Pak. j. sci. ind. res. Ser. B: biol. sci.

2013 56 (2) 98-104

Quantification and Detoxification of Aflatoxin in Food Items

Alim-un-Nisaa*, Naseem Zahrab, Sajila Hinaa, Rizwan Hayatc and Nusrat Ejaza
a
Food and Biotechnology Research Centre, PCSIR Laboratories Complex, Lahore-54600, Pakistan
b
Pakistan Institute of Technology for Minerals & Advanced Engineering Materials (PITMAEM),
PCSIR Laboratories Complex, Ferozepur Road, Lahore-54600, Pakistan
c
Institute of Molecular Biology and Biotechnology, The University of Lahore, Lahore, Pakistan
(received August 1, 2012; revised December 23, 2012; accepted January 24, 2013)

Abstract. The present study was conducted to quantify and detoxify the aflatoxins in food items. For this
purpose, total 30 samples of food were collected. The samples were quantified using thin layer chromatography
(TLC) for the presence of aflatoxin level in food items. Out of them aflatoxins were not found in 10
samples. Remaining 20 aflatoxins +ve samples were treated with various chemical solutions i.e. 0.1% HCl,
0.3% HCl, 0.5% HCl, 10% citric acid, 30% citric acid, 50% calcium hydroxide, 0.2 and 0.3% NaOCl, 96%
ethanol and 99% acetone for detoxification. The aflatoxins were reduced to 55.1%, 90.9%, 28.08% and
80.0% in Super Sella rice, Super Basmati rice, Brown rice and White rice, respectively. The aflatoxin level
was reduced in maize grain, damaged wheat, peanut, figs and dates upto 31.3 %, 64.3 %, 63.6%, 42.7%
and 19.8%, respectively. Aflatoxins were detoxified in cereals Dal Chana, Dal Mash, Dal Masoor), turmeric
(Haldi) and Nigela seeds (Kalwangi) upto 70.5%, 83.0%, 46.2%, 82.09% and 36.9%, respectively. Reduction
of aflatoxins was carried out 39.7 %,7.1 % 39.5% 82.0% and 62.0% in red chilli, makhana, corn flakes,
desert (Kheer Mix) and pistachio. The significant results (p =0.042) of detoxification of aflatoxins in food
items were obtained from present study.
Keywords: pepsin extraction, enzyme activity, stomach mucosa, buffalo

Introduction poisoning of farm animals such as pigs (Loosmore and

Harding, 1961) and calves (Loosmore and Markson,
Aflatoxins are toxic and carcinogenic metabolites
1961). Bioassay test on duckling also helped in furthering
produced by species of Aspergillus, especially Asper-
and establishing the toxic factor (Sargeant et al., 1961).
gillus flavus and Aspergillus parasiticus. The toxic
Aflatoxins producing ability is confined to A. flavus
effects include acute hepatitis, immune-suppression. In
and A. parasiticus. Strains of these two species are
humans, the risks associated with aflatoxin consumption
common and wide-spread and have been isolated from
are well documented and the International Agency for
a number of different host materials. Colonies of A.
Research on Cancer (IARC) has designated aflatoxin
flavus are green-yellow to yellow-green and that of A.
as a human liver carcinogen. Because of these toxic
parasiticus are dark green. The toxin is produced by
effects, the Food and Drug Administration regulates
mycelium and secreted into the medium or substrate,
the aflatoxin concentration in food with aflatoxin.
spores contain very little aflatoxin. Different strains of
Commodities or food with aflatoxin exceeding 20 ppb
A. flavus produce varying amount of aflatoxin and same
(mg/kg) cannot move in trade (Wogan, 1999). Very little
strains also produce varying amount of aflatoxins.
was known about mould metabolites prior to 1961. In
Aflatoxin production is a genetical process depending
that year some alarming reports of a mysterious disease
on specific nutrient and environmental factors (Patterson,
of Turkey poults came from South East of England,
1973). Investigation carried out by various researchers
tentatively named as Turkey X disease. In 500 such
at Tropical Development and Research Institute, London
outbreaks about 1, 00,000 poults, mostly between three
indicated that A. flavus is found in the soil and air
to six weeks of ages, died. Similarly 5000 partridges
throughout the world. Both A. flavus and A. parasiticus
and pheasants from one farm and 14000 ducklings from
are more prevalent in warmer climate and these moulds
another farm died (Asplin and Carnaghan, 1961). Reports
can be isolated from stores, dried stuff and tropical soil
from other places also accumulated on outbreak of acute
(Christensen, 1957). Aflatoxins naturally occur in rice
*Author for correspondence; E-mail: nisaalim64@yahoo.com (including brown, white, black, red and basmati) of
98
Aflatoxin Detoxification in Food Items 99

different countries, including United State, Canada, plan was adopted. These commodities were found
Pakistan, India and Thailand (Bansal et al., 2011). stacked in jute bags and stored in house type godown.
Aflatoxins (AFB1, AFB2, AFG1 and AFG2) also occur In order to obtain a more representative portion of these
in freshly harvested corn grains in different regions of samples, 500 g were collected through a sample probe
Brazil (Liliana et al., 2009). The presence of aflatoxins directly in plastic bags piercing jute bags diagonally
in red chillis may be a great threat to the health of from 2 to 3 places. They were passed through sample
populations. Total 183 samples of red chilli were divider and reduced to approximately 200 g for the
screened out for aflatoxin determination. 48 samples purpose of analysis and thus a greater homogeneity of
were positive for Aflatoxins B1 with the range from 1.2 contaminated portion was achieved. Each sample was
ppb to 968.3 ppb. Aflatoxin B2 was detected only in 3 then thoroughly mixed, ground and made into fine
samples with the range of 0.3 ppb - 159.8 ppb. Aflatoxin
powder for experimental analysis.
G1 and G2 were absent in all chilli samples (Nisa et al.,
2012). Chronic poisoning of aflatoxin results in cancer Extraction. Extraction procedures and analytical
(hepatocellular carcinoma) because liver is the target methods vary from one commodity to another because
organ of aflatoxins. Acute intoxication of aflatoxins in of diverse chemical composition, preventing the
human body is also lethal (Milita et al., 2010). Many development of any one method which could be applied
countries regulate aflatoxin levels in their foods. USA uniformly to all products. However, extraction with
and EU (Europe Union) permit level lower than 20 ppb chloroform is most suitable method for aflatoxins (B1,
and Korea and Japan 10 ppb (Chiavaro et al., 2001). B2, G1, G2, M1 and M2).
Due to its importance different food items were selected Then test portion was taken from mixture. 50 g of
for this study and different chemicals were used for ground sample was kept into 500 mL conical flask and
detoxification of aflatoxins in these food samples. 25 mL water and 150 mL chloroform was added into
flask. Conical flask was shaken on wrist action shaker
Materials and Methods for 30 min and sample was filtered through filter paper.
This study was conducted in Food and Biotechnology 50 mL chloroform was taken into beaker and put on
Research Centre of PCSIR Laboratories Lahore. The steam bath for evaporation.
food samples were prepared for aflatoxin analysis (Begum Chromatographic tank. The dilutions for spotting
et al., 1985). Aflatoxins were detected by Romers’ method were got in micro liter. The 25 mL spot of test solution
(Romer, 1975). Estimation of aflatoxins in toxic extracts was applied on thin layer chromatography plate with
was made by comparison with standard technique micro syringe. Spot of 5 or 10 mL of aflatoxins (B1, B2,
(AOAC, 2005). In this study, TLC technique was used G1, G2, M1 and M2) standard was spotted on same plate
for the determination of Aflatoxin in all samples. as an internal standard. The plate was developed with
Nature of samples. Samples of food such as Corn, anhydrous ether in thin layer chromatographic tank
Wheat, Wheat Flour, White Rice, Brown Rice, Super upto half then removed and dried. Then plate was
Basmati, Super Kernal Rice, Red Chilli, Pistachio, redeveloped in the same direction in thin layer chromato-
Cornflakes, Figs, Haldi, Garam Masala, Peanut, graphic tank with acetone-chloroform (1:9). Plate was
Kalwangi, Makhana and Dates were selected for the removed and test solution spot was observed for presence
present study. or absence of aflatoxins under UV light. If preliminary
plate would show that new concentration of test solution
Sample collection. During research work food samples required then new concentration were prepared for
were collected from local market of the city and brought spotting. Different 1 to 25 mL spots of test solution
to the Laboratory for quantitative determination and (3.5, 10.5, 24.9 mL) were spotted on new thin layer
detoxification. chromatographic plate and on the same plate 1 to 25
Sampling. Since the aflatoxins are not uniformly mL aflatoxins standard was spotted (Braicu et al., 2008).
distributed in commodities, grains were likely to have Detection and Estimation. Fluorescing intensities of
pockets of high aflatoxin concentration, firstly due to sample spots were compared with those of standard
highly heterogeneous distribution of aflatoxins and aflatoxin spots. In case, fluorescing spot of sample lied
secondly due to marketing in lumps of various sizes. between the standard spots, the average value of two
To obtain most representative sample, a suitable sampling standard spots was taken into consideration.
100 Alim-un-Nisa et al.

Confirmation. Another very important step in the 20 samples of food in which aflatoxins had been found
aflatoxins analysis was the fluorescing sample spots. were treated with chemical solutions.
This was carried out by spraying, evenly the thin layer
Chemical solutions 0.1%, 0.3% and 0.5% of hydrochloric
chromatographic plate with aqueous sulphuric acid
acid reduced aflatoxins to 39.7%, 55.1%, 90.9%, 39.5%,
(50/50 v/v). After the spraying, thin layer chromato-
62.0% and 82.0% in food items (Table 4) which are in
graphic plate was allowed to dry and then viewed under
line with work of Aly and Hathout (2011) who reduced
UV light (365 nm).
aflatoxins 27.6%, 42.5% and 90% in food with concen-
Calculation. Concentration of aflatoxins (mg/kg) present trations of hydrochloric acid at different hours. Aflatoxins
in sample was calculated as follows. also reduced to 49.3%, 86.5% and 71.39% with
concentration of 0.1%, 0.3% and 0.5% of hydrochloric
S´Y´V
Aflatoxins (µg/kg) = acid which is same work as Aly and Hathout (2011) who
Z´W
did reduction of food items.
Where:
Aflatoxins reduced to 31.3%, 64.3%, 19.8%, 28.08%,
S = volume in µL of aflatoxins standard of equivalent 70.5% and 83.05% with treatment of 10% citric acid, 30%
intensity to Z (µL of sample) citric acid, 1% sodium bisulphate, 2% sodium bisulphate,
Y = concentration of aflatoxins standard in µg/ mL 0.2% sodium hypochlorite and 0.3% sodium hypochlorite
V = volume in µL of solvents required to dilute final in food items and Aflatoxins reduced to 63.0%, 70.0%,
extract 69.16%, 53.9%, 10.0% and 35.05% with treatment of 10%
Z = volume in µL of sample extract required to give citric acid, 30 % citric acid, 1% sodium bisulphate, 2%
fluorescence intensity comparable to that of S = µL of sodium bisulphate 0.2% sodium hypochlorite and 0.3%
aflatoxins standard sodium Hypochlorite in food items which are in line with
W = weight in g of original sample contained in final work of Mukendi et al. (1991). They had detoxified
extract
Table 1. Solutions for detoxification of Aflatoxins in
Treatment for detoxification. Fifty grams of grinded food items
samples in which aflatoxins had been detected were kept
in separate 500 mL conical flasks. Chemical solutions of Food product Chemical solution
0.1% HCl, 0.3% HCl, 0.5% HCl, 10% citric acid, 30% for detoxification
citric acid, 05% calcium hydroxide, 0.2 and 0.3% NaOCl, Red chilli 0.1 % HCl
96% ethanol and 99% acetone were added into different 0.3% HCl
flasks (Table 1). Conical flasks were shaken on wrist action Red chilli 5 % NaOH
shaker for 2 h and sample was filtered through filter paper Super Sella rice 0.3 % HCl
and dried for two days. Super Basmati rice 0.5 % HCl
Quantification after detoxification. Quantification of White rice 5% Ca(OH)2
detoxified sample for aflatoxins was carried out by Maize grain 10 % Citric acid
same method such as chloroform extraction, detection Wheat damage 30 % Citric acid
by thin layer chromatography, estimation through UV Peanut 99 % Acetone
light and calculation by formula. Figs 96 % Ethanol
Dates 1 % Sodium bisulphate
Statistical analysis. The statistical significance of the Brown rice 2 % Sodium bisulphate
data was analyzed (p=0.042) using pair t-test (Steel Makhana 5 % KOH
et al., 1997). Dal chana 0.2 % NaOCl
Results and Discussion Dal mash 0.3 % NaOCl
Corn flakes 0.1 % HCl
Aflatoxins were detoxified by the treatment of different
Kheer mix 0.3 % HCl
chemical solutions. For this purpose, total 30 samples
Pistachio 0.5 % HCl
were collected. These 30 samples were quantified using
Haldi 5 % Ca(OH)2
thin layer chromatography (TLC) for the presence of
Kalwangi 10 % Citric acid
aflatoxins level in food items (Table 2). The aflatoxins
Dal masoor 99 % Acetone
were not found in 10 samples of food product, remaining
Aflatoxin Detoxification in Food Items 101

Table 2. Detection and estimation of Aflatoxins in food products

Sample Food Aflatoxin S Y V Z W ppb
No. product
1. Red chilli B1 0.5 2.02 0.5 4.7 15.04 7.14
(1000)
2. Red chilli B1 2 2.02 0.51 4.9 16.72 25.15
(1000)
3. Super Sella B1 2 2.02 0.51 2.9 16.78 42.
rice (1000)
4. Super Basmati B1 5 2.02 0.99 0.9 16.92 656.9
rice (1000)
5. Dal mash Absent _ _ _ _ _ _
6. White rice B1 8.5 2.02 0.51 24.9 16.70 125.53
(1000)
7. Pistachio Absent _ _ _ _ _ _
8. Haldi Absent _ _ _ _ _ _
9. Maize grain G1 0.5 2.03 0.51 0.9 16.65 34.48
(1000)
10. Wheat damage B1 0.9 2.02 1470 0.9 16.67 174.32
11. Peanut B1 8.5 2.02 0.51 4.9 16.70 21.08
(1000)
12. Figs B1 1.0 2.02 0.50 1.0 16.77 14.9
(1000)
13. Dates B1 0.5 2.02 1980 3.9 16.68 30.76
14. Brown rice B1 2 2.02 0.51 1.0 16.76 123.58
(1000)
15. Makhana B1 0.5 2.02 0.51 14.9 16.73 2.09
(1000)
16. Dal chana B1 0.5 2.02 0.99 14.9 16.72 8.02
(1000)
17. Figs Absent _ _ _ _ _ _
18. Peanut Absent _ _ _ _ _ _
19. Dal mash B1 0.5 2.02 0.5 4.7 15.04 7.14
(1000)
20. Corn flakes B1 1.0 2.02 1 9.5 16.27 13.0
(1000)
21. White rice Absent _ _ _ _ _ _
22. Kheer mix B1 0.9 2.02 0.51 4.9 16.70 11.32
(1000)
23. Pistachio B1 1.0 2.02 3.0 2.9 16.51 167.17
(1000)
24. Haldi B1 1 2.02 1980 2.9 16.68 82.68
25. Peanut Absent _ _ _ _ _ _
26. Brown rice Absent _ _ _ _ _ _
27. Kalwangi B1 4.0 2.02 0.51 5.0 16.72 49.2
(1000)
28. Super Sella rice Absent _ _ _ _ _ _
29. Super Basmati rice Absent _ _ _ _ _ _
30. Dal masoor B1 2.0 2.02 0.91 2.9 16.67 76.05
(1000)
102 Alim-un-Nisa et al.

aflatoxins by comparing chemicals citric acid, sodium been reduced statistically when aflatoxins were compared
bisulphate, sodium hypochlorite (Table 3 and Table 4). before and after detoxification.
The present study showed significant detoxification in Present study also showed that thin layer chromatography
aflatoxins (p = 0.042) of food items when pair T-test is a reliable method for detection and quantification of
was applied to quantified aflatoxins of food items before aflatoxins in food items before and after detoxification.
and after detoxification. It means that aflatoxins had (Okwu et al., 2010, Olufunmilayo and Oyefolu, 2010).
Table 3. Detection and estimation in Aflatoxins in food products after detoxification
Sample Food Aflatoxin S Y V Z W ppb
No. product
1. Red chilli B1 0.9 2.02 0.99 24.9 16.67 4.3
(1000)
2. Red chilli B1 0.9 2.02 0.51 4.9 16.71 11.34
(1000)
3. Super Sella B1 0.5 2.02 0.91 2.9 16.67 19.01
rice (1000)
4. Super Basmati B1 2.5 2.02 1975 2.0 16.70 59.68
rice (1000)
6. White rice B1 2.0 2.02 0.51 4.9 16.72 25.51
9. Maize grain G1 9.5 2.03 0.51 24.9 16.71 23.67
(1000)
10. Wheat damage B1 1.0 2.02 0.515 1.0 16.69 62.31
(1000)
11. Peanut B1 1.5 2.02 0.99 24.9 16.77 7.2
(1000)
12. Figs B2 0.5 0.5 0.48 0.9 16.56 8.54
(1000)
13. Dates B1 2 2.02 0.50 4.9 16.72 24.66
(1000)
14. Brown rice B1 8.5 2.02 0.51 5.9 16.71 88.87
(1000)
15. Makhana B1 0.5 2.02 0.51 5.9 16.72 1.94
(1000)
16. Dal chana B1 1 2.02 487 24.9 16.68 2.36
19. Dal mash B1 0.5 2.02 0.50 24.9 16.69 1.21
(1000)
20. Corn flakes B1 0.5 2.02 0.51 3.9 16.69 7.9
(1000)
22. Kheer mix B1 0.9 2.02 0.99 24.9 16.77 4.3
(1000)
23. Pistachio B1 0.5 2.02 1980 3.9 16.68 30.7
24. Haldi B1 6.0 2.02 0.51 24.9 16.69 14.8
(1000)
27. Kalwangi B1 7.5 2.02 0.51 14.9 16.72 31.0
(1000)
30. Dal masoor B1 3.0 2.02 0.50 4.7 15.90 40.9
(1000)
Aflatoxin Detoxification in Food Items 103

Table 4. Comparison of Aflatoxins estimation of food products before and after detoxification

Sample Food Aflatoxin Estimation Chemical ppb Reduction

No. product before solution for after in %age
detoxification detoxification detoxification
(ppb)

1. Red chilli B1 7.14 0.1 % HCl 4.3 39.7

2. Red chilli B1 25.15 5% NaOH 11.34 54.9
3. Super Sella rice B1 42.34 0.3% HCl 19.01 55.1
4. Super Basmati rice B1 656.9 0.5% HCl 59.68 90.9
6. White rice B1 125.53 5% Ca(OH) 25.51 80.0
9. Maize grain G1 34.48 10% Citric acid 23.67 31.3
10. Wheat damage B1 174.32 30% Citric acid 62.31 64.3
11. Peanut B1 21.08 99% Acetone 7.2 63.67
12. Figs B2 14.9 96% Ethanol 8.54 42.7
13. Dates B1 30.76 1% Sodium bisulphate 24.66 19.8
14. Brown rice B1 123.58 2 % Sodium bisulphate 88.87 28.08
15. Makhana B1 2.09 5% KOH 1.94 7.1
16. Dal chana B1 8.02 0.2% NaOCl 2.36 70.5
19. Dal mash B1 7.14 0.3% NaOCl 1.21 83.05
20. Corn flakes B1 13.0 0.1% HCl 7.9 39.5
22. Kheer mix B1 11.32 0.3% HCl 4.3 62.0
23. Pistachio B1 167.17 0.5% HCl 30.7 82.0
24. Haldi B1 82.68 50% Ca(OH)2 14.8 82.09
27. Kalwangi B1 49.2 10% Citric acid 31.0 36.9
30. Dal masoor B1 76.05 99% Acetone 40.9 46.2

Ultra-violet (UV) light, a standard procedure was used to Chiavaro, E., Dall., A.Sta, C., Galaverna, G., Biancardi,
differentiate the toxin from non-toxin forms of A. spergillus A., Gambarelli, E., Dossena, A., Marchelli, R.
species. 2001. New reversed-phase liquid chromatographic
method to detect aflatoxins in food and feed with
Conclusion cyclodextrins as fluorescence enhancers added to
the eluent. Journal of Chromatography A, 937:
It was concluded that significant detoxification i.e. 90.9%
31-40.
was observed when 0.5% HCl was used as detoxifying
Christensen, C.M. 1957. Deterioration of stored grains
agent for aflatoxin B1 in Super Basmati rice. Similarly,
by fungi. The Botanical Review, 23: 108-134.
83.05% detoxification of Aflatoxin B1 was observed in
Liliana, O.R., Viviane, K.N., Raquel, B., Tatiana, A.R.,
Dal Mash, 82.09% in Haldi, 82% in Pistachio and 80% Estela, K., Benedito, C. 2009. Mycoflora and co-
in White rice when 0.3% NaOCl, 50% Ca(OH)2, 0.5% occurrence of fumonisins and aflatoxins in freshly
HCl and 5% Ca(OH)2 were used, respectively. harvested corn in different regions of Brazil.
International Journal of Molecular Sciences, 10:
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