aflatoxin

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


CornPeanuts

CottonseedBrazil nuts

Pistachio nutsCopra

FigsAlmonds

PecansWalnutsSultanas

Spices

SoybeansPulses

SorghumMilletWheatOats

BarleyRice

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 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


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 [email protected]

[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

aflatoxin

Documents

dietary aflatoxins

aflatoxin b1

aflatoxin exposure

aflatoxins includecereal

world food crops

developing world

world countries

aflatoxin m1dna conjugate