production of aflatoxin by aspergillus flavus

8/4/2019 production of aflatoxin by aspergillus flavus

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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

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production of aflatoxin by aspergillus flavus