Indian Phytopath.

50 (1) : 77-82 (1997)

Aflatoxin contamination in oil seeds, oil cakes and oil

samples

R.1. VERMA, D.N. MEHTA, P.J. RAVAL and H.C. DUBE*

Department of Zoology, University School of Sciences, Gujarat University, Ahmedabad 380 009
"Departmem of Life Sciences, Bhavnagar University, Bhavnagar 364 002

ABSTRACT : Samples of oil seeds, oil cakes and oils of peanut, cotton, sesamum and castor were analyzed
for the incidence of aflatoxigenic fungi and aflatoxin contamination. The incidence of Aspergillus flavus was
higher in peanut seeds followed by castor, sesamum and cotton. The isolates from peanut seeds were most
potent aflatoxin producers followed by those from sesamum, cotton and castor. Aflatoxin contamination was also
highest in peanut seeds followed by cotton, sesamum and castor. Amongst oil cakes, Aspergillus flavus was
more prevalent on cotton followed by peanut, castor and sesamum. However, sesamum isolates were most
toxigenic followed by those isolated from peanut, castor and sesamum. But aflatoxin content was highest in
peanut cakes than in castor, cotton or sesamum. Analysis of oil samples revealed that aflatoxin contamination
was highest in peanut followed by sesamum, cotton and castor oil.

Keywords : Aflatoxin, Aspergillus flovus, cotton, groundnut, castor sesamum

In western parts of India, 100% contamina-
tion of maize samples with aflatoxin in the range
of 6.250 to 15,600 ug/kg was recorded by
Krishnamachari et a/. (1975). This contamination
caused an acute aflatoxicosis among the tribals of
Panchmahal district of Gujarat and Banaswada
district in Rajasthan during 1974 (Krishnamachari
et aI., 1975, 1977). Consumption of aflatoxin con-
taminated food has also been correlated with the
occurrence of Indian childhood cirrhosis (Amla et
al., 1974) and hepatomegaly (Sreenivasamurthy,
1975).
Though some studies on the incidence of
aflatoxigenic fungi and natural occurrence of af-
latoxins in oil seeds, oil cakes and chewing prod-
ucts have been made earlier by Verma et al. (1991,
1995), more comprehensive studies are needed
Received for publication May 13, 1996.
from other areas of Gujarat. The present investi-
gation is an attempt to collect and analyze the
samples of oil seeds, oil cakes and oils from
Surendranagar district of Saurashtra, Gujarat.
MATERIALS AND METHODS
Samples of oil seeds, oil cakes, and oils of
peanut, cotton, sesamum and castor were collected
during October-November 1992 from
Surendranagar district of Gujarat. Ten grams of
each sample (seeds and cakes) were taken in 250
ml conical flasks containing 100 ml of sterilized
distilled water and subjected to horizontal shaking
for 30 minutes on a shaker. Thereafter, suitable
spore suspensions in 0.5 ml sterilized water were
aseptically added to peptone - glucose-rose bengal-
agar medium containing tetracycline (Booth, ·1971)
and incubated at 28±2°C for 3-5 days. Fungal
colonies formed were identified and per cent in-
cidence of each fungus was calculated.
 78 Indian Phytopathology [Vol. 50(1) 1997]

Table 1. Percent incidence of fungi on different varieties of oil seeds

Per cent incidence

Isolates
Peanut Cotton Sesamum Castor

Aspergillus niger 37 34 34 31
A. flavus 11 8 9 10
A. tamarii 18 5
Arochraceus 2 5
A. candidus

Aspergillussp. 26 10 6 12
Penicillium citrinum 7
Penicillium expansum 1.5
Penicillium sp. 17 21.5 2 41
Mucor sp. 6 2 2 3

Rhizopus sp. 0.8 4

Fusarium solani 2
Fusarium moniliforme 1.8 2 4
Fusarium oxysporum 13
• Carvularia lunata

Curvularia sp. 0.5

Alternaria sp. 0.6
Helminthosporium sp. 0.5
Rhizoctonia solani

Cladosporium sp. 0.4

Usti/ago sp. 0.5
n = S.

Isolates of Aspergillus flavus obtained were
screened for their aflatoxin-producing potentials
in SMKY liquid medium (Diener and Davis, 1966).
25 ml of SMKY liquid medium was taken in 250
ml Erlenmeyer flasks and autoc1aved. 0.5 ml of
spore suspension (105 spores/ml) prepared from 5-
day old culture was used for inoculating the me-
dium under aseptic condition (in triplicate). After
completion of incubation period (10 days at
28±2°C), flasks were autoc1aved and the culture
media filtered through Whatman filter paper No.
1. Aqueous culture filtrates were extracted twice
with analytical grade chloroform (1:2, v/v) and
qualitatively analyzed for different types of afla-
toxins on TLC plates (Reddy et al., 1970). Quan-
titative estimation of aflatoxins was done accord-
ing to the method of Nabney and Nesbitt (1965)
using Shimadzu UV 160A spectrophotometer.
[Vol. 50(1) 1997] Indian Phytopathology 79

Table 2. Aflatoxin production by Aspergillus flavus isolates from different varieties of oil seeds in SMKY medium

Number of isolates Amount of

Varieties Aflatoxin aflatoxin
Screened Toxigenic (%) produced BI pro-
duced (ppm)

Peanut 19 4 (21) BI + B2 + 01 6.1

Cotton 12 2 (16) BI + B2 + 01 + 02 1.7

Sesamum 10 3 (30) BI + B2 + 01 2.6

Castor 11 (18) BI + 01 1.5

Table 3. Aflatoxin content in diffferent varieties of oil seeds

Aflatoxin (ppm)
Samples
Total BI B2 °1 G~

Peanut 4.16 1.37 0.97 1.28 0.54

Cotton 0.29 0.18 0.11

Sesamum 0.20 0.09 0.05 0.05 0.01

Castor 0.17 0.07 0.04 0.06

n - 5.

To study aflatoxin contamination in collected sesamum and cotton.

samples of oil seeds, cakes and oils, known amount
Fifty two isolates of A. jlavus were obtained
of each sample (25 g powdered sample in case of
from oil seeds (Table 2) of which 11 produced
oil seeds/cakes and 25 ml in case of oil, each in
aflatoxin in SMKY liquid medium. Seven isolates
triplicates) were extracted with methanol:water
~roduced a mixture of BI' B2 and GI' while two
(~5:45, v/v) and sodium chloride (Anon. 1975).
Isolates produced only BI and GI. Two isolates
Filtered aqueous methanolic extract was defatted
obtained from cotton seeds produced all four types
using n_hexane followed by its extraction for af-
of aflatoxins (BI' B2, G.; G2). However,
latoxin with chloroform (1:2, v/v; twice) which
aflatoxigenic potentials of A. flavus varied with
was processed for qualitative analysis of aflatox-
the isolates. Peanut isolates were most potent af-
~s o~ TLC plates (Reddy et al., 1970) and quan-
latoxin producers followed by sesamum, cotton
tification was made using Shizmadzu UV 160A
and castor (Table 2).
spectrophotometer (Nabney and Nesbitt, 1965).
Results in Table 3 show that aflatoxin con-
RESULTS tamination was highest in peanut seeds followed
by cotton, sesamum and castor. All four types of
Amongst 21 fungi isolated from the seeds
aflatoxins were present in peanuts and sesamum
(Table 1), A. niger, A. flavus, Aspergillus, Peni-
samples while cotton and castor seeds contained
cillium, Rhizopus, Mucor and' Fusarium species
BI + GI and BI + B2 + GI respectively.
were more frequent. The incidence of A. jlavus
was highest in peanut seeds followed by castor, Fifteen fungi were recovered from oil cakes
80 Indian Phytopathology [Vol. 50(1) 1997]

Table 4. Per cent incidenceof fungi on differentvarietiesof oil cakes

Isolates
Peanut Castor
Aspergillus niger 25 9
A. jlavus 12 7
A. ochraceus 2 2

Aspergillus sp. 6 4
Fusarium moniliforme 4

Fusarium rigidiuscula
Fusarium oxysporum 19

Penicillium citrinum 7
Penicillium expansum
Penicilium sp. 2 46

Mucor sp. 32

Rhizopus sp. 100

Curvularia lunata 2

Alternaria tenuis 2

Ijelminthosparium sp. 6

n=5.

(Table 4). Aspergillus niger, A. flavus, Aspergil-
lus, Fusarium, Penicillium, Mucor and Rhizopus
species were common. Incidence of A. jlavus was
highest in cotton followed by peanut, castor and
sesamum.
Thirty eight isolates of A. jlavus were ob-
tained from oil cakes of which 20 were
aflatoxigenic. Sesamum isolates were maximum
toxigenic followed by peanut and cotton/castor.
Four isolates from cotton cakes produced BI and
G1, while all others produced BI + B2 + G1 only.
Aflatoxigenic potential measured in terms of AFBI
concentration revealed that peanut isolates were
maximum aflatoxigenic followed by sesamum, cas-
tor and cotton cake isolates (Table 5).
The results (Table 6) revealed that concentra-
. tion of aflatoxin was highest in peanut oil cake
followed by castor, cotton and sesamum.
Again, total aflatoxin content in oil sample
was also highest in peanuts followed by sesamum,
cotton and castor oils (Table 7). In all samples,
concentration of BI was always highest followed
by GI' B2 and G2. All samples contained BI' B2
and G1 but castor and sesamum showed G2 also.
DISCUSSION
Investigations on the microflora of oil seeds
and oil cakes revaled a wide variety of fungi. In
general, the number of fungi associated with oil
seeds were more than on oil cakes. Analysis of
microflora of individual oil seeds and cakes re-
vealed that peanut and cotton seeds as well as
 [Vol.50(1) 1997] Indian Phytopathology 81

Table 5. Aflatoxin produced by Aspergillus jlavus isolates from different varieties of oil cakes

Number of isolates Amount of

Varieties Aflatoxin aflatoxin
Screened Toxigenic (%) produced BI pro-
duced (ppm)

Peanut 8 4 (50) BI + B2 + GI 1.9

Cotton 10 4 (40) BI + GI 0.13

Sesamum 10 8 (80) BI + B2 + GI 0.42

Castor 10 4 (40) BI + BI+ GI 0.28

Table 6. Aflatoxin content in different varieties of oil cakes

Aflatoxin (ppm)
Samples
Total BI B2 GI G2

Peanut 0.60 0.30 0.14 0.10 0.05

Cotton 0.15 0.07 0.08

Sesamum 0.08 0.06 0.02

Castor 0.26 0.13 0.03 0.10

n = 5.

Table 7. Aflatoxin content in different varieties of oil samples

Aflatoxin (ppm)
Samples
Total BI B2 GI G2

Peanut 3.87 1.98 0.56 1.33

Cotton 1.34 0.58 0.37 0.39

Sesamum 1.77 0.67 0.41 0.59 0.10

Castor 0.40 0.27 0.05 0.07 0.01

n = 5.

their cakes were more prone to fungal contamina-
tion.
The incidence of A. flavus differed with the
variety of seeds and cakes. All of them were not
able to produce aflatoxins.
Raper and fennell (1965) also reported that
all A. flavus isolates are not toxigenic. In 1965,
Hiscocks noted that some isolates of A. flavus
produced either B or G toxins, but majority of
them produced both toxins. None of the isolates
produced B2, GI and G2 in absence of BI (Lillehoj
et aI., 1977). It has been suggested that toxigenic
nature of the isolates is possibly governed by their
genetic make up (Ciegler, 1977).
82 Indian Phytopathology
The presence of variable amount of aflatoxin
in different types of samples could be due to en-
vironmental factors, toxigenic potential of the
fungal strains and composition of substratum
(Nagarajan and Bhat, 1973; Bilgrami, 1984). Some
isolates that produce aflatoxin under cultural con-
ditions, fail to do so under natural conditions.
This could be due to unfavourable and changing
conditions and the effect of interaction with other
microorganisms.
Besides, genetical factors might also be re-
sponsible for the variations in aflatoxin produc-
tion by different strains of A. flavus (Maggon et
al., 1969; Ciegler, 1977).
ACKNOWLEDGEMENT
Financial assistance from Gujarat Council of
Science and Technology is thankfully acknowl-
edged.
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