Aflatoxin.pdf

Mycotoxins = Toxic metabolites of fungi.
Mycotoxins contaminate the wide variety of food
as a result of fungal infection in crops, during
growth or in storage.
Mycotoxins
Chandra Prakash Singh - Analytical Diligence Services

Mycotoxins Main Producing Fungi
Aflatoxins B1, B2, G1, G2 Aspergillus flavus, A.
parasiticus, A. nomius
Ochratoxin A Penicillium verrucosum, A.
alutaceus, A.carbonarius
Patulin P. expansum, A. clavatus,
Byssochlamys nivea
Fumonisins Fusarium moniliforme, F.
proliferatum
Deoxynivalenol
(trichothecenes)
F. graminearum, F. culmorum,
F. crookwellense
Zearalenone F. graminearum, F. culmorum,
F. crookwellense
In summary: 6 major chemical types of mycotoxins

The name Aflatoxin comes from
A (Aspergillus)
FLA (flavus)
toxin.
Aflatoxins

Aflatoxins are a group of structurally related toxic
secondary metabolites produced by three species:
Aspergillus flavus, Aspergillus parasiticus and the rare
species A. nomius (Kurtzman et aL., 1987) and known to
be highly toxic and potential carcinogens.
Aflatoxins were first identified in 1961 in animal feed
contaminated by Aspergillus parasiticus (Sargeant et al.,
1961).
Aflatoxins

The things which evoke scientist to study Aflatoxin
Consequently, more than five billion people in
developing country worldwide are at risk of chronic
exposure to Aflatoxins through contaminated foods
and medicinal plants. Aflatoxin-associated health
effects pervade the developing world.
Aflatoxins are the only mycotoxins currently
regulated by the U.S. Food and Drug Administration.

According to International Agency for Research on
Cancer(IARC), Evidence of acute aflatoxicosis in
humans has been reported from many parts of the
world, namely the Third World Countries, like Taiwan,
Ouganda, India, and many others. And In 1988, the
IARC placed aflatoxin B1 on the list of human
carcinogens.
This is supported by a number of epidemiological
studies done in Asia and Africa that have demonstrated
a positive association between dietary aflatoxins and
Liver Cell Cancer (LCC).

Studies have shown that concurrent infection with the
Hepatitis B virus (HBV) during aflatoxin exposure
increases the risk of hepatocellular carcinoma (HCC).
As HBV interferes with the ability of hepatocytes to
metabolize aflatoxins, an aflatoxin M1-DNA conjugate
exists for a longer period of time in the liver, increasing
the probability of damage to tumor suppressor genes
such as p53.

FAO estimates, 25% of the world food crops are
affected by mycotoxins each year.
Crop loss due to aflatoxins contamination costs US
producers more than $100 million per year on average
including $ 26 millions to peanuts ($69.34/ha).

Food products contaminated with aflatoxins include
Cereal (maize, sorghum, pearl millet, rice, wheat),
Oil seeds (groundnut, soybean, sunflower, cotton),
Spices (chillies, black pepper, coriander, turmeric,
zinger),
Tree nuts (almonds, pistachio, walnuts, coconut)
Natural occurrence of Aflatoxin

Dairy products (Milk, Cheese, Fluid milk)
Green coffee
Dry fruits
Human biological fluids (human urine, milk
and blood samples.)
Eggs
Liver
Medicinal plant

Corn
Peanuts
Cottonseed
Brazil nuts
Pistachio nuts
Copra
Figs
Almonds
Pecans
Walnuts
Sultanas
Spices
Soybeans
Pulses
Sorghum
Millet
Wheat
Oats
Barley
Rice
Natural occurrence and Aflatoxin risk
High risk
Moderate risk
Low risk

The aflatoxin-producing Aspergillus species, and
consequently dietary aflatoxin contamination, are
ubiquitous in are as of the world with hot, humid
climates, including sub-Saharan Africa and
Southeast Asia. Exposure in those countries results
from contamination of dietary staples and is
therefore likely to be chronic.
Geographical Occurrence

Origin No. of lots Lots %
Determinable > 26µ g/kg
China 2585 15 2.5
India 1453 92 58.0
Sudan 932 94 78.0
Argentina 446 40 4.0
South Africa 112 41 95.0
Malawi 80 60 2.0
FAO/WO/UNEP Monitoring Program: Afatoxins in raw,
shelled groundnuts imported into the USA, 1981

It is probably not possible to eliminate completely
exposure of humans to aflatoxins.
In 1987, at least 50 countries had existing or
proposed regulations for aflatoxins in foodstuffs. And
the maximum limits range from none detectable to 50
µg/kg of food for either the sum of Aflatoxins B1, B2,
G1 and G2 or for Aflatoxin B1 alone; 5 µg/kg is the
commonest maximal limit.
Regulations and guidelines

ln 1987, aflatoxin M1 levels in dairy products were
regulated in 14 countries. The tolerances in infants' and
children's food were 0.05-0.5 µg/kg milk.
Aflatoxins were reviewed by a joint FAO/WHO
Expert Committee on Food Additives in 1987 (WHO,
1987).
No acceptable daily intake was given; it was
recommended that human intake be reduced to the
lowest practicable level.

20 ppb For corn and other grains intended for immature
animals (including immature poultry) and for dairy
animals, or when its destination is not known;
20 ppb For animal feeds, other than corn or cottonseed
meal;
100 ppb For corn and other grains intended for breeding
beef cattle, breeding swine, or mature poultry;
200 ppb For corn and other grains intended for finishing
swine of 100 pounds or greater;
200 ppb For corn and other grains intended for finishing (i.e.
feedlot) beef cattle and for cottonseed meal
intended for beef cattle, swine or poultry.
The FDA will consider action if Aflatoxin levels exceed

Aflatoxins are normally refers to the group of
difuranocoumarins and classified in two broad groups
according to their chemical structure.
A. Difurocoumarocyclopentenone series (AFB1, AFB2,
AFB2A, AFM1, AFM2, AFM2A and aflatoxicol)
B. Difurocoumarolactone series (AFG1, AFG2, AFG2A,
AFGM1, AFGM2, AFGM2A and AFB3).
Aflatoxins and its Physio-chemical dimensions

Main type
Aflatoxin B1
Aflatoxin B2
Aflatoxin G1
Aflatoxin G2
Major metabolites of Aflatoxin B1
Aflatoxin M1
Aflatoxin D1
Aflatoxin P1
Aflatoxin Q1
Aflatoxin M2
Aflatoxin B2a
Aflatoxicol
Aflatoxicol H1
Aflatoxcol M1

The relative proportions of Aflatoxin B1, Aflatoxin G1,
Aflatoxin B2 and Aflatoxin G2 on crops depend on the
particular Aspergillus species present.
A. flavus produces aflatoxins B1 and B2, whereas
 A. parasiticus produces aflatoxins B1, B2, G1 and G2
(Dorner et al., 1984).

ELISA technique (Enzyme linked immunosorbent assay)
Fluorometry
TLC (Thin Layer Chromatography)
HPLC - FLD (High Performance Liquid Chromatography
– Fluorescence Detector)
HPTLC (High Performance Thin Layer Chromatography)
LC-MS
Method of analysis

Colourless to pale-yellow crystals.
Intensely fluorescent in ultraviolet light.
Aflatoxins B1 and B1 = blue fluorescence
Aflatoxins G1, G1 = yellow-green fluorescence
Aflatoxin M1 = blue-violet fluorescence
Description

Aflatoxin Melting-point (ºC)
B1 268-269
B2 287-289
G1 244-246
G2 237-239
M1 299
Melting-point

Very slightly soluble in water (10-30 µg/ml)
Insoluble in non-polar solvents
Freely soluble in moderately polar organic solvents
(e.g., chloroform and methanol)
Especially in dimethyl sulfoxide.
Unstable to ultraviolet light in the presence of oxygen,
to extremes of pH (< 3, >10) and to oxidizing agents.
Solubility
Stability

The lactone ring is susceptible to alkaline hydrolysis.
Aflatoxins are also degraded by reaction with ammonia
or sodium hypochlorite.
Reactivity
Structurally the dihydrofuran moiety, containing double
bond, and the constituents liked to the coumarin moiety
are of importance in producing biological effects.
Biological effects

“No animal species is resistant to the acute toxic effects
of aflatoxins; hence it is logical to assume that humans
may be similarly affected.”
The aflatoxins display potency of toxicity,
carcinogenicity, mutagenicity in the order of AFB1 >
AFG1 > AFB2 > AFG2 as illustrated by their LD50
values for day-old ducklings.
Life-threatening effect of Aflatoxins and its mechanism

1. Liver damage 2. Liver necrosis
3. Liver cirrhosis 4. Fever
5. Progressive jaundice 6. Limb swelling
7. Pain Vomiting 8. Enlarged liver
Symptoms of Aflatoxin B1 exposure
Aflatoxicosis in humans
The syndrome is characterized by vomiting, abdominal
pain, pulmonary edema, convulsions, coma, and death
with cerebral edema and fatty involvement of the liver,
kidneys, and heart.

Schematic representation of AFB1 metabolism highlighting the
formation of its critical product AFB1-exo-8,9-epoxide, its DNA-
and protein adducts and major urinary metabolites.

Farming Storage Processing
Transport
Distribution
Retail
Consumer
ENVIRONMENT
Natural Toxins
Mycotoxins
Veterinary
drugs
Mycotoxins
/ Aflatoxin
In situ formation
due to heat, pH,
etc.
Migration
from
packaging
Heat-induced
carcinogens
e.g. heterocyclic
aromatic amines,
acrylamide
Routes of Aflatoxin contamination

Pre- Harvest Harvesting
Storage
Preventing or Reducing Aflatoxin Exposure
1. Pre-Harvest
2. Post-Harvest : Drying & Storage

Timing of planting;
Crop planted;
Genotype of seed planted;
Irrigation;
Insecticides;
Competitive exclusion;
Timing of harvest;
Pre-Harvest

Hand sorting
Drying on mats
Sun drying
Rodent control
Storing bags on
wooden pallets or
elevated off ground
Insecticides
Post-Harvest: Drying & Storage
Hand sorting
Winnowing
Washing
Nixtamalization
Acidification
Chemoprotectant
Enterosorption
Crushing and dehulling

Thanks for your attention
Analytical Diligence Services
analyticaldiligenceservices@gmail.com
singhprakash06@gmail.com
Special thanks
to
Mr. Rajesh Garg, Mrs. Anurekha Jain and all BRNCOP family
B.R. Nahata College of Pharmacy, Mandsaur (M.P) 458001
&
Ashwagandha Technology Development & Extension Center
National Medicinal Plant Board, Department of AYUSH, GOI

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