Aflatoxin (1)

INTRODUCTION
A toxin can be defined as a substance that is synthesised by a plant
species, an animal, or by micro-organisms, that is harmful to another
organism
Mycotoxins are toxic metabolises produced by fungi, mostly by
saprophytic moulds growing on a variety of foodstuffs including that of
animal feeds and also by many plant pathogens.
Mycotoxins are potentially hazardous to man and domestic animals

Aflatoxins(AFTs)
AFTs are a group of closely related widely researched mycotoxins that are
produced by fungi A. Flavus and A. parasiticus.
The AFT were first heavily researched and understood after the death of
more than 100,000 young turkeys on poultry farms in
England,(turkey×disease) that were found to be related to the
consumption of Brazilian peanut meal
Many agricultural commodities are vulnerable to attack by fungi that
produce mycotoxins
Most mycotoxins are stable compounds that are not destroyed during
food processing or cooking.
Among mycotoxins, four main aflatoxins B1, B2, G1, and G2 are extremely
potent carcinogens and can have significant economic impacts, making them
important targets for detection and quantitation.

Structures of naturally occurring aflatoxins

AIM AND OBJECTIVES
 Identification of fungal infected food materials for isolation
of aflatoxin producing fungal species.
 Culturing of each sample on potato dextrose agar for active
culture of fungal species.
 Extraction of aflatoxins from culture media and detection by
UV method.
 Production of aflotoxin by using the synthetic medium and
quantification of aflotoxin levels in fermented broth.
 Control of fungal growth by using root extract of ginger

MATERIALS REQUIRED
Equipment and glassware:
 Autoclave
 Incubator
 Laminar airflow cabinet
 Micropipettes
 pH meter
 Compound Microscope
 Silica gel-G sheets
 Chromatography chamber
Glassware: (Borosilicate make)
 Conical flasks
 Volumetric flasks
 Beakers
 Petri dishes
 Test tubes

Fungal culture media:
Czapek-Dox Agar (pH 7.0):
Composition/litre:
 Sodium Nitrate-2.0g
 Potassium Chloride-0.5g
 Magnesium sulphate-0.5g
 Dipotassium Hydrogen Phosphate-1.0g
 Ferrous Sulphate-0.01g
 Sucrose-30.0g
 Agar-15.0g
 Distilled water-1000 ml
Czapecks broth (pH 6.4-7.0)Composition/litre:
 Sodium nitrate-2.0 gm
 Potassium chloride-0.5 gm
 Magnesium sulphate-0.5 gm
 Dipotassium hydrogen phosphate-10 gm
 Ferrous sulphate-0.01 gm
 Dextrose (or) cellulose-10.0 gm
 Distilled water-1000 ml

Aspergillus differentiation agar (AFPA):
 yeast extract, 20 g/L,
 peptic digest of animal tissue, 10 g/L, ferric
ammonium citrate, 0.5 g/L, dichloran, 0.002 g/L
&agar, 15 g/L
Coconut cream agar: which comprised coconut
cream (50%) and agar (1.5%).

Chemicals for extraction of aflotoxin from mycelium:
 Methanol, Potassium chloride
Solvent system for TLC of extracted aflotoxins:
 Benzene:Methanol:Acetone(80:15:5)
 ELISA Kit(Commercially available)
 Fungal infected food grains
 Roots of Zingiber officinale

METHODS
Collection of fungal infected food materials:
Identified and collected fungal infected ground nuts, maize, and coconut
materials based on their colour appearance from the farmers of nearby
villages of Visakhapatnam. Each sample carefully stored in clean
polythene bags.

Isolation of aflatoxin producing Aspergillus species
from infected food materials:
Prepared Potato dextrose agar medium, autoclaved the
medium at 1200C and 15 lbs pressure for 20 minute.
↓
Poured media in a sterilized Petri dishes, after
solidification the fungal infected food materials are
inoculated in the centre of plates.
↓
The plates have been incubated at room temperature for
about 6 days.

Extraction of aflatoxin from strains grown on agar
media:
Aflatoxin has been isolated by grinding the mouldy agar along
with grown fungal culture (20gm) in mortar and pestle with
a mixture of KCl (0.5%) and methanol(100ml).
↓
Then the mixture has been filtered with the help of whatman
No.1 filter paper.
↓
The filtrate thus obtained has been subjected to evaporation in a
boiling water bath such that 1ml of the total volume is
remained
↓
Subjected to thin layer chromatography(TLC)for the presence or
absence of fluorescence.

Production of aflatoxins from isolated fungal species:
The spores that have been grown on agar and showing
fluorescence when exposed to UV light are subjected to
fermentation for production aflatoxins.
↓
Prepared 100 ml of Czapeck’s broth, autoclaved the
medium at 1200C and 15 lbs pressure for 20 minutes.
↓
Cooled the media and inoculated with isolated fungal
species who is showed fluorescence when exposed to UV
light and on TLC plates
↓
Incubated for about 4 days and observed for the growth then
the sample has been taken and is subjected to TLC.

Identification of Aflotoxin on TLC:
The production of Aflatoxin has been evaluated with the help of TLC to
confirm the presence or absence of Aflatoxin.
↓
Readymade silica gel coated plates have been used for TLC. The solvent
mixture have been used for TLC is Benzene:Methanol:Acetone
(80:15:5).
↓
The sample is spotted on the TLC sheet and kept in chromatographic
chamber
↓
After the solvent reached to top edge, removed plates from chamber and air
dried
↓
Then the sheet is observed under UV-light for the fluorescence.

Identification and Characterization of aflatoxin
producing fungal species
Morphological Studies by selected media:
 Coconut cream agar (CCA):
The CCA is used to detect aflatoxin producer strains. The
production of aflatoxin is detected by a blue fluorescence when
exposed to a UV-light.
 Aspergillus differentiation agar (AFPA):
AFPA is a selective identification medium for the detection
of A. flavus group strains. With this method is possible to
distinguish these species from other Aspergillus based on the
development of orange colour on the reverse of the plates.


 Czapek Dox agar (CZ). When grown on CZ, colonies taxonomically between
the two species can also be separated. Those of A. flavus being yellow-green
and those of A. parasiticus a distinctly darker green, referred to as near Ivy
green.
 AFPA, CCA and CZ media suing their respected components, autoclaved and
cooled to approximately 50oC and poured into petridishes. After solidified
the media inoculated each isolated fungal species into all media and then
subjected incubation at room temperature for about one week. After
completion of incubation period observed the morphology of the fungi.

Growth Control of aflatoxin producing fungi by root extract
of Zingiber officinale
Preparation of water Extract from roots of Zingiber officinale
Fifty grams rhizome of Zingiber officinale (Ginger) was treated with 500 ml
of distilled water with constant stirring for 5 hours.
↓
After stirring, the solution was filtered through 2 layers of cheese- cloth
gauze and Whitman’s (No.1) filter paper
↓
Evaporated the solvent, air dried the extract and stored in small, sterilized 5
ml screw-capped glass bottles and kept in the refrigerator (4oC) until
further usage.

Determination of Antifungal activity of water extract of Zingiber
officinale root
Taken four 250ml conical flasks and labelled as A,B,C,and D.
↓
To each flask transferred 100ml of cZpek dox broth, autoclaved the media at 1200C
and 15lbs pressure for 15 minutes.
↓
After completion of autoclaving process cooled the media to room temperature and
added 100 mgs(1mg/ml) of water extract powder to flask A, 200 mg to flask
B(2mg/ml), 300mg to flask C(3mg/ml) and 400 mg to flask D(4mg/ml).
↓
Mixed thorourely and inoculated loopful of active spores of isolated aspergillus species
to each flask who is confirmed producing aflotoxins.
↓
Incubated fungal inoculated flasks at room temperature for six days.

RESULTS
Sample
Colour of infected
seeds
Seeds Showing
fluorescence
Growth on PDA
medium
Fluorescence of
growth medium
Sample-1
Green Yes Green Yes
Sample-2
Black No Black No
Sample-3
Brown No Brown No
Sample-4
White No White N o
Sample-5
Yellowish green Yes Yellowish green Yes
Ground nut
Sample
Colour of infected
seeds
Seeds Showing
fluorescence
Growth on PDA
medium
Fluorescence of
growth medium
Sample-1
Green Yes Green Yes
Sample-2
Black No Black No
Sample-3
Yellowish green Yes Green Yes
Sample-4
White No White No
Maize

Sample
Colour of
Endosperm
Endosperm
Showing
fluorescence
Growth on PDA
medium
Fluorescence of
growth medium
Sample-1
Green Yes Yellow to green Yes
Sample-2
Black No Black No
Sample-3
White No White No
Maize

Growth on PDA medium and Emitting fluorescence exposed to UV light

Growth on CCA Medium Showing fluorescence when exposed to UV Light

Growth on Aspergillus differentiation agar (AFPA) development of orange colour on the reverse of the plates

Growth on Czapek Dox agar (CZ): Those of A. flavus being yellow-green

TLC of growth media extracts samples showing fluorescence
Ground nuts:
Sample
Fluorescence
Sample-1
Yes
Sample-5
Yes
Maize:
Sample
Fluorescence
Sample-1
Yes
Sample-3
Yes
Coconut:
Sample Fluorescence
Sample-1
Yes

TLC image of aflatoxins showing fluorescence when exposed to UV light

Conclusion:
 The food materials, ground nuts, maize and coconut endosperm are infected with
different types of fungal species in which some are contaminated with aflatoxin
producing aspergillus species.
 The aflatoxin production was observed the plates are exposed to UV light followed TLC
method and ELISA.
 Then the aflatoxin producing species are identified by their growth on coconut agar
medium blue fluorescence was observed when exposed to a UV-light, on Czapek Dox
agar medium the yellow-green colour growth was observed and development of orange
colour on the reverse of the plates when the organism grown on Aspergillus
differentiation agar.
 Based on the above results the food materials are infected with Aspergillus Flavus.
From the quantitative ELISA method, estimated the quantities of aflatoxins in infected
food material, solid medium and enrichment medium.
 Based on ELISA results the aflatoxin production rate was increased with enrichment
medium.
 When the root extract of Zingiber officinale was used to control the growth of
Aspergillus species, the extract successfully controlled sporulation of the fungi.


Aflatoxin (1)

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