. A post- column derivatization was used with a zero dead
volume T-piece and PTFE reaction tube (30cm × 0.3mm i.d.)
and pyridine hydrobromide perbromide (PBPB) reagent was
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Effect ofCo‑60 gamma irradiation onAspergillus flavus, Aflatoxin B1 andqualitative c…
1 3
added at the flow rate of 0.3ml/min. The retention time of
AFB1 was observed at 13.55min [4].
Total phenolic content (TPC) andantioxidant
activity (AOA)
Methanol solution (80%, 10ml) was used to extract one-
gram of each ground pistachio samples. The mixture was
agitated (20min) and sonicated (Sonorex Digitech DT 1028
H, Bandelin, Germany) two times for 15min. The mixture
was placed in the dark at 25°C for 24h. The extract was
centrifuged (5000×g at 4°C for 4min) (Httich Refrigerated
Centrifuge Universal 320R, Germany) and the supernatant
was used to determine total phenolic compounds (TPC) and
antioxidant activity (AOA) using Folin-Ciocalteu reagent
method and DPPH free radical scavenging activity, respec-
tively [14].
Determination ofmalondialdehyde (MDA)
Malondialdehyde determination was done by spectropho-
tometry method [15].About 7g of pistachio nuts powder
was mixed with 15ml of 7.5% Trichloroacetic acid (TCA)
(w/v) [containing 0.1% EDTA (w/v) and 0.1% propylgallate
(w/v)] and homogenized (18,000rpm for 1min) and filtered.
2.5ml of the filtrated and 2.5ml of TCA reagent (46mM)
was transferred to a test tube and heated in boiling bath, then
cooled. Absorbance of the extracts was read at 532nm. The
results were expressed as nano molar malondialdehyde per
gram pistachio powder.
Chlorophylls andtotal carotenoids content
Chlorophylls and carotenoids of pistachio nuts were
extracted by mixing about 0.2g of ground pistachios and
5ml of 80% (v/v) acetone solution. The extract was placed in
the darkness (15min) and centrifuged (1500×g for 15min)
and passed through a filter paper. The filtrated extract was
quantified spectrophotometrically at 470 (A470), 663 (A663)
and 645 (A645) nm. The carotenoids (µg/g), chlorophyll a
(µg/g) and chlorophyll b (µg/g) concentrations were meas-
ured using following equations [16]:
C
a=12.21
(
A663
)
−2.81
(
A645
).
C
b=21.13
(
A645
)
−5.03(A663)
.
C
t=
[
1000
(
A470
)
−3.27Ca−104Cb
]
∕
229
Instrumental color
The color parameters of Pistachio nuts powder samples were
directly determined utilizing a Minolta Colorimeter CR-400
(Konica Minolta, Inc., Osaka, Japan) with a D65 illuminant
as and an observation angle of 10 at 25°C. The color val-
ues of L*, a*, b* and C* or chroma were measured based
on International Commission on Illumination (CIE). L*
value represents lightness-darkness, and a* and b* describe
redness-greenness and yellowness-blueness of color, respec-
tively. C* or chroma is 0 at the center of a color sphere and
increasing according to the distance from the center. The
L*, a* and b*color values measurement was carried out in
five replicates.
Total soluble protein
Total soluble protein determination was carried out using
Bradford method [17]. The pistachio nut protein extract
was prepared by mixing (30min) pistachio powder with
10% Phosphate-buffered saline solution (pH = 6.8, W/V)
with glycerol. Then the mixture was centrifuged (5000×g
for 5min) and the supernatant was collected and stored at
− 20°C for further use. The collected supernatant (100µl)
and Bradford reagent (5ml) were transferred to a test tube
and kept for 30min. The absorbance was measured at
595nm and bovine serum albumin (BSA) was used as the
standard. The results were reported as mg of protein per g
of pistachio nuts (mg/g).
Electrophoretic pattern ofproteins
The electrophoretic pattern of pistachio proteins in SDS-
PAGE was performed according to Laemmli method [18]
under non-reducing and reducing conditions using a Mini
Protean ΙΙ Cell system (Bio-Rad, Hercules, USA). Proteins
precipitation was done with cold acetone and after centri-
fuging (5000×g for 4min at 4°C) the precipitate was col-
lected. For reducing condition, samples were prepared in
sample buffer (65mM Tris, pH 6.8, 10% glycin, 2 SDS,
0.2% bromophenol blue and 5% 2-mercaptoethanol) and
heated in water boiling bath. For the non-reducing condition
β-mercaptoethanol was excluded. 50µg of each sample were
loaded onto the gels containing of 12.5% acrylamide sepa-
rating gel and 5% acrylamide stacking gel. Both gels were
stained overnight with Coomassie Brilliant Blue R-250 and
de-stained in a methanol-acetic acid–water (1:1:8 v/v) mix-
ture until the background appeared clear. The gels images
were captured using Gel Doc XR + and Quantity One 1-D
Analysis software (Bio-Rad, Hercules, USA) and Gel-Pro
Analyzer (ver.6.0) was used to analyze the molecular weight,
relative density, and intensity of the bands.
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M.Makari et al.
1 3
Statistical analysis
Data were statically analyzed by means of a SPSS Software
v.26.0 (SPSS Inc., Chicago, IL, USA) based on a one-way
analysis of variance (ANOVA). Differences were significant
at the p < 0.05 using Duncan’s test. All experiments were
conducted in a completely randomized design and the data
were expressed as the means ± standard deviations of three
replicate experiments (SD).
Results anddiscussion
Effect ofgamma irradiation oninactivation ofA.
avus spores
The effect of different doses of gamma irradiation (0.5,
1, 1.5, 2, 4 and 6kGy) on A. flavus viable spore popula-
tion is shown on Fig.1. According to the results with the
increase of gamma ray dose the spore population signifi-
cantly decreased in all samples (p < 0.05). However, the
viable spore population effectively decreased at doses of 4
and 6kGy by 99.999% (5 log).
Iqbal etal. [4] observed that gamma irradiation at dose
of 6kGy showed a 4–7 log reduction in the viable spore
population of A. flavus in red chilli samples. Aquino etal.
[10] observed that gamma irradiation reduced the viable
spore population of A. flavus on maize. Boonchoo etal. [19]
reported 88% reduction in spore population of A. flavus by
gamma irradiation at dose of 6kGy in brown rice samples.
Irradiation lead to microorganism inactivation through dif-
ferent fatal mechanisms such as DNA damage, cell mem-
brane rupture, and/or damage to the cell wall [8]. The main
inactivation mechanism by ionizing photons is the direct
or indirect damage to DNA affected by free radicals gener-
ated via radiolysis of water, which lead to microorganism
inactivation by inhibiting the cells replicate [20]. However,
fungal spores are more resistant to ionizing radiation in
compare with the bacterial spores because of their very low
DNA content and melanized hyphae [8, 20]. Therefore, the
appropriate irradiation dose for control and inactivation of
microorganisms in food products should be selected accord-
ing to the type and strain, state of microbial development
and population [8].
Effect ofgamma irradiation on AFB1 concentration
The effect of gamma irradiation on AFB1 contaminated pis-
tachio nuts are presented in Table1 and Fig.2. The results
indicated that by increasing gamma irradiation does, the
AFB1 concentration in pistachio samples decreased com-
pared to the control. The maximum reduction in AFB1
concentration was 73.27% and 86.36% and was observed at
doses of 4 ≤ kGy. According to the results, there is a posi-
tive correlation between the rate of AFB1 degradation and
increase in doses of gamma radiation [5]. Similar results
were observed by Iqbal etal. [4], gamma irradiation (2, 4
and 6kGy) showed 86%-98% reduction in AFB1 concen-
tration in whole and ground chillies. In a study Ghanem
etal. [5] investigated the effect of gamma irradiation (4, 6
and 10kGy) on AFB1 in rice, pistachio, peanut, corn and
feed (corn, wheat and wheat bran) and observed that with
increase in doses of irradiation the rate of AFB1 degrada-
tion increased, and stated that there is a negative correlation
between the oil content of food and percentage of AFB1 deg-
radation and the percentage of AFB1 degradation decreases
with the increase of oil percentage in food.
Fig. 1 Decontamination of A. flavus by different dose of gamma radi-
ation on contaminated pistachio nuts. Data are means of three repli-
cates. Different letters indicate significant differences between treat-
ments according to Duncan’s test (p < 0.05)
Table 1 Effect of different doses of gamma irradiation (0.5–6kGy) on aflatoxin B1 concentration (ppb) in contaminated pistachio nuts powder
Data are the mean ± SD. Means with different superscripts letters (a, b, c, d, e, f, g) in the same row are statistically different (p < 0.05) according
to Duncan’s multiple range test
Dose of gamma irradiation (KGy)
0 0.5 1.0 1.5 2.0 4 6
493.11g ± 0.43 488.64f ± 0.88 410.36e ± 0.78 355.11d ± 0.54 284.34c ± 0.74 131.83b ± 0.36 67.25a ± 0.44
Reduction (%) 0 0.91 16.78 27.99 42.34 73.27 86.36
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Effect ofCo‑60 gamma irradiation onAspergillus flavus, Aflatoxin B1 andqualitative c…
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In another study, Sen etal. [11] reported that the AFB1
reduction was 47% in hazelnut samples after gamma irradia-
tion (10kGy, 10min) and expressed that oil content, type of
fatty acids and antioxidant compounds in the treated sam-
ples may affect the efficiency of gamma rays on the toxin.
Jalili etal. [21] reported that the AFB1 concentration showed
50.6%. Reduction in black and white peppers after gamma
irradiation (30kGy, 30% moisture).
Aquino etal. [10] reported that gamma irradiation of
maize samples at doses of 2 and 5kGy reduced 68.9% and
47% of AFB1, respectively. Radiolysis of water and pro-
duction of hydroxyl radicals, ions and atoms of hydrogen
following the irradiation of food and the reaction or addi-
tion of these free radicals to the double bonds in aromatic
and heterocyclic rings or carbon groups of lactone and
furan rings in the structure of aflatoxins can reduce the
Fig. 2 Chromatograms: effect of
different doses of gamma irra-
diation (0–6kGy) on detoxifica-
tion of contaminated pistachio
nuts powder with aflatoxin B1
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M.Makari et al.
1 3
biological activity of these toxins and/or convert them into
less toxic compounds [10, 21]. On the other hand, radioly-
sis of water and formation of free radicals increase with
the increase in doses of gamma irradiation [21].
Total phenolic content (TPC) andantioxidant
activity (AOA)
The results of TPC and AOA of gamma irradiated pistachio
samples are provided in Table2. All of the data were sig-
nificantly different (p < 0.05). The increase in doses of irra-
diation significantly reduced the TPC of pistachio samples
in compared to the control. Radiation doses of 4 and 6kGy
remarkably declined the TPC, while the highest reduction
was observed at dose of 6kGy. The highest level of TPC
was observed at doses of 0.5 and 2kGy. The TPC remained
unchanged at dose of 1.5kGy in compare to the control.
Moreover, gamma irradiation improved the AOA of the
pistachio samples at doses up to 4kGy, but gamma irradia-
tion at dose of 6kGy reduced the AOA of the samples in
compare to the control. The lowest and the highest AOA of
pistachio samples were observed at doses of 2 and 6kGy,
respectively.
Similar to our results, Akbari etal. [2] reported the TPC
of gamma irradiated pistachio nuts remained unchanged at
dose of 1kGy, radiation dose of 2kGy enhanced the TPC
of all pistachio samples, but the TPC of irradiated samples
decreased at dose of 4kGy and the AOA of all samples
increased upon irradiation. Also in a similar study Alinezhad
etal. [16] reported that the levels of TPC in gamma irradi-
ated pistachios decreased at doses up to 2kGy, but it reduced
at doses of 4 and 6kGy and AOA increased at doses up
to 2kGy and then diminished at higher doses of irradia-
tion (≥ 4kGy). The TPC and AOA of different genotypes of
gamma irradiated soybeans enhanced at dose of 2kGy and
the TPC of samples decreased with the increase of irradia-
tion doses up to 5kGy [22]. Kim etal. [23] reported that
the TPC and AOA of the irradiated peaches improved with
the increase of irradiation doses up to 2kGy (0.5, 1, 1.5 and
2kGy). The increment in TPC of pistachio samples at low
doses of irradiation can be because of the increase in the
activity of key enzymes of the phenylpropanoid metabolic
pathway like phenylalanine ammonia lyase [22, 23]. On the
other hand, several phenolic compounds are recognized as
antioxidant agents because of having hydrogen with activ-
ity which leads to the hydrogen exchange reaction with free
radicals and form a resonance-stabilized structure [23].
Therefore, the increase of AOA of pistachio samples by
low doses of gamma irradiation can be due to the release
and increase of phenolic compounds, as well as upregula-
tion the pathway involves in antioxidative defences, forma-
tion and release of free flavonoids due to the degradation
of glycosides by radiation [2, 22]. However, increase in the
formation of free radicals at higher doses of gamma irra-
diation causes adverse effects and reduces the antioxidant
activity [22].
Table 2 The effect of different doses of γ-irradiation on the values of the total phenolic content (TPC), antioxidant activity, pigments, instrumen-
tal color parameters and total soluble protein (TSP) of pistachio nuts
Data are the mean ± SD. Means with different superscripts letters (a, b, c, d) in the same row are statistically different (p < 0.05) according to
Duncan’s multiple range test
Properties Dose of gamma irradiation (kGy)
0 0.5 1 1.5 2 4 6
TPC (mg GAE/g) 1.52ab ± 0.05 1.60a ± 0.06 1.52ab ± 0.06 1.49b ± 0.04 1.61a ± 0.06 1.49b ± 0.05 1.38c ± 0.07
Inhibitory of DPPH
activity (%)
81.80d ± 0.19 90.45a ± 0.45 86.70b ± 0.40 85.54c ± 0.59 91.29a ± 0.19 85.57c ± 0.48 77.02e ± 0.99
MDA (n mol/g) 8.23a ± 0.16 8.61a ± 0.16 8.61a ± 0.16 10.26a ± 0.13 10.06a ± 0.29 12.52a ± 0.39 12.71a ± 0.26
TSP (mg/g) 2.92d ± 0.06 3.29a ± 0.08 3.23a ± 0.05 3.05b ± 0.06 3.13bc ± 0.02 3.00cd ± 0.04 3.07bc ± 0.02
Pigments
Chlorophyll a 23.33a ± 0.90 21.24b ± 1.58 19.21bc ± 1.10 17.89c ± 1.25 17.72c ± 1.14 14.94d ± 1.26 12.43e ± 0.72
Chlorophyll b 22.01a ± 1.87 18.21b ± 2.35 15.39bc ± 2.15 12.27d ± 0.83 12.84cd ± 1.51 11.32de ± 0.50 9.19e ± 1.59
Total chlorophyll 45.34a ± 2.78 39.45b ± 3.92 34.60c ± 2.98 30.16cd ± 1.94 30.56cd ± 2.48 26.26de ± 1.73 21.63e ± 2.31
Total carotenoid 25.66b ± 0.40 26.76a ± 1.09 25.38b ± 0.51 26.01ab ± 0.20 26.01ab ± 0.50 23.31c ± 0.18 22.98c ± 0.24
Instrumental color
Lightness (L) 61.88a ± 0.83 62.05a ± 0.44 61.55a ± 0.32 61.53a ± 0.43 61.02a ± 1.50 60.66a ± 0.66 60.89a ± 0.53
Redness (a*) − 6.44d ± 0.25 − 6.28cd ± 0.22 − 5.85bc ± 0.15 − 6.07cd ± 0.40 − 6.17cd ± 0.22 − 5.24a ± 0.28 − 5.40ab ± 0.21
Yellowness (b*) 32.81a ± 0.31 32.52ab ± 0.12 31.42bc ± 0.48 32.38ab ± 0.43 31.59bc ± 0.69 30.77cd ± 0.34 30.18d ± 1.21
Chroma (C*) 33.44a ± 0.28 − 33.12ab ± 0.13 31.97bc ± 0.50 32.94ab ± 0.49 32.19bc ± 0.69 31.21cd ± 0.37 30.66d ± 1.22
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Effect ofCo‑60 gamma irradiation onAspergillus flavus, Aflatoxin B1 andqualitative c…
1 3
Detemination ofmalondialdehyde
The results of malondialdehyde content of gamma irradi-
ated pistachio nuts are set out in Table2. According to the
results levels of the malondialdehyde in irradiated pistachio
samples significantly increased with the increase in doses of
irradiation (p < 0.05). Malondialdehyde in gamma irradiated
pistachios at doses of 0.5 and 1.5kGy remained unaffected
compared to the control and the highest levels of malon-
dialdehyde was observed in gamma irradiated samples at
doses of 4 and 6kGy. However, irradiation at dose of 6kGy
showed the highest increase in malondialdehyde in compare
with the control.
Mexis and Kontominas [1] reported that the peroxide
value of pistachio nuts and peanuts increased with the
increase in doses of gamma irradiation (1, 1.5, 3, 5 and
kGy). Also in another study increase in thiobarbituric acid
value (TBA) with increasing gamma ray doses was observed
in rice samples [24]. In two separate studies, a significant
increase in peroxide value with increasing gamma irradia-
tion doses in pine nuts (0.5, 1, 3 and 5kGy) [25] and pista-
chios, hazelnuts, almonds and walnuts (1, 3, 5 and 7kGy)
[7] was reported. Boonchoo etal. [19] observed that TBA
value of gamma irradiated brown rice samples increased at
dose of 6kGy. Gamma rays cause lipid oxidation via oxida-
tion, dehydration, decarboxylation and polymerization reac-
tions of fat molecules, on the other hand, hydroxyl radicals
generated in the irradiated food can alter the chemical nature
of fatty acids by initiating lipid oxidation [25]. The oxidation
of unsaturated fatty acids of pistachio nuts such as linoleic
acid and oleic acid to peroxides and convert to carbonyl
compounds such as acetaldehyde, pentanal, propanol and
hexanal can result in a color reaction between these com-
pounds and TBA [24].
Chlorophylls andcarotenoids
Table2 shows the effect of gamma irradiation on chloro-
phylls and total carotenoids of pistachio nuts. All data were
statistically significant (p < 0.05). The results indicated that
the chlorophyll a and b in pistachio samples decreased with
the increase in doses of gamma irradiation. Nevertheless,
the degradation rate of chlorophyll b was higher than chloro-
phyll a. The highest reduction of chlorophyll a was observed
at doses of 4 and 6kGy, which reduced 35.96% and 46.72%
of chlorophyll a, respectively. The lowest amount of chloro-
phyll a was observed at dose of 0.5kGy, which was 96.8%
compared to the control sample. The chlorophyll b consid-
erably decreased with increasing the dose of gamma irra-
diation and this reduction was 48.57% and 58.25% at doses
of 4 and 6kGy, respectively, that the highest reduction of
chlorophyll b was at dose of 6kGy compare to the control.
Gamma irradiation at dose of 0.5kGy with 17.2% reduction
in chlorophyll b showed the lowest reduction compare to
the control. Total chlorophyll also decreased significantly
with increasing gamma irradiation dose (Table2). Gamma
irradiation of pistachio nuts at doses of 4 and 6kGy reduced
total chlorophyll by 42.08% and 52.29% compared to the
control sample. The highest amount of total chlorophyll
was at dose of 0.5kGy and was 12.99% compare to the
control. In accordance with Table2 the increase in doses of
gamma irradiation declined the amount of total carotenoids
compared to the control sample. The highest reduction in
total carotenoids was 9.16% and 10.44% compare to the con-
trol, which was observed in irradiated pistachio samples at
doses of 4 and 6kGy, respectively. The carotenoid content
in irradiated pistachio samples was not significant at doses
up to 2kGy compare to the control. Ramamurthy etal. [26]
observed that increasing gamma irradiation dose (1, 2 and
3kGy) reduced the chlorophyll content in capsicum samples.
Byun etal. [27] reported that gamma irradiation (20kGy)
can degrade the chlorophyll b and reduce its content. In a
study Kyung etal. [28] reported that the reduction in chloro-
phyll b is because of the selective devastation of chlorophyll
b biosynthesis or the degradation of its precursors.
Instrumental color
The L*, a* and b*color parameters of irradiated and non-
irradiated pistachio nuts are presented in Table2. All of the
data were statistically significant (p < 0.05). According to
the results, gamma irradiation at doses up to 6kGy had no
effect on L* parameter or lightness of pistachio nut samples
and did not changed the L* parameter values compare to
the control sample. On the other hand, a* parameter that
represent the red color increased slightly after irradiation
at doses of 4 and 6kGy and tended to move towards the
red spectrum. The b* parameter decreased at doses higher
than 4kGy compared to the control sample and tended to
move towards blue spectrum. The C* parameter or chroma
decreased at gamma irradiation doses higher than 1kGy,
so that the highest reduction was observed at dose of 6kGy
compare to the control. These changes in color parameters
after gamma irradiation resulted in darkening of the color
of pistachio samples. The reason for the darkening of pis-
tachios can be explain by the increase in the destruction of
glycoside and peptidic bonds during irradiation, which leads
to maillard reaction and formation of color compounds by
producing products such as carbonyl and amino compounds
and their reactions [1]. Similar to the results, Alinezhad etal.
[16] reported darkening of pistachio samples with increas-
ing the dose of gamma irradiation (1, 1.5, 2, 4 and 6kGy).
Mexis and Kontominas [1] observed that after gamma irra-
diation the L* and b* parameters decreased at irradiation
doses higher than 5kGy and a* parameter increased and
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

M.Makari et al.
1 3
led to darker pistachio nuts. Sánchez-bel etal. [3] reported
that the darkness of pistachio samples gradually increased
with increasing gamma radiation dose (1–10kGy). Gölge
and Ova [25] reported that increase in gamma irradiation
dose led to darker color of pine nuts. Boonchoo etal. [19]
reported gamma irradiation at dose of 6kGy resulted in
yellowing of brown rice samples due to a non-enzymatic
browning reaction.
Soluble protein
The effect of gamma irradiation on total soluble protein of
pistachio nuts is summarized on Table2. Results suggested
that gamma irradiation slightly enhanced the protein solu-
bility of pistachio samples (p < 0.05), so that the highest
amount of solubility was observed at dose of 0.5kGy and
the lowest amount of solubility was at dose of 4kGy. In gen-
eral, there were no significant differences between gamma
irradiated samples. In contrast to our findings, in a similar
study Alinezhad etal. [16] observed that total protein solu-
bility of pistachio nut reached to its maximum reduction
with increasing the dose of gamma irradiation (1 < kGy).
Afify etal.[29]reported that the protein solubility of pea-
nut, soybean, and sesame was affected by gamma irradiation
and decreased. They alsostated that radiation, as a factor
in the degradation and aggregation of proteins could be the
reason for the decrease in solubility of the protein. Gamma
irradiation reduces the solubility via direct the disintegration
of proteins, improving the activity of lysosomal enzymes
or increasing disulfide bonds and rearrangement of the low
molecular weight proteins into high molecular weight pro-
teins and aggregate protein [29]. Noorbakhsh etal. [30]
observed that the solubility of proteins in steam-roasted
pistachio samples decrease and explained that structural
and chemical protein modifications can reduce the solubility
of protein. On the other hand, gamma irradiation increases
hydrophobic interactions and decreases protein solubility by
cross-linking and aggregating protein chains [31].
Electrophoretic pattern ofproteins
The effect of different doses of gamma irradiation on pis-
tachio soluble protein pattern are illustrated in Figs.3 and
4. Protein bands in non-reducing and reducing conditions
were in the molecular weight range from 3 to 272 and
1 to 132kDa respectively. According to the results the
reducing condition (containing beta-mercaptoethanol)
caused breakdown of disulfide bonds and separation of
protein subunits and formation of low molecular weight
proteins. While the proteins remained unchanged under
non-reducing condition. The results obtained from both
gels in non-reducing and reducing conditions showed a
conversion in the pattern of the pistachio kernel proteins
Fig. 3 Profiles of proteins extracted from non-irradiated (lane 1) and
γ-irradiated pistachios (lane numbers of 2, 3, 4, 5, 6 and 7 for 0.5, 1,
1.5, 2, 4, and 6kGy, respectively) under non-reducing and reducing
conditions. “M” indicates a molecular weight marker (SinaClon Bio-
Science, Prestained protein ladder, PR901641)
Fig. 4 Densitometry analysis of the proteins present in SDS-PAGE
profile of soluble proteins extracted from non-irradiated (0.0 kGy)
and irradiated (1.0–6.0 kGy) pistachio nuts in non-reducing (a) and
reducing conditions (b)
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Effect ofCo‑60 gamma irradiation onAspergillus flavus, Aflatoxin B1 andqualitative c…
1 3
under the influence of different doses of gamma irradia-
tion. Therefore, the changes in pattern of the proteins or
protein–protein interactions due to gamma irradiation can
be understood by comparing the results. According to the
analysis of the densitometry in non-reducing condition,
the intensity of protein bands increased with increasing
the gamma irradiation doses up to 2kGy, but irradiation
at doses of 4 and 6kGy reduced the intensity of protein
bands in the molecular weight of 43 to 50kDa, so that the
lowest intensity was achieved at dose of 4kGy (Fig.4).
Alinezhad etal. [16] reported that gamma irradiation
altered the electrophoretic pattern of soluble proteins
of pistachio nuts, so that gamma irradiation doses up to
6kGy reduced the intensity of protein bands in all sam-
ples, while gamma irradiation at dose of 4kGy led to a
reduction in protein bands in molecular weight of 7kDa.
In another study Naei etal. [32] reported that gamma irra-
diation (1, 10 and 100kGy) drastically changed the pattern
and intensity of proteins extracted from pistachio samples
and caused reduction or absence of some protein bands.
Krishnan etal. [31] observed the formation of new bands
and disappearance of some of the protein bands related
to two varieties of soybeans after gamma irradiation. The
appearance ordisappearance of these bands could be due
to themodification of the physicochemical characteristics
of proteins such as oxidation, which causes condensation,
polymerization or aggregation of proteins and reduces the
solubility of the proteins.
Conclusion
The results of this study showed that gamma irradiation at
doses of 4 and 6kGy significantly reduced the viable spore
population of A. flavus (5 log) in pistachio samples and the
AFB1 reduction in these absorbed doses was 73.27% and
86.36%, respectively. The TPC was increased at doses up to
2kGy and decreased with the increase in irradiation dose
(4 ≤ kGy). Increasing gamma irradiation doses up to 4kGy
enhanced the AOA of the samples, but it declined at dose of
6kGy. Lipid oxidation remarkably increased with increasing
gamma irradiation dose and the highest levels of malondi-
aldehyde was observed at dose of 6kGy. The increase in
irradiation dose decreased the chlorophyll a and b content
and by affecting the color parameters resulted in darker color
of pistachios. Solubility of proteins slightly increased with
the increase in irradiation dose and changed the pattern and
intensity of proteins bands. In accordance with the obtained
results changes in spore population, AFB1 concentration and
quality characteristics of pistachio nuts were dose depend-
ent. Gamma irradiation at doses of 4 and 6 considerably
reduced the microbial contamination and toxin concentration
and lower doses effectively maintained the quality charac-
teristics of pistachio nuts.
Acknowledgements We are grateful to the respected authorities of
Agricultural Sciences and Natural Resources University of Khuzestan
for supporting this project.
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