International Journal of Agriculture and Crop Sciences.
Available online at www.ijagcs.com
IJACS/2013/6-17/1199-1202
ISSN 2227-670X ©2013 IJACS Journal
Determination of AFB1 in Peanut, Almond, Walnut,
and Hazelnut in Kermanshah Markets, Iran
Amiri MJ1, Karami M2, Sadeghi E3*
1. Department of chemical engineering- Food Sciences, Kermanshah Science and Research branch, Islamic
Azad University, Kermanshah, Iran.
2. Assistant Professor, BuAli Sina University, Faculty of Food Science & Technology, Hamedan, Iran.
3. Assistant Professor, Research Center for Environmental Determinants of Health (RCEDH), Kermanshah
University of Medical Sciences, Kermanshah, Iran.
*Corresponding author email: [email protected]
ABSTRACT: Aflatoxins are toxic and carcinogenic substance produced by certain strains of the
molds Aspergillus flavus, Aspergillus parasitic us and the rare Aspergillus nomius. There are four
principle types of Aflatoxin: B1, B2, G1 and G2 in food stuff. Aflatoxin B1 is the most frequently and
most toxic. To determine the Aflatoxin B1 contamination status in for nuts including Peanut, Almond,
Walnut and Hazelnut, 80 samples were collected in five region in Kermanshah, Iran in 2013, then
utilized for Aflatoxin B1 testing using a determination scheme consisting of enzyme-linked immune
sorbent assay (ELISA). The result showed contamination in two samples of roasted Peanut in
comparing with EU standard (2.5% of total samples). The highest amount of Aflatoxin B1 were found
in this Peanut samples (15.744 µg/kg, 7.776 µg/kg). The interval range for Aflatoxin B1 in Peanuts
were from 0.048µg/kg up to 15.744 µg/kg , for Almonds from 0.016 µg/kg up to 0.696, for Walnuts
from 0. 632 µg/kg up to 1.704 µg/kg and for Hazelnuts from 0.040 µg/kg up to 0.736 µg/kg. The best
average were for Almonds(0.26 µg/kg) followed by Hazelnuts (0.423 µg/kg) and then Walnuts (1.178
µg/kg) and Peanuts (1.608 µg/kg). There was no difference detected between roasted and raw
samples of Almond and Hazelnut and also content of Aflatoxin B1 in tree nuts samples did not reach
to maximum tolerable limit for Aflatoxin B1 in EU standard (p>0.05). Thus in future is recommended
systematic and continues monitoring system for nuts.
Keywords: Aflatoxin B1, Nuts, Aspergillus, ELISA,Iran.
INTRODUCTION
Nowadays Aflatoxins are known as carcinogen and toxic metabolites. Among the four Aflatoxin
categories, Aflatoxin B1 (AFB1) is the most frequent and toxic metabolite in contaminated samples such as nut
and nutty products. Aflatoxin B2 (AFB2), G1 (AFG1) and G2 (AFG2) are commonly not found in the absence of
AFB1 (Sadeghi et al., 2013; Sook et al., 2007). AFs bearable limit are regulated in more than 75 countries.
Currently, the worldwide tolerable limits for AFB1 and total AFs are 1–20 µg/kg 0–35 µg/kg, respectively (FAO,
2004). European union Commission regulations set limits for AFB1 and total AFs of 2ppb and 4ppb, respectively
in groundnuts, nuts, dried fruit and cereals since1998 (Commission Directive, 1998; Commission
Regulation,2001; Leonga et al., 2010). The Codex Alimentarius Commission (CAC) Joint Food and Agricultural
Organization of the United Nations and the World Health Organization food standards program determine a
level of 15 mg/kg for AFs for unprocessed peanuts and 10 mg/kg for ready-to-eat tree nuts (CAC, 2001). AFS
produced by Aspergillus flavus, which produce only Aflatoxins B, Aspergillus parasiticus which produces both
Aflatoxins B and G and the rare Aspergillus nomius. Liver is the main attacking organ for AFB1 toxicity and
carcinogenicity (Ding et al., 2012). Aflatoxins M1 and M2 are oxidative metabolic products of AFB1 and B2
produced by mammals and are exist in milk and other excretion (Aycicek et al., 2005; Sadeghi et al., 2013).
Lack of technology is one of important parameter in AFB1 content because of mechanical damage during
harvest (Mutegi et al., 2009). Because of moisture is the most influent parameters that affect fungal growth and
mycotoxin production, drying should take place very soon after harvest and as rapidly as possible (Reis et al.,
2012). A group of scientists showed that the Aflatoxin concentrations increased when aw of the kernels
decreased, and the highest concentration was at aw=0.71 (Gonçalez et al., 2008). Aspergillus parasiticus occurs
rather commonly in peanuts, but is apparently quite rare in other foods and is more restricted geographically as
compared to Aspergillus flavus (Frisvad et al., 2007; Luttfullah and Hussain., 2011). Fungal growth on nuts not
only produce mycotoxin product but also can decrease quality of nuts and found decrease in nutritive quality of
Intl J Agri Crop Sci. Vol., 6 (17), 1199-1202, 2013
walnut kernel due to fungal infection(Singh& Shukla, 2008). AFB1 is classified by the International Agency of
Research on Cancer (IARC) as human carcinogen (group 1) (IARC, 1993) with a role in etiology of liver cancer,
notably among subjects who are carriers of hepatitis B virus surface antigens (IARC, 2002) (R. Imperato et al
2013). The United Nations-Food and Agriculture Organization (FAO) as well as the World Health Organization
(WHO) were faced with the dilemma of setting the limits for aflatoxin in human foods against high background
levels of malnutrition. (Kamika & Takoy, 2011; Moss, 1996). Saleemullah found that the aflatoxin contents of
both cereals and nuts were significantly (p<0.05) affected by storage period (Saleemullah et al., 2006).
However, this four nuts have never been tested for Aflatoxin B1 in west of Iran in 2013. The aim of this study
was to determine prevalence and levels of AFB1 in four nuts which most consumed by people in Kermanshah,
Iran and comparing the result with worldwide standards.
MATERIAL AND METHODS
Sampling
80 samples consist of different types of nuts were purchased randomly from retail and wholesale shop
among five districts of Kermanshah. The retail shops were randomly selected in the most populated areas in the
city of Kermanshah. The shop types ranged from wholesale to supermarkets. The samples were commonly the
most consumed nuts including: roasted hazelnut (10 sample), raw hazelnut (9 sample), roasted hazelnut in shell
(1sample), raw walnut (20 sample), raw almond (10 sample), roasted almond (10 sample), raw peanut (2
sample), roasted peanut (17 sample), roasted peanut in shell (1sample). The samples were mostly packed as
100 g weight. Samples were purchased and were ground and thoroughly mixed using blender. Hulled peanuts
and hazelnut were de hulled before grinding. Until analysis all of the samples were placed in Zip pack
containers and stored at -20 Ċ.
Enzyme-linked immune sorbent assay (ELISA) analysis
Samples were analyzed for AFB1 by using the Quantitative Aflatoxin Test Kit (EuroProxima, Netherland)
according to the protocol of the manufacturer. Multiskan Microplate Reader (Multiskan, labsystem) was used to
read the absorbance at 450 nm. 3 g of ground sample was extracted using 9ml of 80% methanol (Merck,
Germany) in a high speed blender for 10 min. Then the mash was filtrated with filter paper (Whatman No. 1,
England). Placed the standards solution (wells A1, B1 to G1: Zero, 0.0313, 0.0625, 0.125, 0.5 and 1.0ng/ml) in
micro titer plate. 150 µl of dilution buffer and 50 µl of filtrate were mixed together and 50 micro liter of mixture
placed into the each mixing well of the micro titer plate (H1 to G11). Added 25 micro liter of conjugate (AflatoxinHRP) and 25 micro liter of antibody solution to all wells. After mixing for a few second incubated the wells for
one hour in the dark at 37Ċ. The contents were emptied out and washed 3 times with rinsing buffer. After
removing the rinsing buffer, 100 micro liter of substrate was added to each well and incubated for 30 min at
room temperature (20Ċ -25Ċ) . At last step, 100 micro liter of stop solution was added to each well and mixed
prior to absorbance reading at 450nm for a few seconds.
Precautions in contact with Aflatoxins
Utilize of standards and solutions done in fume hood and worn suitable protective clothes (laboratory
coat, mask and gloves). All of the laboratory instruments pre washed with 10% sodium hypochlorite before
cleaning or discarding and after use. Laboratory surfaces cleaned by 1% sodium hypochlorite if accidental spills
(Leonga et al., 2010). Aflatoxin standard solutions and associated analytical work were protected from daylight
(Bacaloni et al., 2008). AFs are deactivated by autoclaving in the presence of ammonium and by treatment with
hypochlorite (World Health Organization, 1979; Nakai et al., 2008).
RESULTS
AFB1 contamination in the nuts (Peanut, Almond, Walnut and Hazelnut) is shown in Table 1. Two
samples of Peanut were more than 2 ppb [the maximum permitted levels of AFB1 contained in European Union]
(Commission Regulation, 2001). The highest amounts of Aflatoxin B1 were found in peanut samples (15.8µg/kg,
7.8µg/kg). The interval range for Aflatoxin B1 in Peanuts were from 0.048µg/kg up to 15.8 µg/kg , for Almonds
from 0.000 µg/kg up to 0.692 ,for Walnuts from 0.0632µg/kg up to 1.704µg/kg and for Hazelnuts from 0.006
µg/kg up to 0.74 µg/kg . The lowest mean of AFB1were found in Almonds (0.260 µg/kg), Hazelnuts (0.390
µg/kg) Walnuts (1.200 µg/kg) and Peanuts (1.610 µg/kg) and shown in Figure 1.
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Intl J Agri Crop Sci. Vol., 6 (17), 1199-1202, 2013
Table 1. AFB1 levels in Peanut, Almond, Walnut and Hazelnut
Samples
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Peanut(Mean±STD)
0.112±3.709
0.488±3.709
15.744±3.709
0.792±3.709
0.048±3.709
0.200±3.709
0.464±3.709
0.584±3.709
0.360±3.709
0.672±3.709
0.384±3.709
0.528±3.709
0.688±3.709
0.496±3.709
0.504±3.709
0.608±3.709
0.584±3.709
0.552±3.709
7.776±3.709
0.584±3.709
Almond(Mean±STD)
0.056±0.187
0.312±0.187
0.152±0.187
0.144±0.187
0.472±0.187
0.136±0.187
0.032±0.187
0.016±0.187
0.040±0.187
0.088±0.187
0.480±0.187
0.408±0.187
0.312±0.187
0.304±0.187
0.480±0.187
0.360±0.187
0.696±0.187
0.160±0.187
0.296±0.187
0.376±0.187
Hazelnut(Mean±STD)
0.504±0.188
0.280±0.188
0.040±0.188
0.296±0.188
0.504±0.188
0.096±0.188
0.296±0.188
0.600±0.188
0.112±0.188
0.384±0.188
0.552±0.188
0.576±0.188
0.320±0.188
0.472±0.188
0.488±0.188
0.528±0.188
0.496±0.188
0.600±0.188
0.576±0.188
0.736±0.188
Walnut(Mean±STD)
1.368±0.275
1.152±0.275
0.952±0.275
0.920±0.275
0.728±0.275
1.368±0.275
1.560±0.275
0.944±0.275
0.632±0.275
1.064±0.275
0.904±0.275
1.152±0.275
1.704±0.275
1.272±0.275
1.528±0.275
1.344±0.275
1.216±0.275
1.248±0.275
1.200±0.275
1.296±0.275
2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
1.608
1.178
0.423
EU STD
peanut
walnut
Hazelnut
0.266
Almond
Figure 1. The mean of AFB1 in peanut, Almond, Walnut and Hazelnut in compare of EU Standard.
There was no difference in comparing four nuts AFB1 levels (p>0.05). Also there was no difference in
comparing roasted and raw Almond and Hazelnut samples together (p>0.05).
The difference between raw and roasted almond samples, raw and roasted hazelnut samples were significant
(p<0.05).
AFB1 levels in peanut sample were not confirmed by EU standard, whereas confirmed by Iran standard (5ppb).
AFB1 levels in Almond, Walnut and Hazelnut were found safe as EU and Iran standard.
DISCUSSION
AFB1 levels range in tree nuts: Almond, Walnut and Hazelnut, found: 0.00 - 1.704ppb, and the range for
Peanuts, were: 0.048-15.776 ppb, which had a low prevalence: 10%. AFB1 levels in all of the samples were
confirmed by national standard of Iran (Max AFB1=5ppb) and all of samples except Peanut could pass the EU
standard (Max AFB1=2ppb). These results showed that roasting didn’t have any effect on AFB1 content in nuts
and approved the lasts result by (Midio et al., 2011; Nakai et al., 2008). Sook analyzed AFB1 levels in 85 nut
samples and found 9 samples (10.6%) were contaminated and 2 Peanut samples contained over AFB1 standard
in South Korea: 10 ppb (Sook et al., 2007) that is similar to the result of our study. In investigation in Malaysia
the highest level of AFB1 incidence found in raw groundnut without shell, with a total concentration of 711 ppb,
respectively which is higher than the result of our study. Walnut showed lowest AFB1 contamination (Leong et
al., 2010). In china AFB1 was detected in 25% of the nut samples, with the range 0.01 to 720 ppb (Ding et al.,
2012) which is higher than the result of present study. In Turkey reported that AFB1 contamination was detected
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Intl J Agri Crop Sci. Vol., 6 (17), 1199-1202, 2013
in 43 sample (84.32%) of de hulled hazelnut samples: ranging from <1 to 10 ppb and in 38 samples (95%) of
cacao hazelnut cream: ranging from <1 to 13 ppb, (Aycicek et al., 2005) which is higher than the result of our
study. In other investigation Aflatoxins B1 were analyzed, in a 603 samples of peanuts, pistachios, dried figs,
sweet corn, breakfast cereals, corn snacks, red pepper, free-gluten food, ethnic food and baby food. The
highest percentages of positive samples were found in red pepper, pistachios and peanuts: 57%, 20% and
11%, respectively (Cano-Sancho et al., 2013) that is similar to the result of our study.
CONCLUSIONS
In this study, the incidence AFB1 levels in peanut, almond, walnut and hazelnut from five districts in
Kermanshah-Iran was investigated. This study provides a view about the potential risk of AFB1 hazard in
different kinds of nuts. Now the risk of AFB1 is not high in Kermanshah but the high carcinogenic of AFB1
approve the need for regular monitoring and more applied food quality control system in order to control the
AFB1 at the lowest possible levels. Additionally, advanced and faster analytical methods will be essential to
achieve this aim. We recommend this study should be repeated on all seasons of year. Thus in future is
recommended systematic and continues monitoring system for nuts.
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