DESCRIPTION DETERMINATION AND CONTROL

1. MADE BY
ASMA NASIR
TYBPHARM.

2. Group of secondary metabolites derived from
polyketides produced by fungal species such as:
A. Aspergillus flavus
B. Parasiticus
C. rarely A. nomius (Payne and Brown, 1998).
Under humid conditions, these fungi are produced in
 Groundnuts, corn/ maize, sorghum, rice, spices
and other agricultural products
 Livestock feeds.
 Medicinal herbs.

3. Aflatoxin contamination
 can occur at any stage of food production
from pre-harvest to storage stages of the food chain
Aflatoxin accumulation is dependent on
 Environmental factors such as:
Moisture
Temperature
Plant density
 poor harvest practices and
 Improper grain storage.

4.  The removal of aflatoxins is very difficult due to
their stability and thermal resistance in dried
products
 Melting points > 250C
 stable at a pH range of 3 to 10.
 resistant to food processing
 Thus remain unchanged throughout the food
chain.

5. There are more than 20 known aflatoxins, but the
four main ones are:
 aflatoxin B1 (AFT-B1)
 aflatoxin B2 (AFT-B2)
 aflatoxin G1 (AFT-G1)
 aflatoxin G2 (AFT-G2)
AFTs B series are produced by A. flavus, A. parasiticus and A.
nomius
AFTs G series are produced by A. parasiticus and A. nomius
The level of toxicity associated with aflatoxin varies with the types present
AFT-B1 > AFT-G1 > AFT-B2 > AFT-G2

6. Aflatoxins are of great concern to public health
because of their ability to:
 accumulate in the body.
 found in edible tissues of animals, such as liver
and muscles.
 found in animal food products such as milk and
eggs.
 found in human maternal breast milk and
maternal cord blood.

7. Exposure to aflatoxin can lead to several health related
conditions including
 acute and chronic aflatoxicosis
 aflatoxin-related immune suppression
 liver disease (liver cancer and liver cirrhosis)
 adverse pregnancy outcomes, including
intrauterine growth restriction
premature delivery
pregnancy loss
 nutrition-related problems in children such as stunted
growth
 mutagenesis

8. The major sources of aflatoxins are fungi such as
Aspergillus flavus
Aspergillus parasiticus
Aspergillus nomius
Taxonomically, these fungal species are from:
Phylum: Ascomycota
Class: Eurotiomycetes
Order: Eurotiales
Family: Trichocomaceae

9. The fungal species infect many crops in the field,
during harvest, in storage,
and during processing.
 A. flavus is dominant in corn, cottonseed, and tree
nuts
 A. parasiticus is dominant in peanuts.
 A. nomius has been reported from tree nuts,
sugarcane and insects such as alkali bees
They can grow at temperatures ranging between 12
and 48C and 40degrees latitude north and south

10.  TESTS FOR AFLATOXINS
 These tests are designed to detect the possible
presence of aflatoxins B1, B2, G1 and G2, which
are highly toxic contaminants in any material of
plant origin.

11.  RECOMMENDED PROCEDURE
 It uses a multifunctional column, which contains lipophilic
and charged active sites, and high-performance liquid
chromatography
 (HPLC) using fluorescence detection to determine
aflatoxins B1, B2, G1 and G2.
 Standard solutions of aflatoxin B1, B2, G1 and G2 (2.5
ng/ml)
 Stock standard solution. Weigh exactly 1.0 mg each of
crystalline material of aflatoxins
 B1, B2, G1 and G2 and dissolve in 50 ml of toluene-
acetonitrile (9:1) solution by
 shaking vigorously in a glass flask to obtain a standard
stock solution (20 μg/ml).
 This standard solution should be kept in a tightly sealed
container, covered with
 aluminium foil, and kept in a refrigerator at 4 °C in the
dark.

12. WORKING STANDARD SOLUTION.
0.5 ml of stock standard solution is added to tolueneacetonitrile
(9:1) solution to give 200 ml (working standard solution (50 ng/ml).
Standard solution. Take 1.0 ml of working standard solution and
add to tolueneacetonitrile (9:1) solution to give 20 ml (final
standard solution (2.5 ng/ml).
STANDARD SOLUTION FOR LIQUID CHROMATOGRAPHY
ANALYSIS.
Transfer 0.25 ml of the final standard solution (as described above)
into a glass centrifuge tube and evaporate to dryness at 40 °C or
by using a nitrogen air stream. To derivatize1 aflatoxins B1 and
G1 (precolumn derivatization), add 0.1 ml of trifluoroacetic acid
(TFA) solution to the residue in the tube, tightly seal the tube
and shake vigorously. Allow the tube to stand at room
temperature for 15 min in the dark. Add 0.4 ml of
acetonitrile:water
(1:9) solution to the tube. A 20-μl portion of the sample solution in
the tube is
subjected to liquid chromatography analysis.

13. PREPARATION OF SAMPLE.
Grind the herbal material for testing to a uniform consistency using a coffee mill,
and extract a 50-g test sample with 400 ml of acetonitrile-water (9:1) by shaking
vigorously in a glass flask fitted with a stopper for 30 minutes or by using a
mechanical blender for 5 minutes. Filter the solution through a filter paper
or centrifuge. Transfer a 5-ml portion of the filtrate, or the top clean layer, to a
multifunctional column (such as a MultiSep #228 cartridge column (Romer Labs)
or an Autoprep MF-A [Showa-denko]) and pass through at a flow rate of 1 ml/
minute. The aflatoxins present in a sample are passed through the column as the
first eluate. Obtain the first 1-ml of the eluate as the test solution.
Evaporate 0.5 ml of the test solution in a glass centrifuge tube to dryness at 40 °C
or by using a nitrogen air stream to remove solvent.
To derivatize aflatoxins B1 and G1 (precolumn derivatization), add 0.1 ml of
trifluoroacetic acid (TFA) solution to the residue in the tube, tightly seal the tube
and shake vigorously. Allow the tube to stand at room temperature for 15 minutes
in the dark. Add 0.4 ml of acetonitrile-water (1:9) solution to the tube. Subject a
20-μl portion of the sample solution in the tube to liquid chromatography analysis.

14. INTERPRETATION OF THE RESULTS.
Compare the retention time of peak area or peak
heights of the aflatoxin under study in the
chromatograms. If they are bigger or higher than
those obtained in a standard solution of the
aflatoxin under investigation, it should be
regarded as a positive result for the presence of
aflatoxin in the sample solution.

15.  PHYSICAL TREATMENT
1) Drying seeds and commodities to the safe moisture level
(<9-11%).
2) Maintenance of the container or store house at low
temperature and humidity.
3) Keep out insects and pests from the storage.
4) Gamma-irradiation of large-scale commodities.
5) Dilution of the contaminated feed with safe feed.

16. CHEMICAL TREATMENT
1. Use of fungicides:
– Acetic acid, propionic acid, benzoic acid, citric acid and their
sodium
salts, copper sulfate at rate of 0.2–0.4 % in feed.
2. Use of fumigants
– Ammonia at rate of 0.2-0.4%
3. Addition of herbal extracts
– Garlic, onion, clove oil, turmeric powder, thyme at rate of
0.25-0.5%

17. BIOLOGICAL TREATMENT
a) Enzymes
– Chitin and glucan, as constituents of fungal cell
wall, could be enzymatically hydrolyzed resulting
in killing of mycelia or spore of fungi.
– Anti-fungal enzymes, chitinase and Beta -1,3
glucanase:
• They are found in plant seeds
• They act as defense against pathogenic fungi
– Plant seeds rich in these anti-fungal enzymes likely
to resist the infestation of fungi.

18. b) Fungal bio-competition.
– Application of non-toxigenic strains of Aspergillus
flavus and Aspergillus parasiticus to soil in maize
plots, lead to reduction in colonization of toxigenic
fungi in subsequent years.
– The non-toxigenic biocompetitive Aspergillus
strains out-compete the toxigenic isolates,
resulting in reducing pre-harvest contamination
with aflatoxin in peanut and cotton.

19. c) Lactic acid bacterial metabolites
Some bacterial cultures may produce certain
metabolites having antifungal activity as;
1. Cyclic dipeptides
2. Phenylactic acid
3. Caproic acid
4. Reuterin
5. Lactic acid
6. Acetic acid
7. Fungicin

20. THANKYOUFORYOUR
ATTENTION.