Isolation and identification of aflatoxin producing fungal strains from groundnuts

UNDER THE GUIDANCE OF : COMPILED BY :

Introduction
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. parasiticus on variety of food products. These toxins are toxic, carcinogenic, mutagenic and immunosuppressive agents.

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.

Objectives
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
tools.

about the aflatoxin by using bioinformatics

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.

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 were
examined under Ultra- violet radiation

The toxins produced were characterized by comparing their Rf values with the standard (Aflatoxin B1) and fluorescence

The spots with blue fluorescence under UV light were scrapped and dissolved in methanol.

Quantification of aflatoxin was done by Spectrophotometer by using methanol as reference.

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

Bioinformatics tools used

Pfam & My Hits Protparam tool

GOR tool PSI- BLAST

Prosite scanner

Tables
Growth of Aspergillus Flavus on their selective media
Media Total no. of Samples No. of positive Percentage of plates (olive green colonies) positive plates

Czapek Dox media

12

75

Biochemical Analysis
Test performed Test result No. of positive results for biochemical test Total no. of positive samples

(positive or
negative)

Catalase

Sugar

fermentation

Rf values of the samples along with standard

SAMPLE M G E R A N F I
H Standard B1 Solvent: Benzene : methanol : acetic acid (24 :2 :1) Toluene : ethyl acetate : formic acid (6 :3 :1)

Rf Value 0.32 0.30 0.31 0.32 0.32 0.30 0.31 0.33

0.32 0.31

Developer : p-anisaldehyde :methanol: glacial acetic acid : sulphuric acid (0.5 : 85 :10 :5)

Spectrophotometeric Analysis

Sample name

Absorbance at 363 nm

(in nm)
A E F G 0.071 0.009 0.147 0.014

H
I M N R

0.017
0.018 0.160 0.017 0.008

Readings for standard curve

Concentration of the aflatoxin (g/mL.) 0 2 4 6 8

Absorbance at 363 nm (in nm)

0 0.19 0.287 0.47 0.572

10
12 14 16 18 20

0.77
0.872 1.069 1.181 1.377 1.468

Standard curve

Determination of aflatoxin content of samples by comparison to the standard curve.

samples

Concentration of aflatoxin (g/mL.)

A
E F G H I M

1.0
1.2 2.1 2.3 2.4 2.5 3.2

N
R

2.4
1.1

Partial characterization of Aflatoxin

On the basis of Rf values
samples Rf values Type of Aflatoxin (analyzed from standard values ) B1 B1 B1

M G E

O.32 0.30 O.31

R
A N F

0.32
0.32 0.30 0.31

B1
B1 B1 B1

I
H STANDARD B1

0.33
O.32 0.31

B1
B1 B1

On the basis of the color of fluorescence

Red fluorescence- Ochratoxin Green fluorescence- Aflatoxin G Blue fluorescence- Aflatoxin B

BIOINFORMATICS RESULTS

Retrieved sequence was analyzed by bioinformatics tools which are as follows-

Sequence retrievalThe sequence of aflR, Aflatoxin regulatory protein was retrieved from the NCBI

Domain predictionOn 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)

 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.

 Protparam tool results

The retrieved sequence was analyzed by Protparam tool for computation of physicochemical parameters of the enzyme Oxidoreductase. The sequence has total 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.

Photos
Isolated colonies on Czapek agar

Aspergillus flavus

Aspergillus flavus

Slide culture technique

Lacto Phenol Cotton Blue stained slides

Biochemical test
Sugar fermentation test

Gas production positive result

Slants preparation

Tip culture technique

Thin layer chromatography

Aflatoxin fluorescence yellow under the visible light

Aflatoxin B fluorescence blue under UV light

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).

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.

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)-Cx(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.

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.flavus was 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 flavus but there were no results for A.parasiticus

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.

THANKS