J Sci Food Agric 1990,53, 169-174

Postharvest Incidence of Aflatoxins in Linum

usitatissimum Seeds

S S Sahay, T Prasad and K K Sinha

Mycotoxin Laboratory, University Department of Botany, Bhagalpur University,
Bhagalpur-812007, India

(Received 4 July 1989; revised version received 15 February 1990;

accepted 28 March 1990)

ABSTRACT

Linseed (Linum usitatissimum L),collected from kothi and gunny bag, had a
total of 18 fingal species. Aspergillus spp were the more jiequent and
accounted for more than 40% of the total count. The gunny bag storage
system exhibited higher dominance of A flavus than occurred in the kothi
system. Out of 251 isolates screened, 44% were toxigenic and produced
different components of alfatoxins in the range 105-3000 p g litre-'. Of 105
samples extracted for the natural presence of alfatoxin, 46 had afatoxin
contamination. The concentration of alfatoxin Bl in contaminated samples
ranged @om 120 to 810 pg k g - ' .

Key words: Post harvest, linseed, aflatoxin, kothi, gunny bag.

INTRODUCIION

Among oil crops, linseed (Linum usitatissimum L) is one of India's main economic
products, and is widely grown in the plains of northern India. It is grown
predominantly as a rain-fed cold-season rabi crop. In Bihar state, linseed is also
grown as a mixed crop with wheat, gram and barley.
The bulk of linseed produced in India is utilised for the expression of oil and is
preferred for edible purposes. In Bihar, until the expression of oil, linseed is stored
largely in a kothi or in a gunny bag. A kothi is a cylindrical structure made of mud
and paddy straw mixture having an inlet opening at the top and an outlet opening at
the bottom. These openings are also closed after keeping the seeds, with the same
mixture of mud and husk. A gunny bag is a porous sack made up of jute (Crotolaria
juncea) fibres with a capacity of about 100 kg of grain. Sacks are piled in rows in the
169

J Sci Food Agric 0022-5142/90/$03.50 0 1990 SCI. Printed in Great Britain

170 S S Snhoy, T Pmsad, K K Sinha

storehouse. Besides oil, linseed cake is used as a protein supplement for livestock. It
has medicinal value too and is used as an emollient, expectorant and diuretic.
Although linseed has been reported to be a susceptible substrate for aflatoxin
contamination in field conditions (Sinha and Ranjan 1989), no report is available
with regard to aflatoxin contamination of linseed in storage. An attempt has been
made in the present investigation to record the variations in the mycoflora and
aflatoxin contamination in linseed stored in different storage systems.

EXPERIMENTAL

A regular survey of different areas of Bihar for collection of linseed samples was
made from March 1984 to February 1985 in order to assess the fungal load and
natural Occurrence of aflatoxin in them. Samples were collected randomly (Drew and
Granovasky 1977) in sterilised polythene bags from kothi and gunny bags. The
temperature and humidity of kothi and gunny bag were determined with a dip-shaft
hygrometer and electronic moisture meter before each collection of seed.
Assay of fungal flora
Fungal flora from the stored seeds was assessed in three ways, viz dilution plating,
blotter test as per the recommendations of the International Seed Testing
Association (1966), and direct plating of the seeds on Czapek Dox agar medium.
The number of fungi was expressed quantitatively as the total count (lo3 g-'
sample) by dilution plating (1ml of lo3 dilution plated per petri dish) and
qualitatively as the number of fungal species isolated from 50 seeds plated on the
blotting paper or directly on the agar medium. Pure cultures of Aspergillus flauus
were maintained on PDA and Czapek Dox media.
Assay of aflatoxins
The aflatoxin-producing potentiality of different isolates of A flauus was tested in
liquid rice-flour medium (Misra and Sinha 1979). For the natural presence of
aflatoxin, extraction of samples was done by the method of Thomas et a1 (1975).
Qualitative detection of aflatoxin was done by a thin layer chromatography
(TLC) technique with a toluene/isoamyl alcohol/methanol (90:30:2, v) solvent
system (Reddy et a1 1970). The presence of aflatoxin B, was confirmed chemically by
using trifluoracetic acid (Stack and Pohland 1975). Quantitation of aflatoxin B1 was
done spectrophotometrically (Nabney and Nesbitt 1965).

RESULTS AND DISCUSSION

Table 1 presents a quantitative analysis and lists the fungal species isolated from
kothi and gunny bags. The total count of fungi in gunny bags was higher than in
kothi. Species of Aspergillus accounted for more than 40% of the total count
followed by Penicillium citrinum (24%) and Fusarium sp (18%). The incidence of
other fungi was only 16%. Microbial decomposition of organic matter and the
Postharvest incidence of afatoxins in linseed 171

TABLE 1
Total counts (lo3 g-' sample), percentage counts and frequency of occurrence of fungal
genera and species

Genus and species Kothi Gunny bag

Total Count Frequency Total Count Frequency

count (%I (%) count (%I (%)

Alternaria lini 1.15 3.28 66.0 1.16 3.56 77.3

Aspergillus jlavus 2.30 8.83 52.00 3.05 9.39 84.6
A niger 4.38 29.00 76.0 6.81 21.34 75.0
A ochraceus 1.05 3.13 26.3 1.15 3.60 45.0
Colletotrichum linicola 0.60 2.28 23.33 026 1.75 26.66
Curvularia lunata 0.64 2.44 9.33 0.82 2.52 10.66
C pallescens 0.28 1.06 8.0 0.60 1.84 9.33
Fusarium equisiti 3.92 14.91 19.33 2.30 7.06 44.66
F lini 0.80 2.45 8.0 2.26 15.18 19.33
F moniliforme 1.42 4.36 17.33 0-26 1.75 26.66
Memnoniella echinute 0.01 0.03 2.66 0.18 0.68 5.33
Monilia sitophila 0.01 0.04 5.33 0.02 0.06 17.3
Mucor sp 0.60 2.29 24.6 1.16 3.79 76.3
Penicillium citrinum 3.80 14.46 30.66 6-36 19.53 52.0
Trichoderma viride 0.06 0.23 3.3 0.16 0.49 12.0
Total count (lo3g - ' sample) 21.56 26.55

association between the majority of saprophytic/pathogenic fungi and the crops,

especially during the postharvest/storage period, have been well documented
(Hesseltine 1974; Jhala 1976; Bhat et al1978; Anon 1981). Storage fungi in the three
recognised categories are important since they colonise stored products and
produce toxic metabolites (Christensen and Kaufmann 1965). Over 150 species of
storage fungi belonging to 10-15 groups are known, with species of Aspergillus and
Penicillium being most common (Hesseltine 1976). In our study of fungal
contamination of linseed from two types of storage, total counts of fungi were higher
in gunny bag samples than in the kothi samples, and aspergilli were the most
dominant group of fungi. Fungal incidence on the substrate has, in general, been
correlated with a number of factors such as temperature and humidity in tropical
countries. Observations on the higher fungal count in gunny bag samples may be
partly related to the higher moisture percentage and ambient temperature range
available. This is in conformity with other reports in the literature (WHO 1979).
The dominance of A flauus was higher in gunny bags than in kothi (Table 2).
However, this difference was not important during the winter and summer seasons
but significant in the monsoon season. The high per cent incidence of A fraous in
gunny bags during monsoons might be attributed to the porous nature of the sacks,
which allows exposure of the seeds to the moist conditions of the prevailing
atmosphere. Kothi, being non-porous unlike gunny bag and closed from all sides,
might not allow free exchange of the air.
Out of 251 isolates of A flauus obtained from 105 samples of linseed, 44% were
toxigenic (Table 3). Qualitatively 35 isolates produced B, alone, and B, and B, were
172 S S Sahay, T Prasad, K K Sinho

TABLE 2
Percentage incidence of A jlavus in relation to moisture (%) and temperature (“C)
Type of storage Season Moisture Temperature A flavus
( %) (“C) incidence
( %)

Winter 14-20 8-20 10

Kothi Summer 17-24 16-40 56
Monsoon 22-33 16-26 67
Winter 18-22 1&18 13
Gunny bag Summer 2&26 20-40 74
Monsoon 25-35 22-28 86

TABLE 3
Qualitative and quantitative estimations of aflatoxins
Positive to Qualitative estimation Quantity of
ajlatoxin ajlatoxin B ,
B, B , , Bz Bi, B,, G i Bi, Bz, G I, Gz

A flavus
isolates
screened
(251) 111 35 48 16 12 105-3000 pg
litre-
Linseed
samples
extracted
46 22 10 8 6 12CL810 pg
kg-’

elaborated by 48 isolates. Sixteen isolates yielded G, along with B, and B,, whileall
four, B,, B,, G, and G,, were synthesised by 12 isolates only. On the basis of the
quantity of aflatoxin B, elaborated, these toxigenic isolates were assigned to the
categories of high toxin producers (140&3000 pg litre-’ : 17 isolates), moderate
toxin producers (603-1 208 pg litre - : 26 isolates) and low producers (105s406 pg
litre-’: 68 isolates). From Table 3 it is apparent that only some of the isolates were
toxigenic. Morphologically it has not been possible to distinguish between the
toxigenic and non-toxigenic strains of A frcluus (Mehan and Chouhan 1974; Diener
et al 1987). It is possible that some genetic factor enables some strains to produce
toxin. The behaviour of different isolates producing different types of aflatoxin
might also be assigned to genetical differentiation (Ciegler 1975). Quantitative
variation of the different isolates may be related to the differences in the nature of
strains of the various isolates (Codner et a1 1963). Maggon et a1 (1969) explained it
on the basis of a genetic hypothesis.
Postharvest incidence of aflatoxins in linseed 173

Of 105 samples of linseed extracted for the natural presence of aflatoxin, 46 were
positive to aflatoxin contamination. Aflatoxin B, alone was found in 22 samples,
and both B, and B, were observed in 10 samples. Eight samples showed G , along
with B1 and B,, and six samples exhibited B,, B,, G, and G,. The quantity of
aflatoxin ranged from 120 to 810pg kg-'. Aflatoxin production in natural and
synthetic substrates is influenced by several factors, such as strain of the fungus,
temperature, moisture, aeration and length of incubation periods etc. Naturally
contaminated samples contain aflatoxin B1 more predominantly than others which
may be present in comparatively smaller proportion or even in undetectable
amount (Goldblatt 1968). The formation of all four aflatoxins, B,, B,, G1 and G,,
might be attributable to differences in toxigenic potential of different strains of A
flauus or to the presence of A parasiticus (Hesseltine et a1 1970). Diener and Davis
(1966) have also reported the occurrence of B, and B, and of all four aflatoxins (Bl,
B,, G , and G,) in naturally contaminated corn, soya bean, peanuts and cotton
seed.
The range (120-81Opg kg-') at which aflatoxin occurred in linseed samples
warrants attention because the tolerance level (20 pg kg- ')prescribed by the WHO
is well below the amount detected in the contaminated samples. Aflatoxins have
frequently been associated with certain clinical conditions, eg Indian childhood
cirrhosis (ICC) (Yadgiri et a1 1970; Amla et a1 1974; Rajkumar et a1 1982). Other
toxin activities of aflatoxins are hepatotoxicity, carcinogenicity and mutagenicity
(Bhat et a1 1978; WHO 1979). Elimination of aflatoxin contamination in stored
products will only be possible with the improvement and proper monitoring of
storage practices, especially post harvest, and effective physical or chemical
detoxification mechanisms (Borker et a1 1966; Shanta and Sreenivasamurthy 1975;
Bhat et a1 1978; Niles 1978), thereby reducing the risk to human or animal lives.

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