See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/286740784

Algae as biological detoxificant of aflatoxin produced by Aspergillus flavus

isolated from sunflower seed

Article in Indian Journal of Science and Technology · January 2008

DOI: 10.17485/ijst/2008/v1i3/29226

CITATIONS READS

13 421

4 authors, including:

Narasimhan Banu Sekaran Sridhar

Bharathi Womens College (Autonomous) Kalaignar Karunanidhi Government Arts College, Tiruvannamalai
51 PUBLICATIONS 351 CITATIONS 59 PUBLICATIONS 1,204 CITATIONS

SEE PROFILE SEE PROFILE

Muthumary Johnpaul
University of Madras
84 PUBLICATIONS 3,321 CITATIONS

SEE PROFILE

All content following this page was uploaded by Sekaran Sridhar on 29 July 2020.

The user has requested enhancement of the downloaded file.

 1
Indian Journal of Science and Technology http://www.indjst.org Vol.1 No 3 (Aug. 2008)
Algae as biological detoxificant of aflatoxin produced by Aspergillus flavus isolated from sunflower seed
N. Banu1, S.Sridhar 2, J. Muthumary and R. Rengasamy
Centre for Advanced Studies in Botany,
University of Madras, Guindy Campus, Chennai – 600 025, India.
1
Vel’s college of science, Pallavaram; 2Pepsico India Ltd., Chennai
banunkl@yahoo.com
Abstract: Aspergillus flavus is the widely reported prescribed for appropriate implementation. To
food-borne fungus and the most important achieve such low levels,
mycotoxin source in the world’s food supplies. A decontamination/detoxification procedures are
new approach using algae- Spirulina platensis useful in order to recuperate mycotoxin-
(blue-green alga), Ulva faciata (green alga) and contaminated commodities. The ideal
Sargassum wightii (brown alga) was experimented decontamination procedure should be easy to use,
to control the growth of A. flavus and its aflatoxin inexpensive and should not lead to the formation of
B1 production. Among the three algae, S. wightii compounds that are still toxic or may reverse to
was found as most suitable biocontrolling agent. reform the parent mycotoxin or alter the nutritional
The optimum biomass concentration of the alga to and palatability propertied of the product. Hence
detoxify the aflatoxin B1 and inhibition of the fungal approaches involving physical, chemical and
mycelial growth was worked out in a controlled biological methods have been made to detoxify
condition. An algal biomass of 1.8% (in final aflatoxin in food and feedstuffs by many workers
concentration) of S. wightii effectively eliminated (Landers et al., 1967; Park, 1993; Shannaz &
(100%) aflatoxin B1 and total inhibition of the Ghaffar 1999; Namazi et al., 2002; Vilar et al.,
fungal growth of A. flavus was also achieved 2003).
concomitantly. The experimental results were In the present investigation, different
encouraging for possible application of algal concentrations of the algal biomass (in powder) of
biomass as biocontrolling agent to recuperate Spirulina platensis, Ulva faciata and Sargassum
aflatoxin contaminated food and feed commodities. wightii was tested to prevent the growth of A. flavus
Introduction and the aflatoxin B1 production by the fungus.
Aflatoxins are the potent toxic, mutagenic, Materials and methods
heterogenic and carcinogenic metabolites Spirulina platensis (blue-green alga), Ulva
produced by species of Aspergillus flavus and A. faciata (green alga) and Sargassum wightii (brown
parasiticus in food and feed, especially in the oil alga) were selected to assess their efficacy to
seeds and their products, both at pre- and post- detoxify aflatoxin B1 content produced by A. flavus.
harvest conditions. Their occurrence in food and Ten gram of each dry powder sample of the algae
feed materials has caused not only health hazards was obtained from algal culture collection of Centre
to animals and humans but also economic losses for Advanced Studies in Botany, University of
especially to the exporting countries. They are Madras. The above algal biomaterials were
initially classified as human carcinogens by the dissolved in distilled water to get 3% stock
International Agency on Research in Cancer in solutions.
1993 and further epidemiological experiments Five and ten milliliter of S. platensis and
continue to show a strong link
between aflatoxin exposure Table 1. Quantification of aflatoxin B1 by TLC and HPTLC
and Hepato Cellular
Carcinoma (HCC). The U.S. Mycelial Concentration of
FDA has currently established Algal aflatoxin B1 (ppm) as
dry
action levels (Max.) of Name of the algae biomass (ml) determined by
weight
aflatoxin to be 20 ppb for (3% solution)
(g) TLC HPTLC
human foods (except milk),
0.5 ppb for milk, 20 ppb for Spirulina platensis 5 3.3 12 14
animal feeds, except some S. platensis 10 2.3 28 22
cases of feeds meant for Ulva faciata 5 4.0 24 25
maturing and finishing of meat U. faciata 10 3.6 28 29
animals, which varied from Sargassum wightii 5 3.6 14 16
100 to 300ppb (Park, 1993). S. wightii 10 3.4 28 26
From the viewpoint of S. wightii 20 3.3 1.4 2.5
health and economics, it is S. wightii 40 1.6 0.33 0.65
imperative that such low S. wightii 60 1.6 - -
levels of aflatoxin are S. wightii 80 2.1 1.4 1.4
Control - 3.9 30 36

iSee© category: Research article “Algal decontamination of aflatoxin” by Banu et al.

Indian Society for Education and Environment

Indian Journal of Science and Technology
Fig. 1 a - l. HPTLC Densitogramms
a. Authentic
b. Control
c. Aspergillus flavus with 5ml of
Spirulina platensis
U. faciata and 5, 10, 20, 40, 60 and 80 ml of S.
wightii stock solutions were added separately to a
final volume of 100 ml of Yeast Extract Sucrose
medium (2% yeast, 20% sucrose) (Davis et al.,
1966) and inoculated with a disc of A. flavus. It was
iSee© category: Research article “Algal decontamination of aflatoxin”
Indian Society for Education and Environment
2
http://www.indjst.org Vol.1 No 3 (Aug. 2008)
incubated for a period of 8 days at 30ºC up to the
stage of stationary culture. The fungus was
isolated earlier from the surface sterilized
sundered sunflower seed used for oil crushing.
After incubation, the culture was killed and the
culture filtrate was filtered through Whatmans No.
1 filter paper and the dry weight of the mycelium
was recorded. One hundred milliliter of culture
filtrate was extracted thrice with equal volume of
chloroform. The chloroform extract was dried
over rotary evaporator at 62 ºC. The final residue
was dissolved in 0.2 ml of chloroform. The same
procedure was followed for control without
adding algal suspension.
Quantification of aflatoxin B1 by TLC
Five, 10, 20 and 40 µl of the above
extracts were applied to pre-coated
TLC plates (Merck) along with the standard
aflatoxin B1. The plates were developed in a tank
containing chloroform: acetone in the ratio of
88:12. After the development, the plates were
viewed under long UV light at 365 nm. Blue-
fluorescence similar to standard aflatoxin B1
indicated the presence of aflatoxin B1.
Quantification of aflatoxin B1 was made by
evaluating on the plate itself using long UV light.
The role of algal suspension on detoxification
was determined by quantifying the intensity of
blue fluorescence of aflatoxin B1.
High Performance Thing layer chromatography
Twenty micro liter of the above sample
extracts were loaded onto pre-coated silica gel
plate. The plate was developed in a saturated
tank containing tertiary butyl methyl ether:
methanol: water in a ratio of 9.6:0.3:0.1. The
developing distance of the plate was up to 80
mm. The developed plates were scanned in a
Camag TLC Scanner 3 at 366 nm. The presence
of blue-fluorescence indicated the presence of
aflatoxin and confirmed with authentic sample.
Results and discussion
The blue-green alga, Spirulina platensis
showed marked reduction of aflatoxin B1
(starting concentration was 30 ppm and 36 ppm
as per TLC and HPTLC methods, respectively) to
12 ppm as determined by TLC and 14 ppm by
HPTLC method. At the same time, addition of
higher concentration of the alga (10 ml stock
solution) poorly effected the aflatoxin B1
elimination; at the same time mycelial reduction
of A. flavus was observed. Thus the addition of 5
ml algal stock solution, the weight of the
mycelium was reduced to 3.3 g in comparison with
control (3.9 g). But at 10 ml concentration algal
biomass, the effect was only on the growth of the
mycelium and not inhibited the aflatoxin production
(Table 1 & Fig. 1 a, b, c & d).
by Banu et al.

Indian Journal of Science and Technology
d. Aspergillus flavus with 10ml of Spirulina platensis
f. Aspergillus flavus with 10ml of Ulva faciata
The green alga U. faciata at both 5 and 10 ml
concentrations had not showed any positive control
on aflatoxin B1 production compared with S.
platensis. But the mycelial dry weight showed
reduction at 10 ml concentration of U. faciata. At 5
iSee© category: Research article “Algal decontamination of aflatoxin”
Indian Society for Education and Environment
3
http://www.indjst.org Vol.1 No 3 (Aug. 2008)
ml concentration, the dry weight of the
mycelium was increased by 1 g compared
to control (Table 1 Fig. 1 e & f).
The brown alga S. wightii at 5 ml
concentrations effected reduction in the
production of aflatoxin B1 (to 14 ppm as
determined by TLC and 16 ppm by HPTLC
method) as well as in the growth of the
mycelium. But at 10 ml, not much effect
was noted both on the growth of the
fungus and in aflatoxin B1 production
(Table 1; Fig. 1g- h).
Among the 3 algae tested for their
efficacy on controlling the growth of A.
flavus and its aflatoxin B1 produciton, all
the three were found to be suitable in a
varying degree. Considering the variability,
Spirulina platensis and Sarassum wightii
were found to be better organisms to
decontaminate A. flavus and detoxify the
production of aflatoxin B1. However in the
present investigation, only Sargassum
wightii was selected for further study (at
20, 40, 60 and 80 ml stock solution).
At 20 ml concentration of S. wightii on
A. flavus, a marked reduction on the
growth of the mycelium (3.3 g) as well as
in aflatoxin B1 production was observed
(to 1.4 ppm as recorded by TLC and to 2.5
ppm as recorded by HPTLC method). At
40 ml concentration of the alga, the
mycelial weight was reduced to 3.6 g and
aflatoxin B1 was 0.33 ppm by TLC and
0.65 ppm by HPTLC. At 60 ml of
concentration S. wightii, 100% elimination
of aflatoxin B1 was noted along with the
reduction of mycelial growth. But at 80 ml
concentration of the alga, mycelial growth
was observed along with the production of
aflatoxin B1 at a concentration of 1.4 ppm
as quantified both by TLC and by HPTLC
(Table I and Fig. 1 i, j, k & l).
Thus, among the different
concentrations (5, 10, 20, 40, 60 and 80
ml), 60 ml of S. wightii was found to be
suitable for 100% elimination of aflatoxin
B1 as well as total inhibition of the growth
of A. flavus. Hence the optimum biomass
concentration of S. wightii was found to be
60 ml (1.8% biomass dry/wet in a 100 ml
of YES medium) to detoxify aflatoxin B1 and to
control fungal growth.
by Banu et al.

Indian Journal of Science and Technology
g. Aspergillus flavus with 5ml of Sargassum wightii
h. Aspergillus flavus with 10ml of Sargassum wightii
i. Aspergillus flavus with 20ml of Sargassum wightii
The capacity of the algal biomass to control and
elimination of toxins produced by A. flavus is
mainly due to the presence of algal chlorophyll-
Chlorophyllin. It is basically a water-soluble mixture
of sodium-copper salts of chlorophyll formed from
the saponification of the relatively nonpolar, parent
compound (Kepart, 1955). It acts as an interceptor
forming tight molecular complexes with planar,
iSee© category: Research article “Algal decontamination of aflatoxin”
Indian Society for Education and Environment
4
http://www.indjst.org Vol.1 No 3 (Aug. 2008)
aromatic compound including the potent
hepatocarinogen, aflatoxin B1 (Negishi et al.,
1989; Breinholt et al., 1995). Chlorophyllin
may block the absorption of carcinogens,
thereby reducing their bioavailability in target
tissues ultimately leading to reduced DNA
adduct formation and tumor burden (Egner et
al., 2003).
Conclusion
The preferential inhibition of aflatoxin B1
production and its mould growth by S. wightii
is significant. The investigation supports the
possible role of algae as biological control
agent over the mould and its toxin. Further
investigation on the safe use of algal biomass
to protect the food stuff from mould and
aflatoxin, may lead to an inexpensive and
safe approach to realize the public health
mandate.
Acknowledgements
We greatly acknowledge the Department
of Science and Technology, Govt. of India for
providing the financial support to carryout the
research work.
References
1. Breinholt V, Schimerlik M, Dashwood R
and Bailey G (1995) Mechanisms of
chlorophyllin anticarcinogenesis against
aflatoxin B1 complex formation with the
carcinogen. Chem. Res. Toxicol. 8, 506 –
514.
2. Davis ND, Diener UL and Eldridge DW
(1966) Production of aflatoxin B1 and G1
by Aspergillus flavus in a semisynthetic
medium. Appl. Microbiol. 14, 378–380.
3. Egner PA, Muñoz A and Kensler TW
(2003) Chemoprevention with
clhlorophyllin in individuals exposed to
dietary aflatoxin . Mut. Res. 523- 524,
209– 216.
4. Kephart JC (1955) Chlorophyll derivative,
their chemistry, commercial preparation
and uses. Eco. Bot. 9, 3– 38.
5. Landers KE, Davis ND and Diener UL
(1967) Influence of atmospheric gases on
aflatoxin production by Aspergillus flavus
in peanuts. Phytopathol., 57, 1086–1090.
6. Namazi M, Allameh A, Aminshahidi M,
Nohee A and Malekzadeh F (2002)
Inhibitory effects of ammonia solution on
growth and aflatoxin production by Aspergillus
parasiticus NRRL – 2999. Acta Poloniae
Toxicol. 10, 65–72.
7. Negishi T, Arimoto S, Nishizaki C and Hayatsu
H (1989) Inhibitory effect of chlorophyll on the
genotoxicity of 3-amino-1-methyl-5H-pyrido
(4,3-b) indole (Trp-p-2). Carcino. 10, 145– 149.
by Banu et al.
 5
Indian Journal of Science and Technology http://www.indjst.org Vol.1 No 3 (Aug. 2008)
j. Aspergillus flavus with 40ml of Sargassum wightii 8. Park DL (1993) Perspectives of
mycotoxin decontamination
procedures. Food Add. And Conta.
10, 49 – 60.
9. Shahnaz D and Ghaffar A (1999) Use
of ammonia gas in the control of
Aspergillus flavus infection and
aflatoxin production in sunflower
seeds. Pak.J. Bot. 31, 231– 235.
10. Vilar MS, Kuilman-Wahls MEM and
Fink-Gremmels J (2003) Inhibition of
aflatoxin B1 mutagenicity by
cyclopiazonic acid in the presence of
human liver preparations. Toxicol.
Let. 143, 291–299.

k. Aspergillus flavus with 60ml of Sargassum wightii

l. Aspergillus flavus with 80ml of Sargassum wightii

iSee© category: Research article “Algal decontamination of aflatoxin” by Banu et al.

Indian Society for Education and Environment

View publication stats