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Mycotoxins
Mycotoxins are chemically diversified low
molecular weight compounds produced by secondary
metabolism of fungal genera such as Aspergillus,
Penecillium, Fusarium, Altenaria and
Claviceps over a variety of foodstuffs.
These mycotoxins exhibit a wide array of
biological effects and individual mycotoxins can
be mutagenic, teratogenic carcinogenic,
embryo-toxic, nephro-toxic, oestrogenic and
immunosupressive (Hohler et al 1998). Mycotoxin
contamination of various agricultural crops and
foods and feeds continues to be a serious
quality and safety problem worldwide.
Considerable global attention is being focused
on mycotoxin contamination of foods and feeds is
because of its adverse effects on human and
animal health and economic effects. Mycotoxin
contamination problems seriously affect the
agricultural economies of many developing
countries, particularly the developing ones due
to increasingly stringent regulations imposed
for mycotoxin levels in agricultural products.
The application of different permissible levels
in different countries (e.g. European Union
countries, the USA, and developing countries)
has a tremendous impact on international trade
in various agricultural commodities such as
groundnuts, maize and
chilies.
Aflatoxins
Mycotoxins are of different types such as
aflatoxins, ochratoxins, tricothecens etc. Among
the different types of mycotoxins aflatoxins
have received greater attention than other
mycotoxins because of their established
carcinogenic effect in various animals and their
acute toxicological effects in humans.
Aflatoxins are potent, carcinogenic, mutagenic,
teratogenic and immunosupressive agents produced
as secondary metabolites by the
fungal species Aspergillus
flavus and Aspergillus
parasiticus on a
variety of agricultural commodities like
groundnut, maize, sorghum, wheat, chilies,
pepper, turmeric, dryginger, coriander etc. The
problem was first recognized
following
outbreaks of Turkey "X"
disease in the United Kingdom in 1960. Aflatoxin
contamination has gained significance due to its
deleterious effects on human health and animal
health and consequent effects on international
trade in affected crops. There are more than 15
types of aflatoxins. Among them Aflatoxin
B1, B2, G1, and
G2 are more potent. Aflatoxin
B1 is the most toxic followed by
G1, B2, G2 in
order of decreasing
potency.
Aflatloxin
contamination in different types of agricultural
commodities:
Different types of agricultural
commodities like groundnut, maize, wheatflour,
cornflour, sorghum, redchilies, turmeric,
dryginger, coriander, black pepper are often
contaminated with aflatoxin producing fungi Aspergillus
flavus & Aspergillus
parasiticus and consequently contaminated
with aflatoxins in the field at pre-harvest and
post-harvest stages, during storage and in
transport.
Groundnut:
The
cultivated groundnut (Arachis
hypogaea L.) is the most important oilseed
crop in many developing countries. Groundnut
seed is a valuable protein for human and animal
nutrition. Groundnut contributes significantly
to the public health, export earnings of several
countries and groundnut products are used
locally in various forms of food and feed.
Aflatoxin is considered as one of the
most important groundnut food quality problem in
many countries, especially in tropical Asia.
Aflatoxin-contaminated groundnut cake
contributed to the death of more than 200 000
broiler chickens in 1994 (Ranga reddy district).
Maize:
Maize (Zea Mays
L.) is one of the most important cereals
grown in most countries of the world & is
used as human food, animal feed & industrial
raw material. It is also widely grown as a
forage crop & is fed to the animals both
green & as silage. Maize is used as a human
food & feed for livestock, for fermentation
& for industrial purposes. Every part of the
plant has economic value. In 1963 it was
discovered that aflatoxins are found in maize
(Slocum, 1963). Maize seeds infected with
aflatoxin producing fungus or naturally
contaminated with aflatoxins resulted in reduced
germination. Recently, a poultry farm in
Chitradurgh, Karnataka state, lost more than
2000 baby chickens as a result of feeding them
with aflatoxin contaminated maize
meal.
Sorghum: Sorghum ranks fourth in importance
among the world’s cereals, exceeded in area and
production by wheat, rice, maize only. Sorghum
is used in Africa as a porridge or thick paste,
in India often as a bread or cake (Dogget,1970).
Certain sorghum is used predominantly for
feeding livestock. Usually over a dozen insects
have been recorded as pests of sorghum in India.
These pests make the way easier to Aspergillus
flavus to attack sorghum & leading
further aflatoxin contamination. In sorghum
grain molds (GM) occupies significance in
relation to mycotoxin contamination. Grain molds
have been defined as "fungi that grow on or in
seed" (Williams and Mcdonald 1983). They affect
sorghum in warm and wet conditions between
flowering and harvest
(Williams and Rao 1981; Williams and
Mcdonald 1983). Sorghum seeds are contaminated
with aflatoxins in the field during pre-harvest,
post-harvest, storage conditions and during
transport.
Spices:
Spices are the buds, bark, roots, berries
and aromatic seeds that are harvested for use in
flavouring cooking. Spices are often the
currency of the developing countries. The
primary producers of spices include India (by
far the largest producer and exporter), Egypt,
China, Indonesia, Malaysia, Mexico, Turkey and
Brazil. Since spices are always in demand in the
industrialized world, export of these basic
agricultural commodities by developing countries
can be relied upon to earn valuable foreign
exchange. The major importers are the US,
Europe, Japan, East Asian and Middle Eastern
countries. An added challenge is that exporting
countries with low technology infrastructure
have not only to satisfy highly demanding
customers, but also to comply with the stringent
food laws of the importing country.
India is the biggest producer, consumer
and exporter of spices, with a 45 per cent share
by volume and 23 per cent share by value, in the
world market. The Indian spice export basket
consists of around 50 spices in whole form and
more than 80 products in value added form.
However, a few spices and value added
forms constitute a major segment of the
country’s total export earnings. Spices,
contributing more than 80 per cent in quantity
and 90 per cent by value, based on a three-year
(1997-98 to 1999-2000) average of exports, are
classified as the major
contributors.
In addition to
maize and groundnut, many commodities including
spices (JELINEK et al, 1989; VASANTHI and BHAT,
1998) are contaminated by aflatoxins. Spices
like chilies, turmeric, black pepper, coriander,
dry ginger are contaminated with aflatoxins
during pre-harvest, post-harvest, storage
conditions and during transport. Chilies are
commonly pulled exclusively in countries with
hotter climate. Chilies are used for the
preparation of Gargoyle chili sauce &
Classic chili sauce, Cornish winter sauce.
Aflatoxins tend to pose very serious problems
for chilies. Eighty percent of samples of
chili-based spices imported to Australia have
been found to be contaminated with the potent
cancer-causing agent,
aflatoxin.
Materials
& Methods
In order to conduct surveys for the
occurrence of aflatoxins, it is essential to
develop cost-effective and rapid methods for
their quantitative estimation. ICRISAT has been
investigating the problem of aflatoxin
contamination for over 20 years. Many methods
are available for the estimation of aflatoxins.
They include biological, Physicochemical and
immuno-chemical methods. Immuno-chemical
methods are simple, rapid, sensitive and
specific. In this project the "INDIRECT
COMPETITIVE ELISA" is usedfor the
estimation of aflatoxins in groundnut, maize,
wheat flour, corn flour, coriander, pepper,
chilies, turmeric, sorghum, dry ginger samples.
The basic principle lies in the ELISA procedure
is immobilizing the toxin antigen
on to a solid surface, followed by adding the
sample containing toxin antigen and antiserum to
facilitate a competition and probing with
specific immunoglobulins carrying an enzyme
label. The enzyme retained in case of positive
reaction is detected by adding the suitable
substrate, PNPP. The enzyme converts substrate
to a product, which can be easily recognized by
its colour. The aflatoxin content of the sample
isdetermined by
AFB1
regression analysis
curve.
Results:
Different types of samples such as
groundnut, maize, wheatflour, cornflour,
sorghum, redchilies, turmeric, dryginger,
coriander, black pepper were collected randomly
from A.P markets (Rangareddy, Hyderabad,
Nalgonda, Warangal districts) and were estimated
for aflatoxin content through “INDIRECT
COMPETITIVE ELISA” method. The following results
were observed. The samples were collected in
March & July 2003.
Aflatoxin Contamination in
different types of samples collected from A.P.
markets, March 2003.
Table1
Aflatoxin
Contamination in different types of samples
collected from A.P. markets, July
2003.
Table2
Discussions:
Groundnut:
In
March samples aflatoxin content varied from 0.2
- 785µg/kg, in July samples aflatoxin content
varied from 0.9-8646.5µg/kg. In March 82% of
samples are consumable where as in July 40%
samples are consumable. When we compare the
aflatoxin content between these two periods,
high aflatoxin content was found in July samples
due to the following reasons:
·
Storage
problem
·
Higher humidity during this period
of March to July
·
Remaining bad quality produce from
last year
Maize:
In March samples aflatoxin
content varied from 4.2 - 318µg/kg, in July
samples aflatoxin content varied from
8.6-109.2µg/kg. Maximum aflatoxin content was
found in one of the March samples, as this may
be due to the highly toxigenic strain present in
that sample. In March
63% of samples are consumable where as in July
42% samples are consumable.
Wheatflour:
In March samples aflatoxin
content varied from 0.1 - 34µg/kg, in July
samples aflatoxin content varied from
0.6-235.1µg/kg. In March 98% of samples are
consumable where as in July 44% samples are
consumable. The maximum aflatoxin content found
in March samples is 34µg/kg where as in July
samples it is 235.1µg/kg.
Cornflour:
In March samples aflatoxin content varied
from 0.7 – 8.4µg/kg, in July samples aflatoxin
content varied from 0.4-268µg/kg. In March 100%
of samples are consumable where as in July 44%
samples are consumable. The maximum aflatoxin
content found in March samples is 8.4µg/kg where
as in July samples it is 268µg/kg.
Sorghum:
In March samples aflatoxin
content varied from 1.4– 421µg/kg, in July
samples aflatoxin content varied from
0.1-123µg/kg. In March 95% of samples are
consumable where as in July 63% samples are
consumable. The maximum aflatoxin content found
in March samples is 421µg/kg where as in July
samples it is 123µg/kg. This may be due to the
highly toxigenic strain in that March
sample.
Redchilies:
In March samples aflatoxin content varied
from3.7 – 257µg/kg, in July samples aflatoxin
content varied from 1.0-174.5µg/kg. In March 58%
of samples are consumable where as in July 62%
samples are consumable. The maximum aflatoxin
content found in March samples is 257µg/kg where
as in July samples it is 174.5µg/kg.
Turmeric:
In March samples aflatoxin
content varied from3.7 – 221µg/kg, in July
samples aflatoxin content varied from
0.3-167µg/kg. In March 52% of samples are
consumable where as in July 63% samples are
consumable. The maximum aflatoxin content found
in March samples is 221µg/kg where as in July
samples it is 167µg/kg.
Dryginger:
In March samples aflatoxin
content varied from5.8 – 137µg/kg, in July
samples aflatoxin content varied from
17-72.1µg/kg. In March 64% of samples are
consumable where as in July 28% samples are
consumable. The highest aflatoxin content found
in March samples is 137µg/kg where as in July
samples it is 72.1µg/kg.
Coriander: In March samples aflatoxin
content varied from15.9–71µg/kg, in July samples
aflatoxin content varied from 1.0-65µg/kg. In
March 52% of samples are consumable where as in
July 68% samples are consumable. The maximum
aflatoxin content found in March samples is
71µg/kg where as in July samples it is
65µg/kg.
Blackpepper:
In March samples aflatoxin
content varied from1.2–275µg/kg, in July samples
aflatoxin content varied from 1.1-430µg/kg. In
March 26% of samples are consumable where as in
July 37% samples are consumable. The maximum
aflatoxin content found in March samples is
275µg/kg where as in July samples it is
430µg/kg.
Aflatoxin contamination in the
A.P market samples.
The following table shows the
difference in aflatoxin contamination between
two periods March and July 2003. (According to
Indian
standard).
Aflatoxin contamination in
the A.P market
samples.
The
following table shows the difference in % of
samples with <30mg/kg between
two periods March and July 2003. (According to
Indian
standard).
Conclusions:
Due to the hazards to
humans and livestock from the ingestion of
aflatoxin contaminated food products like
Groundnut (legume crop), Maize, Sorghum,
Wheatflour, Cornflour (cereals & cereal
products), Redchilies, Turmeric, Drygiger,
Coriander, Blackpepper (spices), many countries
have established permissible limits for
aflatoxins in foods and feeds. These permissible
levels vary widely among those countries that
have regulations on aflatoxins. These
permissible levels also vary with the type of
commodity.
The table
below show the current European Union
legislative limits (EU Commission Regulations
446/2001, 257/2002 and
472/2002).
According
to the EU limits, the maximum admissible level
is 4ug/kg for groundnuts and cereals (sorghum,
maize, wheatflour & cornflour) and 10ug/kg
for spices like chilies, chilipowder,
blackpepper, ginger and turmeric. So the samples
satisfying these regulations can be exported to
EEC countries (EEC includes Belgium, Denmark,
France, Germany, Greece, Ireland, Italy,
Luxembourg, The Netherlands and United Kingdom).
The maximum permissible level of aflatoxin in
India is 30m
g/kg for
food and 1000m
g/kg for
feed. So the samples which doesn’t exceed
maximum permissible level for food
(30m
g/kg) are
safe for human consumption and the samples which
doesn’t exceed maximum permissible level for
feed (1000 m
g/kg) are
safe for animal consumption. International
market requirements vary from 0 to 10
m
g/kg of
aflatoxin, so the samples having aflatoxin in
the range of 0 to 10 m
g/kg can be used in international
trade.
Groundnut
March
:
According to the EU
standard, out of 75 samples 17 samples have
<4m
g/kg of aflatoxin & can be exported
to the EU countries.
According to
the Indian standard, out of 75 samples 62
samples (82%) contain <30 m
g/kg & are safe
for human consumption. Remaining 13 samples
(17%) found to exceed the maximum permissible
level (>30ug/kg) & are not safe for human
consumption. All the 75 samples contain <1000
m
g/kg of
aflatoxin & are safe for animal consumption.
36 (48%) samples contain <10 m
g/kg of
aflatoxin and can sold in international trade.
July
:
According to the EU
standard, out of 51 samples 4 samples have
<4m
g/kg and can
be exported to the EU countries. According to
the Indian standard, out of 51 samples 21
samples (41%) contain <30 m
g/kg & are safe
for human consumption. Remaining 30 samples
(58%) found to exceed the maximum permissible
level for food & are not safe for human
consumption. Out of 51 samples 45 samples (88%)
contain <1000 m
g/kg of
aflatoxin & are safe for animal consumption.
8 (15%) samples contain <10 m
g/kg of
aflatoxin and can sold in international trade.
Maize
March
:
None of the maize samples
satisfy the EU standard. According to the Indian
standard,out of 46 samples 29
samples (63%) contain <30 m
g/kg & are safe
for human consumption. Remaining 17 samples
(36%) found to exceed the maximum permissible
level & are not safe for human consumption.
All the 46 samples contain <1000 m
g/kg of aflatoxin & are safe
for animal consumption. 15 (32%) samples contain
<10 m
g/kg of aflatoxin and can sold in
international trade.
July
:
None of the maize
samples satisfy the EU standard. According to
the Indian standard, out of 19
samples 8 samples (42%) contain <30
m
g/kg &
are safe for human
consumption. Remaining 11 samples (57%)
found to exceed the maximum permissible l evel
for food & are not safe
for human consumption. All the 19 samples
contain <1000 m
g/kg of aflatoxin &
are safe for animal consumption. 2 (10%) samples
contain <10
m
g/kg of
aflatoxin and can sold in
international trade.
Cornflour
March
:
According to the EU
standard, out of 28 samples 20 samples have
<4m
g/kg of
aflatoxin & can be exported to the EU
countries According to the Indian standard, all
the 28 cornflour samples contain <30
m
g/kg & are safe
for human consumption.All the
28(100%) samples contain <10 m
g/kg of
aflatoxin and can sold in international
trade.
July
:
According to the EU
standard, out of 34 samples 4 samples have <4
m
g/kg &
can be exported to the EU countries. According
to the Indian standard, out of 34
samples 15 samples (44%) contain <30
m
g/kg & are safe for
human consumption. Remaining 19 samples
(55%) found to exceed the maximum
permissible l evel
for food & are not
safe for human consumption. 6 (17%) samples
contain <10
m
g/kg of
aflatoxin and can sold in
international trade.
Wheatflour
March
:
According to the EU
standard, out of 93 samples 33 samples have
<4m
g/kg of
aflatoxin & can be exported to the EU
countries According to the Indian standard, out
of 93 samples 92 (98%) samples contain
<30m
g/kg & are safe
for human consumption. Remaining 1 sample (1%)
found to exceed the maximum permissible level
& is not safefor human
consumption. 65 samples (69.8%) contain
<10 m
g/kg of
aflatoxin and can sold in international trade.
All the 93 samples are safe for animal
consumption.
July
:
According to the EU
standard, out of 54 samples 5 samples have
<4m
g/kg &
can be exported to the EU countries. According
to the Indian standard, out of 54 samples 24
(44%) samples contain <30m
g/kg & are safe
for human consumption. Remaining 30 samples
(55%) found to exceed the maximum permissible
level for food & are not safefor human
consumption. All the 54 samples contain <1000
m
g/kg of
aflatoxin & are safe for animal
consumption. 8 samples (14%) contain
<10 m
g/kg of
aflatoxin and can sold in international trade.
Sorghum
March
:
According to the EU
standard, out of 75 samples 17 samples have
<4m
g/kg of
aflatoxin & can be exported to the EU
countries. According to the Indian standard, out
of 75 samples 72 samples (96%) contain
<30m
g/kg & are safe
for human consumption. Remaining 3 samples (4%)
found to exceed the maximum permissible level
& are not safe for human consumption. All
the 75 samples have <1000ug/kg of aflatoxin
and can be safe for animal consumption. 55
samples (73.3%) contain <10 m
g/kg of
aflatoxin and can sold in international trade.
July
:
According to the EU
standard, out of 47 samples 7 samples have
<4m
g/kg &
can be exported to the EU countries. According
to the Indian standard, out of 47 samples 30
samples (63%) contain <30m
g/kg & are safe for human
consumption. Remaining 17 samples (36%) found to
exceed the maximum permissible level for food &
are not safe for human consumption. All the 47
samples contain <1000m
g/kg of
aflatoxin & are safe for animal consumption.
18 samples (38%) contain <10 m
g/kg of
aflatoxin and can sold in international trade.
Chilies
March
:
According to the EU
standard, out of 50 samples 9 samples have
<10m
g/kg of
aflatoxin & can be exported to the EU
countries According to the Indian standard, out
of 50 samples 29 samples (58%) contain
<30m
g/kg & are safe
for human consumption. Remaining 21 samples
(42%) found to exceed the maximum permissible
level & are not safe for human consumption.
9 (18%) samples contain <10 m
g/kg of
aflatoxin and can sold in international
trade.
July
:
According to the EU
standard, out of 49 samples 16 samples have
<10m
g/kg &
can be exported to the EU countries. According
to the Indian standard, out of 49 samples 31
samples (63%) contain <30m
g/kg &
are safe for human consumption. Remaining 18
samples (36%) found to exceed the maximum
permissible level & are not safe for human
consumption. 16 (32%) samples contain
<10m
g/kg of
aflatoxin and can sold in international trade.
Turmeric
March
:
According to the EU
standard, out of 50 samples 5 samples have
<10m
g/kg of
aflatoxin & can be exported to the EU
countries. According to the Indian standard, out
of 50 samples 26 samples (52%) contain
<30m
g/kg & are safe
for human consumption. Remaining 24 samples
(48%) found to exceed the maximum permissible
level & are not safe for human consumption.
5 (10%) samples contain <10 m
g/kg of
aflatoxin and can sold in international trade.
July
:
According to the EU
standard, out of 47 samples 20 samples have
<10m
g/kg &
can be exported to the EU countries. According
to the Indian standard, out of 47 samples 30
samples (63%) contain <30m
g/kg &
are safe for human consumption. Remaining 17
samples (36%) found to exceed the maximum
permissible level for food & are not safe
for human consumption. 20 (42%) samples contain
<10 m
g/kg of
aflatoxin and can sold in international trade.
Dryginger
March
:
According to the EU
standard, out of 50 samples 1 sample have
<10m
g/kg of
aflatoxin & can be exported to the EU
countries. According to the Indian standard, out
of 50 samples 32 samples (64%) contain
<30m
g/kg & are safe
for human consumption. Remaining 18 samples
(37%) found to exceed the maximum permissible
level & are not safe for human consumption.
1 sample (2%) contain <10 m
g/kg of
aflatoxin and can sold in international trade.
July
:
None of the samples satisfy
the EU regulations. According to the Indian
standard, out of 39 samples 11 samples (28%)
contain <30m
g/kg &
are safe for human consumption. Remaining 28
samples exceed the maximum permissible level
& are not safe for the human
consumption.
Coriander
March
:
None of the samples satisfy
the EU standard. According to the Indian
standard, out of 50 samples 26 samples (52%)
contain <30m
g/kg & are safe
for human consumption. Remaining 24 samples
(48%) found to exceed the maximum permissible
level & are not safe for human consumption.
All the 50 samples can’t sold in international
trade as they exceed the international
requirement.
July
:
According to the EU
standard, out of 56 samples 15 samples have
<10m
g/kg &
can be exported to the EU countries. According
to the Indian standard, out of 56 samples 39
samples (68%) contain <30m
g/kg & are safe
for human consumption. Remaining 17 samples
(30%) found to exceed the maximum permissible
level for food & are not safe for human
consumption. 15 samples (26%) contain <10
m
g/kg of
aflatoxin and can sold in international trade.
Black pepper
March
:
According to the EU
standard, out of 50 samples 6 samples have
<10m
g/kg of
aflatoxin & can be exported to the EU
countries According to the Indian standard, out
of 50 samples 13 samples (26%) contain
<30m
g/kg & are safe
for human consumption. Remaining 37 samples
(74%) found to exceed the maximum permissible
level & are not safe for human consumption.
6 samples (12%) contain <10 m
g/kg of
aflatoxin and can sold in international trade.
July
:
According to the EU
standard, out of 54 samples 10 samples have
<10m
g/kg &
can be exported to the EU countries. According
to the Indian standard, out of 54 samples 20
samples (37%) contain <30m
g/kg &
are safe for human consumption. Remaining 34
samples (62%) found to exceed the maximum
permissible level for food & are not safe
for human consumption. 10 samples (18%) contain
<10 m
g/kg of
aflatoxin and can sold in international trade.
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