ISOLATION OF AFLATOXIN PRODUCING ASPERGILLUS SPECIES IN

FOOD SAMPLES AND CONTROL OF THEIR GROWTH BY RHIZOME

EXTRACT OF ZINGIBER OFFICINALE

SUBMITTED BYB. DURGA RAO

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.

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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).

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Then the mixture has been filtered with the help of whatman No.1 filter paper.

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The filtrate thus obtained has been subjected to evaporation in a boiling water bath such that 1ml of the total volume is remained

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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.

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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).

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The sample is spotted on the TLC sheet and kept in chromatographic chamber

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After the solvent reached to top edge, removed plates from chamber and air dried

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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.

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After stirring, the solution was filtered through 2 layers of cheese- cloth gauze and Whitman’s (No.1) filter paper

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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.

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To each flask transferred 100ml of cZpek dox broth, autoclaved the media at 1200C and 15lbs pressure for 15 minutes.

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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).

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Mixed thorourely and inoculated loopful of active spores of isolated aspergillus species to each flask who is confirmed producing aflotoxins.

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Incubated fungal inoculated flasks at room temperature for six days.

 

RESULTS

SampleColour of infected

seedsSeeds Showing

fluorescenceGrowth on PDA

mediumFluorescence of growth medium

Sample-1Green Yes Green Yes

Sample-2Black No Black No

Sample-3Brown No Brown No

Sample-4White No White N o

Sample-5Yellowish green Yes Yellowish green Yes

Ground nut

SampleColour of infected

seedsSeeds Showing

fluorescenceGrowth on PDA

mediumFluorescence of growth medium

Sample-1Green Yes Green Yes

Sample-2Black No Black No

Sample-3Yellowish green Yes Green Yes

Sample-4White No White No

Maize

SampleColour of

Endosperm Endosperm

Showing fluorescence

Growth on PDA medium

Fluorescence of growth medium

Sample-1Green Yes Yellow to green Yes

Sample-2Black No Black No

Sample-3White 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:

SampleFluorescence

Sample-1Yes

Sample-5Yes

Maize:

SampleFluorescence

Sample-1Yes

Sample-3Yes

Coconut:

Sample Fluorescence

Sample-1Yes

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.  

 

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