Research Article

ISSN: 0974-6943

G.Narendrakumar et al. / Journal of Pharmacy Research 2011,4(3),621-623

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Characterization of aflatoxin B1 from Aspergillus species and biocompatibility studies
G.Narendrakumar *, R.Dhanapani 1
*
Department of Biotechnology, Sathyabama University, Chennai,India
1
Department of Microbiology, Periyar University, Salem,India

Received on: 05-10-2010; Revised on: 14-12-2010; Accepted on:09-02-2011

ABSTRACT
Soil samples were collected and were screened for aflatoxin producing organisms. Aspergillus flavus was isolated from those soil samples and they were cultivated in media.
Simultaneously along with the soil isolates the MTCC strain, Aspergillus parasiticus was also purchased and tested to compare the amount of aflatoxin production by MTCC strain
and the fungal isolate. Groundnuts and corn kernels were used as substrate for growth of aflatoxin producing organisms. Aflatoxin was extracted using chloroform, methanol and
acetonitrile and its comparative study was done. TLC and UV spectrophotometry were used for conformation of presence of aflatoxin B 1 in the extracts. The inhibitory effects on growth
of Aspergillus flavus and Aspergillus parasiticus were done with Aspergillus niger, Bacillus subtilis. Antagonistic effect on the production of aflatoxin was carried out with
Trichoderma viridae and Fusarium spp.

Key words: Aflatoxin, Biocompatibility studies , Antagonism, As.flavus, As.parasiticus

INTRODUCTION
Mycotoxins, the secondary metabolite of moulds, exert toxic effects on animals and
humans, which is referred to as mycotoxicosis. The severity of manifestation of mycotoxins depends on their toxicity; the extent of exposure; age and nutritional status of the
individual. Mycotoxin presence in foods has been an international concern from their date
of discovery. Among the various mycotoxins identified aflatoxin have assumed a great
significance in context to their most devastating effects on human, poultry and animal
livestock (Rodricks and Stoloff, 1977).
MATERIALS AND METHODS
The source of the organisms selected is soil so as to obtain the aflatoxin producing strains
with higher frequency of isolation. One gram of soil was suspended in 9 ml of physiological saline and vigorously mixed. Serial dilutions were made and aliquots were spread on
to SDA plates. The plates were then incubated 48 h at 28oC. The visible morphological
types of single colonies were isolated, maintained on the above medium at 4oC and
transferred monthly.
Screening and extraction of aflatoxin from the isolated samples
Screening for aflatoxin production was carried out by following the method of Naghy et
al., By Haras method; the capability of the isolated fungal strains to produce aflatoxin
was tested using fluorescence technique on the nutrient medium with fungal growth.
The fungi were inoculated on the NA. Further with non-inoculated control, the inoculated
NA was incubated at 28C for 7 days in dark. At the end of the incubation period plates
were inverted and examined under strong UV lamp and the production of bright fluorescence under and around the colonies confirm the presence of aflatoxin.
Extraction of aflatoxin
Two different substrates were used to grow the organisms for production of aflatoxins.
The substrates are a) Oil seed Groundnut, b) Corn Kernel
To these substrates the isolated fungal species
i.Aspergillus flavus and
ii.Aspergillus parasiticus
were inoculated in separate conical flasks.
Inoculation onto substrates
Oil-Seed Substrate
Groundnut was crushed and inoculated with Aspergillus flavus and Aspergillus parasiticus
in separate beakers containing 70g of crushed ground nut in each of the flasks and kept for
incubation at room temperature for 7 days.
Starchy seed substrate
70g of corn-kernels were crushed and inoculated with the organisms Aspergillus flavus
and Aspergillus paraciticus in separate beakers and incubated at room temperature for 7
days.

*Corresponding author.
*G.Narendrakumar
Assistant Professor, Department of Biotechnology,
Sathyabama University, Jeppiaar Nagar, Chennai
Tel.: + 91-9445266138
E-mail:gnaren22@gmail.com

Extraction of aflatoxin
From oil seed inoculum
Reagents:
105ml of Acetone + 20ml of water were used for chloroform extraction
250ml of methanol: water [60:40 v/v] was used for methanol extraction.
100ml of Acetone + water [55+45] was used for Acetonitrile extraction.
Acetone - chloroform - water [12:88:1] solvent system was used for TLC.

From starchy substrate

Chloroform Extraction
50g of powdered sample was blended with 250ml of methanol : water mixture (60:40 w/
v) for 5min at high speed, filtered through Whatmann No:1 filter paper, 125ml of filtrate
with 30ml saturated salt solution and 50ml n-hexane were taken in a separating funnel,
shaken vigorously for 5min and then the hexane layer was discarded. Lower methanolic
layer was taken into another separating funnel and added with 40ml chloroform, shaken
and allowed to rest undisturbed for separation of layers. The chloroform layer was collected in a flask containing 5g of cupric carbonate, agitated and allowed to settle down.
This extract was filtered through a bed of anhydrous sodium sulfate over filter paper and
was evaporated to dryness. The residue was dissolved in 0.2ml of chloroform and poured
onto vials for further analysis.
Acetonitrile extraction
Acetonitrile extraction of aflatoxin from starchy seed (corn kernels) was done with the
same methodology as for the extraction from oilseeds. The extractions were done both for
isolated and standard strains. The aflatoxin thus extracted was analyzed or characterized by
Thin Layer chromatography.
Thin layer chromatography
Two plates (10x10x10/5cm) were taken for the purpose 16g of silica gel was dissolved in
35ml of distilled water and applied on plates using applicators. The thickness of the gel
used was 0.2mm. The plates were allowed to dry. After drying activation of the plates were
done by keeping them in hot-air oven at 110C for 1hr.
After activating the plates, 10l of the extracted samples were spotted on the silica gel
plates together with authentic sample of aflatoxins (5l). Movement of the sample was
aided with a solvent system - a mixture of chloroform and acetone and water in the ratio
88:12:1. When the solvent moved to the top of the plates, the plates were taken out,
allowed to dry and then inspected under the UV light for fluorescence. Calculations were
done to find the amount of toxin in ppb.
Preparation of aflatoxin standard
Benzene: acetonitrile (98+2) mixture was used to prepare the standard at the concentration
of 10g /ml. The concentration of stock solution was standardized using spectrophotometer. From this stock solution, working standard (4g/ml) was prepared using benzene:
acetonitrile. Aflatoxin B1 standard was purchased from Toxin Lab, Tamil Nadu, Veterinary College, Namakkal 1.
Analysis by UV Spectrophotometry (SHIMADZU, Model No : UV 1601;)
For UV spectrophotometry, the dried extracts were mixed with 5ml of chloroform. UV
absorbance spectrum was taken by using chloroform as blank against the test extracts. A
graph was plotted with the values of the absorbance obtained for each sample.

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Table.1. Fungal cultures and substrates used

Inhibitory Effects of microbes on growth of aflatoxin producing fungi

To see the inhibitory effect of two organisms.
Aspergillus niger
Bacillus subtilis
on the growth of aflatoxin producing strains, Pasters method was followed for the former
(fungal inhibition) and Munimbazis method was followed for the later (bacterial inhibition).
Inhibition by fungal counterparts
200ml of synthetic medium was prepared. Medium was inoculated with Aspergillus niger
and incubated for 4 days in room temperature. After incubation the medium was filtered
through membrane filter under aseptic conditions. 50ml of the filtrate was taken in another
flask containing 150ml of sterile synthetic medium and to this Aspergillus flavus was
inoculated. The inoculated flask was kept for incubation at room temperature for 4 days.
As a control Aspergillus flavus was inoculated in 200ml of synthetic medium without the
filtrate and incubated. Growth in control and the test samples were observed after incubation. The same was performed for Aspergillus parasiticus with the extract of Aspergillus
niger.
Dry cell weight
Dry cell weight measurement was estimated by filtering a known volume of culture broth
through pre-weighed whatmann paper No.41 and drying the paper at 60C under partial
vacuum for 10-12 hr. Filter papers were then weighed separately after regular intervals to
a constant weight.
Inhibition by bacteria
Czapeks yeast extract sucrose agar medium was prepared and sterilized. CYESA medium
was poured onto plates. To half the plate Bacillus subtilis was streaked and incubated for
24 hrs at 37C. After growth of Bacillus, the other half of the plate was streaked with
Aspergillus parasiticus. As control, a sterile, CYESA plate without Bacillus subtilis was
also inoculated with Aspergillus parasiticus. The same was done for Aspergillus flavus
and the plates were incubated at room temperature for 4 days. After incubation the growth
or inhibition was observed for the control and test plates respectively.
Antagonism of Trichoderma viridae and Fusarium on production of aflatoxin
Corn-kernels, one set autoclaved and the other set non-autoclaved were inoculated with
Trichoderma viridae and Fusarium each incubated for 3 days. After the incubation the test
culture Aspergillus flavus was inoculated into them. Another set only with Aspergillus
flavus was kept as control and were incubated for 4 days at room temperature.After the
growth, the toxins were extracted and analysis by TLC, was done for estimating the
extent of inhibition of aflatoxin production in the antagonist fungal inoculated cultures.
The same was done for Aspergillus parasiticus also.
RESULTS
Isolation and Identification
(1)Aspergillus flavus, Aspergillus parasiticus
Isolated from soil samples
(2)Aspergillus parasiticus MTCC 2796

Fungal Cultures

Substrates

Aspergillus flavus
Aspergillus flavus
Aspergillus parasiticus
Aspergillus parasiticus

Groundnuts
Corn kernels
Groundnuts
Corn kernels

Table .2 .Source for Aflatoxin Extraction: Aspergillus flavus inoculated Groundnuts

Solvents

Concentration of Aflatoxin in ppb.

Chloroform
Methanol
Acetonitrile

46.6
32
29

Table .3 .Source for Aflatoxin Extraction: Aspergillus flavus inoculated Corn kernels
Solvents

Concentration of Aflatoxin in ppb.

Chloroform
Methanol
Acetonitrile

39
27
21

Table .4. Source for Aflatoxin Extraction: Aspergillus parasiticus inoculated Groundnuts
Solvents

Concentration of Aflatoxin in ppb.

Chloroform
Methanol
Acetonitrile

98
79
58

Table.5. Source for Aflatoxin Extraction: Aspergillus parasiticus inoculated Corn kernels
Solvents

Concentration of Aflatoxin in ppb.

Chloroform
Methanol
Acetonitrile

85
68
42

Spectrophotometric analysis:
The presence of aflatoxin for the cell free extract was detected by comparing the graph
obtained after scanning which the standard graph, and maximum adsorption was found to
be 360nm.
Antagonastic effects
Inhibitory effect of Bacillus subtilis:
(1)An inhibitory zone was observed in CYESA plates, between Aspergillus flavus and Bacillus subtilis.
(2)An inhibitory zone was observed in CYESA plates, between Aspergillus parasiticus and
Bacillus subtilis.
Table .6.Inhibition of aflatoxin production by Trichoderma viridae and Fusarium Sp:

Screening of Isolated fungal strains for Aflatoxin Production:

Bright blue colour fluorescence fungal colonies were observed under strong ultraviolet
lamp and those colonies were selected for Aflatoxin production.
Aflatoxin Production:
Aspergillus flavus inoculated ground nut [oily seeds] and corn kernels [starchy seeds] and
Aspergillus parasiticus inoculated ground nut [oily seeds] and corn kernels [starchy
seeds] were used for Toxin Production.

Fungal Cultures

Concentration of Aflatoxin in ppb.

Autoclaved corn kernels
Non- Autoclaved corn kernels

Aspergillus flavus
Trichoderma viridae
Fusarium sp

20
14
12

37
16
14

Table .7.
Fungal Cultures

Concentration of Aflatoxin in ppb.

Autoclaved corn kernels
Non- Autoclaved corn kernels

Characterization of aflatoxin:

Aspergillus parasiticus
Trichoderma viridae
Fusarium sp

52
15
14

(1)Thin Layer Chromatography:

The sample and the standard were placed adjacently and allowed to move, the distance
travelled by them were calculated, which confirms the presence of toxin in the extract. The
thin layer chromatography developed was given in the plate.

DISCUSSION

Quantitative TLC analysis:

Calculation:
Aflatoxin Concentration in ppb=

x 1000

S-Standard which compares with the sample in fluorescent intensity

C-Concentration of the standard
D-Dilution factor
T-Sample which compares with the sample in fluorescent intensity.
8.57-Effective weight

68
18
16

Aflatoxins are potent mycotoxins and are the major causatives of both human and animal
mycotoxicosis. Research on these toxins is going on for decades. In the present study,
aflatoxin production by the isolates from soil were found to be in higher concentration
(Chang, et al., and Lee, et al., in 1997) than the MTCC strain. The capability of aflatoxin
production by the fungal strains were detected by the flourescence technique (Hara, et
al.,1974), (Naghy et al.,1995) (Cuetnic, et al.,1995)) and screening for aflatoxin production by use of ammonia vapour was done (Saito, et al., 1999).
Production of aflatoxin by Aspergillus flavus does not directly occur in synthetic media
and so, the mycelium is grown on substrates such as groundnuts and corn-kernels to
increase the concentration of aflatoxin produced (Dau-Mgoc-Hao, et al., 1999, Ronald,
1994) Extraction of aflatoxin is done with methanol, chloroform, acetonitrile solvents
(Reddy & Farid, 1994) and the efficiency in extraction was found to be high when

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Methanol/Chloroform/Acetonitrile were used in proper ratio with water (Han-Hong-Yan,
et al., 1999). Comparison of various methods of extraction procedures (Mehan, et al.,
1985) shows in the present study that chloroform extraction (Truchess, et al., 1979)
yields much higher amounts of aflatoxin than methanol (Whitaker, et al., 1986) and
acetonitrile extractions.

3.

After declaration of aflatoxin B1 on the top list human carcinogen (IARC, 1988), characterization of aflatoxin B1 gained importance and chromatography and spectrophotometry
are used to characterize aflatoxin B 1 (Gabal, et al., 1994). TLC is used for isolation, purity
assessment of aflatoxin (Betina, 1985 & Peter, et al., 1998).

6.

4.
5.

7.
8.

As reported by Rex. Gallagher, et al.,1980 presence of aflatoxin B1 is detected by

observing the TLC plate under strong UV illumination and it flourescence as bluish green
spots. Bluish green spots of aflatoxin B1 in TLC is also reported by Pallavi, et al.,1977.
The biological control of aflatoxin production by Aspergillus niger shows that the fungus
reduces the growth of both Aspergillus flavus and Aspergillus parasiticus. This control
is supported by similar result obtained by Paster et al.,1992. Inhibition of growth of
Aspergillus flavus and Aspergillus parasiticus is also seen with the Bacillus subtilis
(Bullerman, et al., 1977).

9.
10.
11.
12.

Antagonistic effect on production of aflatoxin by two fungal species Trichoderma viridae

and Fusarium sp is done. Aziz, et al.,1997, also shows these fungi to inhibit the
production of aflatoxin at a rate of 89 and 57% respectively.

13.
14.

From the above study it is seen that soil isolates of Aspergillus flavus are more prone to
produce Aflatoxin and that these can be antagonised for production of aflatoxin by Trichoderma viridae and Fusarium sp and the growth is inhibited by Bacillus subtilis and
Aspergillus niger . Further studies are needed to extract the element inhibiting the production and growth so as to use them as drugs for the aflatoxicosis afflicted persons.

15.
16.
17.

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Source of support: Nil, Conflict of interest: None Declared

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