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Mukhtar et al., J. Anim. Plant Sci. 26(2):2016
542
DETERMINATION OF AFLATOXINS IN SUPER KERNEL RICE TYPES CONSUMED IN
DIFFERENT REGIONS OF PUNJAB, PAKISTAN
H. Mukhtar, Z. Farooq and M. Manzoor
Department of Food Science and Human Nutrition, University of Veterinary and Animal Sciences, Lahore.
Corresponding Author E-mail: zubzair.farooq@uvas.edu.pk
ABSTRACT
A study was conducted to check the aflatoxins (B1, B2, G1& G2) in super kernel basmati rice types consumed in different
regions (Lahore, Narowal, Faisalabad and Multan) of Punjab, Pakistan. For that purpose, 48 samples of super kernel
basmati rice types (white, brown, parboiled and broken) were collected from local markets of Lahore, Narowal,
Faisalabad and Multan. Moisture contents and aflatoxin levels of the collected samples were determined. Aflatoxins
detection in rice samples were done by HPLC technique. The results showed the presence of aflatoxins in rice samples.
For the total (48) rice samples, 58% were contaminated with total aflatoxins out of which 56% were contaminated with
aflatoxin B1and 33% with aflatoxin B2. Out of 58% of the contaminated rice samples with total aflatoxins, 7% samples
had aflatoxin level lower than the maximum tolerable limit of 4µg/kg and 93% samples were contaminated with total
aflatoxin above the maximum tolerable limit. The 56% contaminated samples with aflatoxin B1, showed 100%
contamination above the maximum tolerable limit (4µg/kg). A positive relationship was also seen between the moisture
content and presence of aflatoxins (B1and B2). The study showed significant (P<0.05) results and it could add to various
approaches leading to aflatoxin management in rice types.
Key words: Rice, Aflatoxins, Moisture content, HPLC, Food safety.
INTRODUCTION
In Pakistan, rice (Oryza sativa) is an important
cereal crop used as staple food after wheat. Basmati
variety of rice is well known globally for its delicious
taste and aroma (Ahmad et al. 2008). Area wise, it is the
3rd largest cultivated cereal crop of Pakistan after wheat
and cotton. Pakistan is the chief exporter of rice as; it is
the major cash crop contributing 6.10% of total value
added in agriculture and 1.3% to the GDP (Iqbal. 2012).
Provincial involvement in production of rice in
percentage is; Punjab 56%, Sindh 34%, Baluchistan 8%
and NWFP 2% wherein Punjab is at the leading position
(Khan & Khan. 2010).
Rice is the primary source of carbohydrate,
protein, fiber, some vitamins like the B complex & E and
minerals. The 75% of the weight of rice grain comprises
of starch, the major carbohydrate (Vaughan and Geissler.
2009). Although the nutritional value of rice is very good
but despite all this the problem is the toxin production
due to fungal attack on crops or during storage. Rice is
not a favorable commodity for growth of Aspergillus and
aflatoxin contamination under normal conditions, but
high humidity and heavy rains could enhance the capacity
of rice grains for risk to aflatoxin contamination (Siruguri
et al. 2012). Aflatoxins are produced as by product
during the growth of fungi, Apergillus flavus and
Aspergillus parasiticus. They are toxic substances
produced by fungi of the genus Aspergillus which grow
on cereals and other agricultural crops (National
Toxicology Program. 2011). When aflatoxins once
produced it is very difficult to get rid of them because
they are chemically stable in nature and extremely
resistant to degradation procedure by normal cooking
methods (Ramesh. 2013). Fungal proliferation depends
upon environmental favorable conditions like, high
humidity and temperature (Choudhary and Kumari.
2010). The permitted limits in cereal grains set by
European Union Commission Regulation (EC) No.
1881/2006 for total aflatoxins are 4.0 µg/kg and for
aflatoxin B1is 2.0 µg/kg (Journal of the European Union.
2006). Aflatoxins adversely affect the human health.
Aflatoxicosis is a disease caused by the aflatoxins.
Sudden death occurs as a result of acute aflatoxicosis and
in chronic aflatoxicosis the prolonged pathological
changes like cancer and immune suppression happens
(Magnussen and Parsi. 2013). Aflatoxin is well known
agent for its hepatocarcinogenic properties. The risk of
liver cancer is almost 30 times higher in the subjects
exposed to aflatoxin than unexposed ones (Liu and Wu.
2010). In order to control aflatoxins in rice, there is a
need to determine and quantify aflatoxin levels for
making a comparison with the permissible levels set by
the food regulatory authorities in a pursuit to ensure safe
food supply. The present study was therefore planned to
determine the aflatoxins levels in different types of super
kernel basmati rice consumed in different regions of the
Punjab, Pakistan. Furthermore, it was aimed to evaluate
the frequency of occurrence of various types of aflatoxins
The Journal of Animal & Plant Sciences, 26(2): 2016, Page: 542-548
ISSN: 1018-7081
Mukhtar et al., J. Anim. Plant Sci. 26(2):2016
543
in rice species/varieties grown in various regions of the
Punjab, Pakistan.
MATERIALS AND METHODS
Sample Collection: Samples of super kernel basmati rice
grains were procured by simple random sampling
technique from local market of Lahore, Narowal,
Faisalabad and Multan districts of Punjab. Total 48
samples of super kernel basmati rice (white, brown,
parboiled and broken), 12 samples of each type were
included in the study. These samples were stored in
appropriate plastic bags at room temperature to prevent
from moisture and other impurities.
Moisture Analysis: Samples of rice were analyzed for
Moisture by AOAC (2000) procedure.
Aflatoxins determination by HPLC:
Chemicals and regents: Standard solutions of 50μg/ml
of aflatoxins were purchased from Sigma-Aldrich (St.
Louis, Mo., USA). MycoSep column 226 (AflaZone) was
purchased from Romer Labs (Union, Mo., USA). HPLC
grade methanol and acetonitrile were purchased from
Merck (Darmstadt, Germany) and trifluoroacetic acid
(TFA) was obtained from Sigma-Aldrich (St. Louis, Mo.,
USA). All other chemicals and organic solvents were of
analytical grade.
Extraction and purification: Extraction and purification
of samples were carried out using a slightly modified
method of Iqbal et al. (2010). The rice samples were
ground to uniform consistency. Samples (25g) were
extracted with 100 ml of acetonitrile/water (80:20 v/v)
and 5 gram of sodium chloride (NaCl) by shaking for 60
min at 50 rpm at room temperature in 250 ml glass flasks
fitted with a stoppers. The solutions were filtered through
Whatman No.5 papers. To 9 ml portions of the filtrates,
70μl acetic acid was added; the mixture was then
transferred to MycoSep columns (product code 226) and
passed through at a flow rate of 2 ml/min. The aflatoxins
passed through the column. 2 ml portion of each eluate
was taken and evaporated to dryness by nitrogen
evaporator in a centrifuge glass tube. After drying the
mixture 200µl n-hexane was added and vortex mix the
sample for 1-2 min. Then 50µl trifluoro acetic acid was
added and mixed by vortex mixer. 1.95 ml of water and
acetonitrile (1:0.95) were added and mixed again. 1 ml of
sample filtrate obtained from syringe filter was collected
in HPLC vials. A 20μl portion of the sample was
subjected to HPLC analysis.
Mobile Phase: The mobile phase was acetonitrile/
methanol/ water (20:20:60 v/v/v), which was degassed by
sonication. The HPLC (Shimadzu, Kyoto, Japan) was
fitted with a Supelco C18 Column (Discovery HS) with a
fluorescence detector (RF-530). Excitation and emission
wavelengths were 360 nm and 440 nm, respectively. The
flow rate was 1 ml/min and the column was maintained at
40°C. The injection volume was 20μl.
Statistical analysis: Data obtained by estimating
aflatoxins analysis was analyzed by using SPSS software
version 16. For comparing groups two way Analysis of
variance (ANOVA) was used. Correlation analysis was
done to see the relationship between parameters.
Completely randomized design (CRD) was applied on the
data to assess the significance level. P-value < 0.05 was
taken as significant.
RESULTS
Moisture Content: Mean moisture content (percentages)
of the rice samples obtained from four Districts of the
Punjab ranged from 9.5±0.92 to 10.2±0.72, 9.8±0.27 to
10.5±0.98, 9.7±0.20 to 9.8±0.26 and 10.1±0.38 to
10.7±1.16 for white, brown, parboiled and broken rice
respectively (Table No.1).
Table 1. Moisture content of different types of super kernel basmati rice
Moisture Mean (%)
White Rice
Brown Rice
Parboiled Rice
Broken Rice
Lahore
9.5 ± 0.92
10.5± 0.98
9.8±0.26
10.7±1.16
Narowal
9.9 ± 0.61
9.9± 0.60
9.8±0.20
10.1±0.38
Faisalabad
10.2 ± 0.72
10.0±0.45
9.8±0.26
10.4±0.68
Multan
9.6 ± 0.15
9.8±0.27
9.7±0.20
10.5±0.50
Aflatoxins Content: Aflatoxins (B1, B2, G1and G2) in
different types of super kernel basmati rice were
determined by using high performance liquid
chromatography (HPLC) separation technique. Aflatoxin
B1was the most commonly occurring toxin in all types of
super kernel basmati rice followed by aflatoxin B2which
was the second common toxin present in super kernel
basmati rice types examined under this study.
Out of 48 samples of different types of super
kernel basmati rice 28 (58%) were contaminated with
total aflatoxins, of these 27 (56%) were contaminated
with aflatoxin B1and 16 (33%) were contaminated with
aflatoxin B2. Out of 28 (58%) of the contaminated rice
Mukhtar et al., J. Anim. Plant Sci. 26(2):2016
544
samples with total aflatoxins the 2 (7%) samples had
aflatoxin level lower than the maximum tolerable limit of
4µg/kg and 26 (93%) samples were contaminated with
total aflatoxin above the maximum tolerable limit.
Similarly, out of 27 (56%) contaminated samples with
aflatoxin B1; all samples were contaminated with
aflatoxin B1above the maximum limit as it shown in
Table No. 2.
Most of the samples contaminated by aflatoxins
were found to fall in 0-20 µg/kg range, some in 21-40
µg/kg, 41-60 µg/kg and 3 samples of each were
contaminated by total aflatoxins whereas aflatoxin B1
was found in the range >60 µg/kg range (Table. No. 3).
Table 2. Contamination of Aflatoxins detected in various rice samples
Aflatoxins
No. of samples
Positive
samples
Contamination
detected (%)
Upper limit
Lower limit
Total Aflatoxin
48
28
58
26
2
B1
48
27
56
27
0
B2
48
16
33
-
-
G1, G2
48
ND
-
-
-
ND-not determined
Table 3. Aflatoxins range in different types of super kernel basmati rice
Aflatoxins range (µg/kg)
0-20
21-40
41-60
>60
Both Aflatoxins (B1+B2)
18
4
2
3
Aflatoxin B1
18
5
1
3
Aflatoxin B2
16
ND
ND
ND
ND-not determined
Total contamination of aflatoxins in white rice
from different regions (Lahore, Narowal, Faisalabad and
Multan) of Punjab was found 41.7%, in brown, parboiled
and broken rice the aflatoxin presence was 75%, 25% and
100% respectively. The mean for total aflatoxins from all
the four regions of Punjab for white rice was 8.24%, for
brown, parboiled and broken rice; it was 17.8%, 2.2%
and 27.42% respectively (Table no. 4).
In super kernel basmati white rice from the
regions of Lahore, Narowal, Faisalabad and Multan, the
aflatoxin B1was detected in a range of 32.16µg/kg to
8.0µg/kg. Almost equal proportion achieved from each
region. The aflatoxin B2was present only in one sample
from Faisalabad at a level of 20µg/kg (Figure No. 1).
Aflatoxin B1presence was seen in super kernel basmati
brown rice in a range of 80.04µg/kg to 8.0µg/kg equally
from all the regions. The aflatoxin B2contamination was
present in a range of 2µg/kg to 8µg/kg (Figure No. 2)
Table 4. Total Aflatoxins contamination in rice samples from different regions.
Rice type
Region
No. of samples
Contaminated samples (%)
Mean ± SD
White
Lahore
3
41.7
8.24 ± 7.0
Narowal
3
Faisalabad
3
Multan
3
Brown
Lahore
3
75
17.8 ± 10.3
Narowal
3
Faisalabad
3
Multan
3
Parboiled
Lahore
3
25
2.2 ± 1.4
Narowal
3
Faisalabad
3
Multan
3
Broken
Lahore
3
100
27.42 ± 19.1
Narowal
3
Faisalabad
3
Multan
3
Mukhtar et al., J. Anim. Plant Sci. 26(2):2016
545
The most important type of super kernel basmati
rice contaminated with aflatoxins was broken rice from
the four regions of Lahore, Narowal, Faisalabad and
Multan where the contamination ranged from 120.6µg/kg
to 8.0µg/kg. The highest value was detected in broken
rice from the Lahore region.
Linearity of HPLC system was checked to inject
different concentrations of reference standards. The
system was calibrated using the working solution of
aflatoxins in the range of 0.5-10 µg/ml in acetronitrile.
HPLC method was validated by testing linearity,
recovery, limit of detection (LOD) and limit of
quantification (LOQ). Results for recovery are given in
Table No.5. LOD and LOQ were 0.5 and 1µg/kg for
AFB1 and AFG1, respectively, and 0.05 and 0.1 µg/kg
for AFB2 and AFG2, respectively (data not shown).
Table 5. Recovery (%) of aflatoxins in rice samples (n = 6).
Aflatoxins
Spike level (µg/kg)
Recovery (%)
RSD (%)
AFB1
10
90 ± 0.17
0.17
AFB2
5
88 ± 0.12
0.14
AFG1
10
89 ± 0.17
0.19
AFG2
5
86 ± 0.12
0.15
AFB1= Aflatoxin B1, AFB2 = Aflatoxin B2, AFG1 = Aflatoxin G1, AFG2= Aflatoxin G2
Table 6. Parameters of linear regression for aflatoxins.
Aflatoxins
Concentration (µg/kg)
Slope
Intercept
R2
AFB1
0.5 –10
1.0907
0.042
0.9996
AFB2
0.05 –5
2.6638
0.0388
0.9993
AFG1
0.5 –10
1.0907
0.042
0.9996
AFG2
0.05 –5
2.6638
0.0388
0.9993
AFB1= Aflatoxin B1, AFB2 = Aflatoxin B2, AFG1 = Aflatoxin G1, AFG2= Aflatoxin G2
It was also observed that aflatoxin
contamination is directly related to the moisture level
present in the rice grains. The rice types with higher
moisture content have aflatoxin B1and B2contamination
(Figure No. 1 and 2).
It is further confirmed by finding out
correlation analysis between moisture content and
aflatoxins (B1& B2) contaminations that correlation was
significant at the 0.01 level for aflatoxin B1and for
aflatoxin B2it was significant at the 0.05 level. This
showed the strong relationship among higher moisture
content and aflatoxins contamination in different types of
super kernel basmati rice.
Figure No. 1 Correlation between moisture content and aflatoxin B1presence in different types of super kernel rice
Mukhtar et al., J. Anim. Plant Sci. 26(2):2016
546
Figure 2. Correlation between moisture content and aflatoxin B2present in different types of super kernel rice
DISCUSSION
The moisture content for some of the samples of
white, brown and broken rice was higher than others. If
the moisture content is not maintained properly during
storage of rice grains it results in ultimate growth of fungi
and production of aflatoxins (Reddy et al. 2009). Reddy
et al. investigated rice samples collected from India.
These samples were assessed for Aspergillus spp. and
aflatoxin B1. Aflatoxin B1contamination was observed by
two percent (2%) of samples from open storage that were
exposed to rain. The most important parameters which
influence the production of aflatoxins are temperature,
storage conditions, moisture level, and insect infestation
(Babu et al. 2011, Siruguri et al. 2012). The mean
moisture percentage for four districts ranged from
9.5±0.92 to 10.7±1.16 (Table No. 1).
In Pakistan contamination of agricultural crops
with fungal and aflatoxins hazards due to environmental
conditions are a major problem. In the second part of the
study aflatoxins (B1, B2, G1 & G2) were determined in
order to see the level of contamination in different rice
samples collected from different areas of Punjab.
Aflatoxin B1was most common in different types of
super kernel basmati rice samples collected from
different areas of Punjab. Nearly 56% of different rice
samples were contaminated by aflatoxin B1and 33%
were found to be contaminated with aflatoxin B2(Table
No. 2). From health safety point of view aflatoxin B1is
the most important toxin because it is commonly present
in food and at the same time is the most toxic for human
health (Deng et al. 2010). Out of 58% contaminated rice
samples with aflatoxin B1, 64.3% samples had aflatoxin
B1 level lower than the maximum tolerable limit of 20
ppb and 35.7% samples were contaminated by aflatoxin
B1 above the maximum tolerable limit. Similarly, out of
33% contaminated samples with aflatoxin B2, 87.5%
samples were above than maximum limit and 12.5%
samples were contaminated by aflatoxin B2 above the
maximum limit as it shown in Table No. 2. Almost same
results were seen in Uganda where Pakistani rice samples
were examined, 80% of samples contaminated with total
aflatoxins ranging between 20-50 ppb. Aflatoxin B1 was
found to be in a range of 16.08 ppb to 120.06 ppb. The
level of aflatoxin B2 was present in a range of 4ppb to 20
ppb (Taligoola et al. 2011). Bansala et al., (2011) found
that mostly basmati rice from India and Pakistan and
black and red rice from Thailand were contaminated with
aflatoxins.
It is obvious from the results that the aflatoxins
B1 and B2 was present in highest amount in broken rice
from each four areas of Punjab and then its presence was
in descending order from brown rice to white rice to
parboiled rice from all the four regions of Punjab. Same
results were shown by the Lutfullah and Hussain in 2012.
In a study conducted by Iqbal et al., (2012), 50% of
broken rice was contaminated by aflatoxins that hardly
give a clue which rice part is more prone to aflatoxin
contamination. Castells et al., (2007) detected that
aflatoxins were found throughout all fractions, but higher
contamination levels were detected in hull and bran
fractions. Regardless of the rice variety, the aflatoxin
distribution pattern depended on the initial contamination
level and type of milled fraction but not on the duration
of polishing.
After manipulating the mean and standard
deviation of all parameters used in this study, two way
Mukhtar et al., J. Anim. Plant Sci. 26(2):2016
547
ANOVA (Analysis of Variance) was used to compare the
means differences between the four areas and four types
of super kernel basmati rice and presence of aflatoxins
B1 and B2. The result showed that for aflatoxin B1 it was
observed that the p value for difference in means for
different regions was > 0.05 which is non-significant.
However, the mean difference between the different rice
types for aflatoxin B1was < 0.05 which was significant.
Similarly, for aflatoxin B2, it was observed that the p
value for difference in means for different regions and
between the different rice types was < 0.05 which was
also non significant.
In Pakistan sub-tropical conditions such as,
temperature and humidity also play a key role in
aflatoxins production. Post-harvest contamination of
aflatoxins can occur if the drying is delayed and moisture
is allowed to go beyond the limits (Asghar et al. 2013). In
the present study it is observed that the rice samples with
higher moisture content are prone to aflatoxins
contamination. It is further confirmed by using
statistically significant technique of calculating
correlation analysis between moisture content and
aflatoxins presence in super kernel basmati rice types.
The p value is less than 0.05 which was significant.
Conclusion: All this discussion concluded that the super
kernel basmati rice types available in Lahore, Narowal,
Faisalabad and Multan have good quality indeed but the
main issue is the aflatoxins B1and B2presence in the
different types of rice from the four areas under study. As
the moisture level directly correlates with the aflatoxins
contamination so it is important from food safety point of
view to control or monitor the temperature during storage
conditions so there is less chances of fungal attack during
storage and it will help in reduction of contamination by
aflatoxins.
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