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Isolation and identification of aflatoxin producing fungal strains
from groundnuts
UNDER THE GUIDANCE OF :
COMPILED BY :
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Aspergillus flavus and Aspergillus parasiticus are fungi which can invade
peanuts in the field before harvest, during post harvest drying and curing,
and in storage and transportation
Penetration of the peanuts by the fungus leads to the production of
Aflatoxin.
These are secondary metabolites produced by Aspergillus flavus and A.
parasiticuson variety of food products.
These toxins are toxic, carcinogenic, mutagenic and immunosuppressive
agents.
Introduction
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These mycotoxins endanger human health to such a degree that the Food and
Drug Administration (FDA) has currently bans the sale and transport of peanuts
when the level of Aflatoxin contamination exceeds 20 parts per billion.
To investigate the effect of variation in temperature and substrate concentration
on the aflatoxin production and ultimately to investigate about the metabolic
pathway. The toxin production and prediction of the enzyme structure and
function is done with the help of the bioinformatics tools.
The study has been executed in such a way that it can pave the way to down
regulate the production of the toxin so that the food stuffs can be prevented
from contamination for this deadly toxin to help the mankind.
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To isolate the aflatoxin producing fungal strains from
groundnuts.
To extract the toxin produced by them.
To partial characterization of Aflatoxin produced.
To determine the content of aflatoxin of the samples.
To study about the aflatoxin by using bioinformatics
tools.
Objectives
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Methodology
12 different samples were collected and incubated for isolation of fungi
.
Microscopic examination was done by Lacto cotton blue staining by considering the
olive green or dark green colonies in the mixed cultures obtained
.
Sugar fermentation test & Catalase test was done in order to identify the species by using
1% xylose in Potato Dextrose broth and hydrogen peroxide respectively
.
The isolates were purified by means of slide culture technique
Cultures were preserved by preparation of slants on Czapek Dox agar.
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Extract of toxin was prepared by tip culture technique
This extract was screened for Aflatoxin B1 by means of Thin Layer chromatography (TLC)
After treating the TLC plates with particular developer, they wereexamined under Ultra- violet radiation
The toxins produced were characterized by comparing their Rf values withthe standard (Aflatoxin B1) and fluorescence
The spots with blue fluorescence under UV light were scrapped anddissolved in methanol.
Quantification of aflatoxin was done by Spectrophotometer by using
methanol as reference.
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The standard curve for aflatoxin B1 was prepared by plotting concentration of
aflatoxin (g/ml) vs. absorbance (nm) at 363 nm
The content of aflatoxin of samples were determined by plotting the
results on the standard curve
The sequence of aflR regulatory protein was retrieved from the NCBI and analyzed
by bioinformatics tools
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Pfam &
My HitsGOR tool
Protparamtool
PSI- BLAST
Prosite
scanner
Bioinformatics tools used
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Tables
Growth of Aspergillus Flavus on their selective
media
Media Total no. of
Samples
No. of positive
plates
(olive green
colonies)
Percentage of
positive plates
Czapek Dox
media
12 9 75
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Biochemical Analysis
Test
performed
Test result
(positive or
negative)
No. of
positive
results for
biochemical
test
Total no. of
positive
samples
Catalase + 8 9
Sugar
fermentation
+ 6 9
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Rf values of the samples along with standard
SAMPLE Rf Value
M 0.32G 0.30
E 0.31
R 0.32
A 0.32
N 0.30F 0.31
I 0.33
H 0.32
Standard B1 0.31
Solvent:Benzene : methanol : acetic acid(24 :2 :1)Toluene : ethyl acetate : formic acid
(6 :3 :1)
Developer :p-anisaldehyde :methanol:glacial acetic acid : sulphuric acid(0.5 : 85 :10 :5)
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Spectrophotometeric Analysis
Sample name Absorbance at 363 nm
(in nm)
A 0.071
E 0.009
F 0.147
G 0.014
H 0.017
I 0.018
M 0.160
N 0.017
R 0.008
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Readings for standard curve
Concentration of the
aflatoxin
(g/mL.)
Absorbance at 363 nm
(in nm)
0 0
2 0.19
4 0.287
6 0.47
8 0.572
10 0.77
12 0.872
14 1.069
16 1.181
18 1.377
20 1.468
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Standard curve
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Determination of aflatoxin content of samples by comparison to the standard curve.
samples Concentration of aflatoxin
(g/mL.)
A 1.0
E 1.2
F 2.1
G 2.3
H 2.4
I 2.5
M 3.2N 2.4
R 1.1
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Partial characterization of Aflatoxin
On the basis of Rf values
samples Rf values Type of Aflatoxin
(analyzed from
standard values )
M O.32 B1
G 0.30 B1
E O.31 B1
R 0.32 B1
A 0.32 B1
N 0.30 B1
F 0.31 B1
I 0.33 B1
H O.32 B1
STANDARD B1 0.31 B1
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Red fluorescence- OchratoxinGreen fluorescence- Aflatoxin G
Blue fluorescence- Aflatoxin B
On the basis of the color of fluorescence
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BIOINFORMATICS RESULTS
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Retrieved sequence was analyzed by bioinformatics tools which
are as follows-
Sequence retrieval-
The sequence of aflR, Aflatoxin regulatory protein was retrieved from the NCBI
Domain prediction-
On performing Pfam and My Hits, two domains- aflR and Zn cluster were
predicted in the retrieved sequence ranging from 92-354 and 27-65 respectively.
Retrieval of FASTA format of the sequence
The FASTA format of the sequence was retrieved from NCBI (fig-.8)
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PSI BLAST results
The retrieved FASTA sequence was analyzed for the BLAST hits distributed over
the retrieved sequence. Total of 7 different BLAST hits were obtained.
Structure prediction
The retrieved sequence was analyzed by GOR tool to predict the secondary
structure. Retrieved structure contains alpha helix, extended strands and
random coils in percentage of 18.18, 15.15 and 66.67 respectively.
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Protparam tool results
The retrieved sequence was analyzed by Protparam tool for computation of
physicochemical parameters of the enzyme Oxidoreductase. The sequence hastotal of 264 amino acids, molecular weight of 27733.8 and isoelectric point of
5.53 . The amino acid composition , atomic composition and has a molecular
formula C1202H1861N339O389S14.
Prosite scanner
This web based tool was used to detect the PROSITE signature matches in the
protein sequence to detect the functional and structural intra-domain residues.
The pattern obtained was GASTPV]-C-x(2)-C-[RKHSTACW]-x(2)-[RKHQ]-x(2)-
C-x(5,12)-C-x(2)-C-x(6,8)- C.
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Photos
Aspergillus flavus
Isolated colonies on Czapek agar
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Aspergillus flavus
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Slide culture technique Lacto Phenol Cotton Blue stained slides
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Biochemical test
Gas production
positive result
Sugar fermentation test
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Slants preparation Tip culture technique
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Aflatoxin fluorescence yellow under the visible light
Thin layer chromatography
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Aflatoxin B fluorescence blue under UV light
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Discussion
12 different groundnut samples were collected and tested for the growth of
Aspergillus species. Out of them 9 have shown positive for Aspergillus flavus
and all of them were capable of producing Aflatoxin. Screening through TLC
indicated that only Aflatoxin B, G and Ochratoxin were produced. But Aflatoxin
B was the most Dominant toxin present in the groundnuts. Only B1 and G1
aflatoxins were detected from the toxigenic isolates while B2 and G2 were not
detected. (Joe et al.,1996, Adegoke et al. 1991, Kivanc 1990, Chourasia 1999,
Park et al., 1983 and Barrios et al, .1997). (Amadi, J. E. and Adeniyi, D. O, 6
April, 2009).
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Out of 9 positive samples, 8 have shown positive results for catalase production
by producing effervescence on addition of hydrogen peroxide which the
capability of the isolates to produce Catalase enzyme which breaks the
hydrogen peroxide to water and oxygen gas. This oxygen gas is responsible for
the effervescence production. Similarly, 6 out of 9 samples have shown positive
results for the sugar fermentation test by producing gas bubbles (A.A.Zohri and
M.A. Ismail, 1994). Aflatoxin has been studied via their regulatory enzyme
named Oxidoreductase by using bioinformatics tools. The FASTA sequence
was retrieved by NCBI server and has been analyzed by Pfam tool in order to
find the domains. Results obtained have shown two domains- aflR and Zn
cluster ranging from 92-354 and 27-56 respectively. (Woloshuk, et.al, 1994).
Structure prediction was done by using GOR expasy tool. It is for the prediction
of secondary structure of the protein sequence.
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Retrieved structure contains alpha helix, extended strands and random coils in
percentage of 18.18, 15.15 and 66.67 respectively (Garnier J, Gibrat J-F,
Robson B, 1996). Total of 7 different BLAST hits were obtained in the sequence
by use of PSI- BLAST tool (Altschul SF, et.al, 1997). Prosite scanner detects
the pattern of GASTPV]-C-x(2)-C-[RKHSTACW]-x(2)-[RKHQ]-x(2)-C-x(5,12)-C-
x(2)-C-x(6,8)- C. (Castro, et.al, 2006). The Physicochemical parameters were
also retrieved by using Protparam tool. This study tells about the function of the
regulatory enzyme which can pave a way to downregulate the aflatoxin
production.
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Summary & conclusion
Aspergillus flavus and Aspergillus parasiticus penetrate the stored grains and
produces Aflatoxins. Aflatoxins are polyketide secondary metabolites produced
by these two important food borne Aspergillus species. The four main Aflatoxins
produced, Aflatoxin B1 (AFB1), Aflatoxin B2 (AFB2), Aflatoxin G1 (AFG1), and
Aflatoxin G2 (AFG2), are furanocoumarin derivatives and potent liver
carcinogens for a wide variety of animal species including humans.
Total of 12 samples of groundnuts were collected in and around Jalandhar. But
only A.flavuswas isolated by producing olive green colonies on the Czapek Dox
media. Out of 12 samples, 9 have shown positive results for the growth of
Aspergillus flavusbut there were no results for A.parasiticus.
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Out of 9 positive samples, 8 have shown positive results for Catalase by
producing effervescence and 6 have shown positive results for sugar
fermentation test by producing gas bubbles under the mycelial mat over PDB
containing 1% xylose sugar. After performing TLC, Aflatoxin B1, Aflatoxin G and
ochratoxin has shown blue, green and red fluorescence respectively. But the
Aflatoxin B was the most dominant one. Spectrophotometric analysis has shown
that sample M produces maximum Aflatoxin production (absorbance of 0.16).
Aflatoxin has been studied by using bioinformatics tools. It has been studied via
their regulatory enzyme Oxidoreductase. This whole study makes us to
understand how this enzyme regulates the production of aflatoxin. By this we
can downregulate the aflatoxin production which helps the mankind.
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THANKS