A HPTLC Method Of Analysis Of Aflatoxins And Its Possible Effect On Gram Positive

By - 10/02/2004

Login or register to post comments
987 reads

Average:

Your rating: None

Ramya Muthukrishnan

Ramya Muthukrishnan

Departmentof Microbiology, Ramnarain Ruia collage, Matunga, Mumbai-400019.

Aflatoxins are a family of mycotoxins
hat are related by the presence of bisfuranocoumarin compounds. They are probably the best
known and most intensively researched mycotoxins in
the world. They are produced mainly by toxigenic
strains of Aspergillus flavus and Aspergillus
parasiticus. Aflatoxins are
a group of secondary fungal metaboilites that are
potent animal toxins and carcinogens.s

Several species of Penicillium and Escherichia coil were found to be inhabited by the toxin. According to
the tests done by me it is observed that it inhabits the growth of
Streptococcus pneumoniae and Staphylococcus aureus to a great extent.

The project work was carried out
as follows:

-In 250ml of aflatoxin
synthetic medium, 2.5ml of the spore suspension of Aspergillus
oryzae producing toxin (sample) was inoculated and
kept under shakes for continuous 8 days at RT.

-The culture filtrate was then
separated from the crowed cells and treated with equal amount of chloroform in
a separating funnel.

-The chloroform layer was heated
to dryness and the dry toxin was resuspended in about
1 ml of chloroform to obtain concentrated toxin.

-HPTLC technique for separation
of aflatoxin, B,B2,G1,and
G2—was developed using chloroform : acetone (90:10 ) as a solvent system and
the plates detected at 366nm.

-The Rf values of the sample were compressed with the
standard values and were found to coincide.

Different qualitative and semi
quantitative techniques were done to see the effect of toxin on gram positive
organisms. They were;

.Preliminary tube method:
Principle of which involved detection of growth of test organisms based on
turbidity of medium.

. Ditch plate technique: Principle of which was to detect the effect of insoluble
toxin on micro-organisms by forming a ditch on a nutrient agar plate containing
toxin and test organisms were cross streaked over it. Inhibition of organisms
was looked for over the ditch.

. Paper disc
method: Principle of which was diffusion of toxin in the media and dedication
of zone of inhibition around the ditch.

Results indicated that
Staphylococcus auras was completely inhabited by the toxin while Streptococcus
was partially inhabited or had the capacity to destroy the biologically
activity of the toxin and grow.

Introduction

The
term aflatoxins refer to a group of secondary fungal
metabolites that are potent animal toxins and carcinogens.

They have been epidemiologically
implicated as environmental carcinogens in man.
Chemically, aflatoxins are highly substituted coumarines. They are
named so because they were first isolated from the fungus Aspergillus
flavus (A.flavus) toxsins. The Aspergillus group of species includes Aspergillus
flavus and Aspergillus parasticus, which produce aflatoxins
as well as Aspergillus oryzae
and Aspergillus tamari, which do not produce it. Recent research has found out that under
specific conditions some strains of Aspergillus oryzae also have the ability to produce toxins.

Aflatoxins
B2 and G2 were established as the dihydroxy
derivatives of B1 and G1 respectively.
They are designated by B and G series because of their fluorescence
characteristics. B1 and B2 show strong
blue fluorescence under UV while G1 and G2 fluoresce
green yellow. Aflatoxin
B1 is generally found in greatest concentration.

Literature Review revealed that Aspergillus flavus produces afflatoxin overa temperature
range from about 12-42
^o
C with optimum production at 25-32
^o C,
the limits depending on the substrate and the specific experimental
conditions. Under laboratory conditions,
at 25-30 ^o C aflatoxin was formed within 48
hours on moistened groundnuts, rice and cotton seed, whereas requirement of a
minimum period of 4-5 days on wheat has been reported. All strains of Aspergillus
flavus are not toxin producers.

Microbiological assays seem to be
well suited at semi quantitative level for aflatoxins. Both genetic and non-genetic effects of aflatoxins are seen in micro-organism.s

Since aflatoxicosis
is poisoing that results from ingestion of aflatoxins in contaminated food or feed, we have proposed a
work to detect the aflatoxin, B1,B2,G1,G2 by HPTLC
technique and other microbial assay methods such as preliminary tube test,
ditch plate technique and paper disc method.

Materials

Cultures used:

Aspergillus oryzae - 702

Aspergillus oryzae - 755

Medium:

Aflatoxin synthetic medium

Potato
dextrose agar

The
solvents used were of analytical grade

Methodology

→The
surface of the matt sporulation of the growth of
organism was scrapped after adding sterile saline

→The spore
density of the spore suspension was checked.

→About 2.5 ml
of spore suspension was added to 250ml aflatoxin
synthetic medium and allowed to sporulate.

→The culture filtrate was obtained and was used for
extraction.

→Aflatoxins were extracted by adding equal amount of chloroform to the
obtained culture filtrate.

→The lower chloroform layer was collected and heated in a
water bath at 75C till dryness.

→About 1ml of chloroform was added to the dry toxin and a
concentrated toxin was obtained.

HPTLC Method For Identification And
Confirmation Of Aflatoxins:

The instrument used was HPTLC CAMAG Linomat IV
(CAT S3) Scanner 3.

The samples and standards were spotted on precoated aluminum plates with a bandwidth of 4nm
each. The solvent system used was chloroform: acetone (9 :
1 ) and developed in
CAMAG toxin trough chamber and scanned at 366 nm. The RF values for standards
aflatoxin are as follows:

B1 – 0.7

B2 – 0.64

G1 – 0.61

G2 – 0.52

The sample showed similar peaks with
identical spectra as that of the standards
which confirmed the presence of aflatoxin
in the extract of the culture filtrate of Aspergillus
oryzae. The method was validated for accuracy,
precision, and repeatability. A linear graph was obtained for standards aflatoxins at a range of 20-120ng/ml with correlation
coefficient of 0.998. Precision was determined by repeating the studies six times. Repeatability and
reproducibility of the method was found to be good with a percentage CV of 2%
Accuracy of the method was established by carrying out recovery studies. A
known amount of varying standards were added to the simple and the percentage
recovery studies were found to be in the range of 98-100%. The toxin was also
analyzed for quantitative and semi-quantitative studies on the inhibition of
gram positive micro-organisms Staphylococcus species. Three methods were used
viz. preliminary tube method – based on turbidity, ditch plate technique –
based on inhibition over the ditch containing toxin, and paper disc method –
based on zone of inhibition.

Results

HPTLC studies
indicated that the method was accurate, precise and sensitive. RF values of aflatoxin in sample corresponded with the standard RF
values. Linearity study indicated that a very small quantity of culture can be used
for detection of aflatoxins. Since RF values of all the four aflatoxin bands were well resolved and sharp this method
can be used as routine technique for analysis of aflatoxins.
The results of inhibition on gram positive organisms showed that Staphylococcus
aureus was completely inhibited and in case of
Streptococcus reduced growth was seen. Thus it could be concluded that
Staphylococcus aureus is completely inhibited by the
toxin whereas Streptococcus might have destroyed the biological activity of the
toxin and growth or is partially inhibited by it.