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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
Chandra Prakash Singh - Analytical Diligence Services
The name Aflatoxin comes from
A (Aspergillus)
FLA (flavus)
toxin.
Aflatoxins
Chandra Prakash Singh - Analytical Diligence Services
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
Chandra Prakash Singh - Analytical Diligence Services
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.
Chandra Prakash Singh - Analytical Diligence Services
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).
Chandra Prakash Singh - Analytical Diligence Services
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.
Chandra Prakash Singh - Analytical Diligence Services
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).
Chandra Prakash Singh - Analytical Diligence Services
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
Chandra Prakash Singh - Analytical Diligence Services
Dairy products (Milk, Cheese, Fluid milk)
Green coffee
Dry fruits
Human biological fluids (human urine, milk
and blood samples.)
Eggs
Liver
Medicinal plant
Chandra Prakash Singh - Analytical Diligence Services
CornPeanuts
CottonseedBrazil nuts
Pistachio nutsCopra
FigsAlmonds
PecansWalnutsSultanas
Spices
SoybeansPulses
SorghumMilletWheatOats
BarleyRice
Natural occurrence and Aflatoxin risk
High risk
Moderate risk
Low risk
Chandra Prakash Singh - Analytical Diligence Services
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
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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
Chandra Prakash Singh - Analytical Diligence Services
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
Chandra Prakash Singh - Analytical Diligence Services
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.
Chandra Prakash Singh - Analytical Diligence Services
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
Chandra Prakash Singh - Analytical Diligence Services
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
Chandra Prakash Singh - Analytical Diligence Services
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
Chandra Prakash Singh - Analytical Diligence Services
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).
Chandra Prakash Singh - Analytical Diligence Services
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
Chandra Prakash Singh - Analytical Diligence Services
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
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Aflatoxin Melting-point (ºC)
B1 268-269
B2 287-289
G1 244-246
G2 237-239
M1 299
Melting-point
Chandra Prakash Singh - Analytical Diligence Services
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
Chandra Prakash Singh - Analytical Diligence Services
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
Chandra Prakash Singh - Analytical Diligence Services
“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
Chandra Prakash Singh - Analytical Diligence Services
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.
Chandra Prakash Singh - Analytical Diligence Services
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.
Chandra Prakash Singh - Analytical Diligence Services
Farming Storage ProcessingTransport
DistributionRetail
Consumer
ENVIRONMENT
Natural ToxinsMycotoxins
Veterinarydrugs
Mycotoxins/ Aflatoxin
In situ formationdue to heat, pH,etc.
Migrationfrompackaging
Heat-inducedcarcinogense.g. heterocyclicaromatic amines,acrylamide
Routes of Aflatoxin contamination
Chandra Prakash Singh - Analytical Diligence Services
Pre- Harvest Harvesting
Storage
Preventing or Reducing Aflatoxin Exposure
1. Pre-Harvest
2. Post-Harvest : Drying & Storage
Chandra Prakash Singh - Analytical Diligence Services
Timing of planting;
Crop planted;
Genotype of seed planted;
Irrigation;
Insecticides;
Competitive exclusion;
Timing of harvest;
Pre-Harvest
Chandra Prakash Singh - Analytical Diligence Services
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
Chandra Prakash Singh - Analytical Diligence Services
Thanks for your attention
Analytical Diligence [email protected]
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