Vol.2, No.8, 973-987 (2010)
doi:10.4236/health.2010.28145
Copyright © 2010 SciRes. http://www.scirp.org/journal/HEALTH/
HEALTH
Openly accessible at
Aflatoxin B1 contamination in sunflower oil collected
from sunflower oil refinery situated in Karnataka
Narasimhan Banu1*, JohnPaul Muthumary2
1Department of Biotechnology, Vels University, Chennai, India; *Corresponding Author: banunkl@yahoo.com,
banu_sivakumar@rediffmail.com
2Centre for Advanced Studies in Botany, University of Madras, Chennai, India
Received 11 March 2010; revised 15 April 2010; accepted 17 April 2010.
ABSTRACT
In the present study, the aflatoxin B1 contami-
nation at various stages of oil refining and in
refined oil were carried out. This was subse-
quently compared with commercial vegetable oil
samples. Among the 23 different sunflower oil
samples were tested, 10 of them showed posi-
tive results to AFB1 and the remaining 13
showed negative results to AFB1. All the refined
oil samples were free from AFB1 contamination.
Keywords: Aflatoxin B1; Refined Oil; Raw Oil;
Filtered Oil; Sunflower Oil; Edible Oil
1. INTRODUCTION
Fats and oils are an essential part of our diet, supplying
nutrients, improving flavor, aiding in the absorption of
vitamins, and providing concentrated sources of energy
for our body. Food fats and oils are derived from oilseed
and animal sources. There is an universal demand for
vegetable oil due to its use in domestic cooking, as an
ingredient for other food production (in baked goods and
fried snack foods), and as a raw material for the manu-
facture of soap, body/hair oils and detergents. Oils and
fats are natural products and that consequently the impu-
rity levels will vary not only with oil type but also with
weather, soil, harvesting, feed, storage and extraction
conditions [1]. Sunflower oil is the preferred oil in most
of Europe, East Europe, Russia, Mexico, countries along
with Mediterranean and several South American coun-
tries.
Oil processing involves three major conventional
processes which include continuous neutralizing, blea-
ching and deodourisation. Neutralisation of crude oil
with caustic soda is still an essential feature for a refin-
ery required to produce a consistently high quality pro-
duct and to handle a number of different oil types. Strong
caustic soda solutions (4N) when mixed with oil will
saponify triglycerides, with consequent increase in neu-
tral oil loss, but this loss can be minimized by suitable
conditions, and concurrent saponification of pigments
with removal via the soap stock is a valuable way of
recovering poorer quality oil. A bleaching step is neces-
sary to remove soap, trace metals, sulphurous com-
pounds and part of the more stable pigments and pig-
ment break-down products which have resulted from
raw materials damage or oxidation. During the bleaching
stage, peroxides are broken down to aldehydes and ke-
tones and these secondary oxidation products are ad-
sorbed on the earth surface such that the filtered oil after
bleaching should have Peroxide Value (PV) of nil. The
deodorization process, involves steam distillation under
vacuum. Its purpose is to remove, so far as possible,
residual free fatty acids, aldehydes and ketones which
are responsible for unacceptable oil odours and flavors
and, more recently to decolourise the oil by heat de-
composition (270ºC) of the pigments, and distillation of
the decomposition products [1]. Traces of aflatoxins
ranging from 3 to 16 μg/Kg in 13 of 28 samples of crude
peanut oil imported to Malaya from Hong Kong and
Singapore [2,3]. They did not detect any aflatoxins in
five samples of refined peanut oil produced by labora-
tory refining of crude oils which had been found to be
contaminated.
Aflatoxin contamination in crude oils extracted from
the seeds has been reported in groundnut [4]. On study-
ing the state of aflatoxin in raw peanut oil it was found
that 65-70% present in the sediment and 30-35% in the
supernatant oil on centrifugation indicating that the toxin
is sparingly soluble in oil [5]. Mixing of 2% Fuller’s
earth with raw oil and centrifuging resulted in adsorption
of toxin to the earth material thereby the clear oil con-
tained only 10-15% of what was originally present.
Based on these data, filter pads to suit plate-and-frame
filter press in pilot plant/oil mills are prepared and afla-
toxin could be removed successfully to the extent of
85% on filtration [6,7]. Low levels of aflatoxin in oils,
except for unrefined peanut oil in India [8].
N. Banu et al. / HEALTH 2 (2010) 974-987
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A simple method was proposed for aflatoxin determi-
nation in vegetable oils [9]. The method was success-
fully applied to both crude and degummed oils. The oils
analysed contained aflatoxin B1 at levels of 5-200 μg/Kg.
Recoveries of AFB1 standards added to aflatoxin-free
oils were between 89.5 and 93.5%, with coefficients of
variation of 6.3-8.0%. During 1984-1985, 60 samples
(corn, peanut, cottonseed, olive, safflower, salad, walnuts
and sesame seed oil) were analysed for aflatoxins,
trichothecenes and zearelenone. Aflatoxins were de-
tected in 5 of 8 samples of peanut oil (aflatoxin B1 0.52-
0.72 ppb, B2 0.09-0.22 ppb, G1 0.07-0.08 ppb and G2
0.01 ppb). Crude oils, obtained from corn germ were
spiked with aflatoxins (0.8-1.0 ppm), deoxynivalenol (8
ppm)/nivalenol (8 ppm) and zearelenones (10 ppm), and
then refined by stimulated commercial procedure. No
detectable mycotoxins remained in the edible oil. In par-
ticular, neutralization process, including alkaline refining
and washing and decolorizing processes, eliminated
mycotoxins [10].
Proper storage of vegetable oil has a shelf life ranging
from 6-12 months. Heat applied during processing de-
stroys enzymes in raw materials, and also any contami-
nating microorganisms, which would cause rancidity.
Additionally, the oil may be heated after extraction to
remove as much water as possible; this lessens the oc-
currence of microbial spoilage during storage. Correct
packaging and storage conditions slow down chemical
changes caused by light and heat, which may lead to
rancidity [11]. The storage of raw sunflower oil in four
different packaging materials viz., plastic, tin, hindolium
and high-density polyethylene (HDPE) pouches [12].
The effect of heat treatment on storability of oil under
airtight and non-airtight conditions was also studied. In
the case of heat treatment, the oil was heated up to
120C for 90 minutes in order to inactivate the lipase
enzyme completely. It was observed that during storage,
different packaging materials and heat treatment affected
the colour, odour, free fatty acids and iodine value of oil.
The airtight HDPE pouches (120 gauge) with heat-
treated oil were found to be the best packaging materials
for storage of oil up to a period of 16 weeks.
The present study was the first report of aflatoxin B1
from sunflower oil, where the oil is being extracted.
2. MATERIALS AND METHODS
2.1. Sample Collection
2.1.1. Sunflower oil Samples
A total of 23 different sunflower oil samples of each
100 ml were collected. It includes 11 Raw oils viz.,
RO1a, RO1b, RO1c, RO1d; RO2a, RO2b, RO2c; RO3a,
RO3b, RO3c and RO3d; 8 Filtered oils viz., FO1a, FO1b,
FO1c, FO1a; FO2a, FO2b, FO2c, FO2d and 4 refined
oils viz., REF3a, REF3b, REF3c and REF3d. (RORaw
oil; FOFiltered oil; REFRefined oil; 1Nayan pro-
teins; 2Lakshmi industries; 3Raviprakash refinery;
a-dnumber of samples) The above samples were col-
lected between 2003 and 2004 and evaluated for AFB1.
2.1.2. Commercial Vegetable oil Samples
One hundred mililitre of different vegetable oils like
Goldwinner, Saffola, Sunland, Sundrop, Gemini, S.V.S.
(Sunflower oil), V.V.S., Idaiyam, Anandam (Gingelly
oil), Ruchi (Palm oil) were obtained from local markets
at Chennai in pouches. Three different samples of peanut
oils (unbranded) were also obtained from the local mar-
ket at Chennai in sterilized vials for aflatoxin B1 evalua-
tion.
2.1.3. Preparation of Column (Miller et al., 1985)
Slurry of silica gel for column chromatography (60-200
mesh) with hexane was prepared. It was tightly pack in
the column (50 cm height and 1.5 cm width). The col-
umn was washed with diethyl ether, toluene and chloro-
form (100: 100: 100, v/v/v).
2.1.4. Column Chromatography
Five milliliter of each oil sample was dissolved in hex-
ane and mixed with silica gel (60-200 mesh). This was
loaded to the column. Aflatoxin B1 in the oil samples
was eluted from the column with chloroform: methanol
in the ratio of 97: 3. Same procedure was followed 2 to 3
times in order to collect the complete elution of AFB1
from the samples.
2.1.5. Quantification of AFB1 by TLC
5, 10, 20 and 40 µl of each fractions (1, 2 and 3) of the
partially purified aflatoxinB1 were applied to pre-coated
TLC plates (Merck) along with the standard AFB1.
The TLC plates were developed in a tank containing
chloroform: acetone in the ratio of 88: 12, for 30 minutes.
After the development, the plates were viewed under
long UV light at 365 nm. Blue-fluorescence similar to
standard AFB1 indicated the presence of aflatoxin B1 in
the fractions of oil collected through the column.
Quantification of AFB1 was done using long UV light
by evaluating the plate itself. The following formula is
used to quantify the aflatoxin B1 in the sample:
Fluorescent intensity of the standard factor × concentrated of the standard × dilution factor
Aflatoxin content = × 1000
Fluorescent intensity of the sample
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2.2. Ultra Violet (UV) Spectroscopic
Analysis
The partially purified sample of AFB1 collected from
column chromatography was analysed by UV absorption
by dissolving in 100% methanol (HPLC grade) and read
at 370 nm in a Beckman DU-40 Spectrophotometer,
compared with authentic AFB1.
2.2.1. Infra-Red (IR) Spectroscopic Analysis
The partially purified AFB1 was mix with IR grade po-
tassium bromide (1:10) pressed into discs under vacuum
using spectra lab Pelletiser. The spectrum of the sample
was recorded (500-4000 cm-1) in a Burker 17S 85 FTIR
Spectroscopy and compared with authentic AFB1.
2.2.2. Quantification of AFB1 by High-Performance
Thin-Layer Chromatography (HPTLC)
Five µl of fractions I and II collected through the column
chromatography of different samples viz., REF3a, REF3b,
REF3c and REF3d were taken for HPTLC to estimate
the amount of AFB1. The fractions of I and II collected
from the branded sunflower oils viz., Gold winner, Saf-
fola, Sundrop were also estimated for AFB1. The frac-
tions of I and II were loaded onto pre-coated silica gel
plate. The plate was developed in a saturated tank con-
taining tertiary butyl methyl ether: methanol: water in a
ratio of 9.6:0.3:0.1. The developing distance of the plate
was up to 80 mm. After the development the plate was
scanned in a Camag TLC scanner 3 at 366nm. The pres-
ence of blue-fluorescence indicated the presence of
AFB1 as similar to that of authentic AFB1.
3. RESULTS AND DISCUSSION
In the present study, it was planned to evaluate the AFB1
contamination at various stages of oil refining and in
refined oil. This was subsequently compared with that of
commercial vegetable oil samples. A total of 23 different
samples of sunflower raw oil, filtered oil and refined oil
were collected from the Nayan proteins, Lakshmi indus-
tries and Raviprakash refinery (Table 1). From the local
markets of Chennai, 13 different vegetable oil samples
(sunflower, gingelly, palm and peanut oil) were collected
and all the oil samples were evaluated for AFB1 con-
tamination.
A simple method of aflatoxin determination in vege-
table oils was proposed by Miller et al., (1985). The
method was successfully applied to both crude and de-
gummed oils. Recoveries of AFB1 standard added to
aflatoxin free oil were between 89.5 and 93.5%, with
coefficients of variation of 6.3-8.0%.
Among the 23 different sunflower oil samples tested,
10 of them showed positive result to AFB1 and the re-
maining 13 showed negative results to AFB1 (Table
2).The sample RO1a, FO1a, RO1b and FO1c collected
from Nayan proteins, RO2a, FO2a and FO2c collected
from Lakshmi industries and RO3a, RO3c, REF a, REF
b, REF c and REF d collected from Raviprakash refinery
free from AFB1 contamination. The samples from the
same factories but at different time of collection showed
minimum level of AFB1 contamination.
The level of AFB1 contamination in fraction I col-
lected through the column of oil samples ranged from
0.08 to 0.6 ppm, in fraction II of oil samples ranged from
0.1 to 0.6 ppm, and in the fraction III of oil samples
ranged from 0.04 to 0.4 ppm quantified by TLC method.
The samples RO1b and FO1b collected from the Nayan
factory AFB1 contamination of 0.1 ppm in first fraction
of RO1b. But in FO1b, it was about 0.2 ppm in first
fraction and 0.3 ppm in second fraction. The sample
RO2b and FO2b collected from Lakshmi industries
showed the 0.3 ppm of AFB1 in second fraction. The
samples RO3b and REF3 b collected from Raviprakash
refinery showed AFB1 contamination in RO3b only (0.4
ppm). The complete conventional type of refining proc-
esses carried by this refinery removes the AFB1 from
Table 1. List of sunflower oil samples collected from the re-
finery.
S.No.Sample Name Name of the Refinery
1 RO1a Nayan proteins
2 FO1a Nayan proteins
3 RO2a Lakshmi industries
4 FO2a Lakshmi industries
5 RO3a Raviprakash refinery
6 REF3a Raviprakash refinery
7 RO1b Nayan proteins
8 FO1b Nayan proteins
9 RO2b Lakshmi industries
10 FO2b Lakshmi industries
11 RO3b Raviprakash refinery
12 REF3b Raviprakash refinery
13 RO1c Nayan proteins
14 FO1c Nayan proteins
15 RO2c Lakshmi industries
16 FO2c Lakshmi industries
17 RO3c Raviprakash refinery
18 REF3c Raviprakash refinery
19 RO1d Nayan proteins
20 FO1d Nayan proteins
21 FO2d Lakshmi industries
22 RO3d Raviprakash refinery
23 REF2d Raviprakash refinery
(RO – Raw oil; FO – Filtered oil; REF – Refined oil)
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Table 2. Quantification of AFB1 content of sunflower oil sam-
ples by TLC.
S.
No
Sample
name
Fraction I
Level of
AFB1
(ppm)
Fraction II
level of
AFB1
(ppm)
Fraction
III level of
AFB1
(ppm)
1 RO 1 a - - -
2 FO 1 a - - -
3 RO 2 a - - -
4 FO 2 a - - -
5 RO 3 a - - -
6 REF 3 a - - -
7 RO 1 b - 0.1 -
8 FO 1 b 0.2 0.3 -
9 RO 2 b 0.08 0.3 -
10 FO 2 b 0.2 0.3 0.4
11 RO 3 b 0.6 0.4 0.2
12 REF 3 b - - -
13 RO 1 c 0.2 - -
14 FO 1 c - 0.4 -
15 RO 2 c 0.08 0.4 -
16 FO 2 c - - -
17 RO 3 c - - -
18 REF 3 c - - -
19 RO 1 d 0.4 0.5 -
20 FO 1 d 0.3 0.6 -
21 FO 2 d 0.2 0.2 0.04
22 RO 3 d 0.5 0.1 -
23 REF 3 d - - -
raw oil. In refined oil there was no contamination of
AFB1. In the case of RO2c and FO2c, the AFB1 was
detected from RO2c but it was absent in FO2c.
The presence and absence of AFB1 was confirmed by
Ultra-Violet spectrophotometry (Figures 16-36) and
Infra-Red spectroscopy (Figures 37-41).
All the refined oil samples were free from AFB1 con-
tamination (Table 3). This was supported by the absence
of fungi in the refined oil samples [13]. This may be due
to oil processing which includes continuous neutraliza-
tion, bleaching and deodorizations. During these proc-
esses, fungal propagules are probably removed from the
oils. The toxic AFB1 have been found to be heat stable
up to their melting points of around 250˚C. Therefore,
AFB1 was not completely destroyed by such processes,
and was carried along the way from seeds to oil samples.
The complete conventional processes remove these
compounds from the raw oil. But the quantity of con-
tamination is very least ranging from 0.1 to 0.4 ppm.
This low level was due to extraction of oil using food
grade hexane. The extraction plays a role of partially
removing aflatoxin from the oil samples.
Since, the Nayan proteins and Lakshmi industry are
sunflower seed crushing units, they are not having much
advancement for complete oil refining. The samples of
raw oil and filtered oil collected from the factories
showed below minimum level of AFB1 contamination.
The Raviprakash refinery is better than Nayan and Lak-
shmi industries with reference to conventional oil proc-
essing. In the latter, the process removes any fungal
propagules and its toxins during oil processing at very
high temperature of 270˚C for 6 hours.
All the refined oil samples (both first and second frac-
tion) analysed by HPTLC showed no contamination of
AFB1 (Figures 2-9).
The other vegetable oil samples which are commer-
cially available like sunflower (gold winner, saffola,
sundrop, Gemini, S.V.S., sunland), gingelly oil (V.V.S.,
idaiyam, anandam), palm oil (ruchi) and peanut oils
(peanut oil 1, peanut oil 2 and peanut oil 3) were also
evaluated for AFB1 contamination by TLC method (Ta-
ble 4).
The sunflower oil samples like gold winner, saffola,
sundrop and gingelly oil-anandam showed complete
absence of AFB1 contamination. The other samples
(Gemini, S.V.S., sunland, V.V.S., idaiyam, ruchi, peanut
oil 1, peanut oil 2 and peanut oil 3) showed contamination
ranging from 0.08 to 0.504 ppm of AFB1. The AFB1
Table 3. Quantification of AFB1 by HPTLC.
Sample
name
Fraction I level of
AFB1 (ppm)
Fraction II level of
AFB1 (ppm)
REF3 a - -
REF3 b - -
REF3 c - -
REF3 d - -
Table 4. Quantification of AFB1 content of commercial vege-
table oils by TLC.
Sample
name
Fraction I
level of
AFB1 (ppm)
Fraction II
level of AFB1
(ppm)
Fraction III
level of
AFB1 (ppm)
Gold winner- - -
Saffola - - -
Sundrop - - -
Gemini 0.24 0.16 -
S.V.S. 0.224 0.36 -
Sunland 0.24 0.208 -
V.V.S. 0.24 0.3 -
Idaiyam 0.224 0.264 -
Anandam - - -
Ruchi 0.24 0.496 -
Peanut oil 10.16 0.08 -
Peanut oil 20.504 0.224 -
Peanut oil 30.24 0.224 -
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mV
Figure 1. Authentic aflatoxin B1.
Openly accessible at
Rf
mV
Rf
Figure 2. First fraction of REF3 a.
mV
Rf
Figure 3. Second fraction of REF3 a.
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978
mV
Rf
Figure 4. First fraction of REF3 b.
mV
Rf
Figure 5. Second fraction of REF3 b.
mV
Rf
Figure 6. First fraction of REF3 c.
Openly accessible at
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mV
Rf
Figure 7. Second fraction of REF3 c.
mV
Rf
Figure 8. First fraction of REF3 d.
mV
Rf
Figure 9. Second fraction of REF3 d.
Openly accessible at
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980
mV
Rf
Figure 10. First fraction of gold winner sunflower oil.
mV
Rf
Figure 11. Second fraction of gold winner sunflower oil.
mV
Rf
Figure 12. First fraction of saffola sunflower oil.
Openly accessible at
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mV
Rf
Figure 13. Second fraction of saffola sunflower oil.
mV
Rf
Figure 14. First fraction of sundrop sunflower oil.
mV
Rf
Figure 15. Second fraction of sundrop sunflower oil.
Openly accessible at
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UVSpectroscopic Analysis
Figure 19. UV spectrum of second fraction of RO2 b.
Figure 16. Authentic aflatoxin B1.
Figure 20. UV spectrum of first fraction of FO2 b.
Figure 17. UV spectrum of second fraction of RO1 b.
Figure 18. UV spectrum of second fraction of FO1 b. Figure 21. UV spectrum of first fraction of RO3 b.
Openly accessible at
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Figure 22. UV spectrum of first fraction of RO1 c.
Figure 23. UV spectrum of second fraction of FO1 c.
Figure 24. UV spectrum of second fraction of RO2 c.
Figure 25. UV spectrum of second fraction of RO1 d.
Figure 26. UV spectrum of second fraction of FO1 d.
Figure 27. UV spectrum of first fraction of FO2 d.
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Figure 28. UV spectrum of first fraction of RO2 b.
Figure 29. UV spectrum of first fraction of V.V.S. sun-
flower oil.
Figure 30. UV spectrum of first fraction of S.V.S. sun-
flower oil.
Figure 31. UV spectrum of second fraction of sunland
sunflower oil.
Figure 32. UV spectrum of first fraction of ruchi palm
oil.
Figure 33. UV spectrum of second fraction of idaiyam
gingelly oil.
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Figure 34. UV spectrum of first fraction of gemini sun-
flower oil.
Figure 35 UV spectrum of first fraction of peanut oil 1.
Figure 36. UV spectrum of first fraction of peanut oil 2.
Figure 37. FTIR of authentic aflatoxin B1.
60.0
80.0
%T
500.01000.01500.02000.03000.04000.0 1/cmk-5.1
721.3
864.0
1110.9
1454.21631.7
2866.0
2923.9
3425.3
Figure 38. FTIR of first fraction of RO 3a.
40.0
60.0
80.0
%T
500.01000.01500.02000.03000.04000.0 1/cmDT-6
871.8
1022.2
1103.2
1161.1
1454.2
1596.9
1739.7
2349.1
2854.4
2923.9
3417.6
Figure 39. FTIR of first fraction of REF 3a.
50.0
100.0
%T
500.01000.01500.02000.03000.04000.0 1/cmK-7.1
Figure 40. FTIR of first fraction of RO 1b.
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50 .0
10 0. 0
%T
50 0. 010 00 .015 00.020 00 .030 00 .040 00 .01/ cmdt -15o i
11 68 .
8
12 38 .2
13 77 .1
14 58 .1
16 04.7
17 39 .7
28 58 .3
29 27.7
34 36 .9
Figure 41. FTIR of first fraction of RO 2c.
was not eluted in fraction three. It was eluted only in
both first and second fractions.
Openly accessible at
The AFB1 contamination of peanut oil was highest
(0.504 ppm) followed by ruchi palm oil (0.496 ppm),
S.V.S. sunflower oil (0.36 ppm) and V.V.S. gingelly oil
(0.3 ppm). Both the fractions of sunflower oils namely,
gold winner, saffola and sundrop showed no contamina-
tion of AFB1 analysed by HPTLC.
The evidence seems unequivocal that conventional
processing practices remove essentially completely any
aflatoxin that may be found in raw sunflower oils. If
properly stored, vegetable oil has a shelf-life ranging
from 6-12 months. Heat applied during processing de-
stroys enzymes in raw materials, and also any contami-
nating microorganisms which would cause rancidity.
Additionally, the oil may be heated after extraction to
remove as much water as possible, this lessens the oc-
currence of microbial spoilage during storage. Correct
packaging and storage conditions slow down chemical
changes caused by light and heat which may lead to ran-
cidity [11]. During storage of raw sunflower oil in dif-
ferent packaging materials and heat treatment affected
the colour, odour, free fatty acid and iodine value of oil.
The airtight high density polyethylene (HDPE) pouches
(120 gauge) with heat-treated oil were found to be the
best packaging materials for storage of oil up to a period
of 16 weeks [12].
4. CONCLUSIONS
In the present study, the level of AFB1 contamination in
all the vegetable oils tested showed very least amount of
contamination and their level was permissible according
to FDA. So this adds advantages to the vegetable oil
refineries and to the consumers. But this low level may
be biomagnified in our body leading to Hepato Cellular
Carcinoma (HCC). So, we have to look for new methods
of detoxification and decontamination of A. flavus and
its toxins.
The research is essential to respond to the needs of our
country in confronting the mycotoxin problem. The re-
search undertaken reflects the current status in the de-
velopment of mycotoxin problem. The work should be
recognized by the industries for transferring the results
into practical applications.
5. ACKNOWLEDGEMENTS
We sincerely thank the Department of Science and Technology, Gov-
ernment of India, for funding this project.
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