Reduction of non-digestible oligosaccharides in horse gram and green gram flours using crude alpha -galactosidase from Streptomyces griseoloalbus


Anisha, G.S.; Prema, P.

Food Chemistry 106: 1175-1179

2008


The effectiveness of using crude extracellular α-galactosidase from Streptomyces griseoloalbus for the treatment of horse gram and green gram flours was investigated by comparing with traditional treatments such as soaking and cooking. The enzymatic treatment was most effective and the raffinose content in horse gram flour was reduced by 97.5% and stachyose content by 93.2%. The reduction in the raffinose content of green gram flour was 96.3% and that for stachyose was 91.8%. The information obtained from the present investigation is advantageous for the large-scale production of horse gram flour and green gram flour free of flatulence-causing oligosaccharides.

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Food
Chemistry
ELSEVIER
Food
Chemistry
106
(2008)
1175-1179
www.elsevier.com/locate/foodchem
Reduction
of
non-digestible
oligosaccharides
in
horse
gram
and
green
gram
flours
using
crude
a-galactosidase
from
Streptomyces
griseoloalbus
G.S.
Anisha,
P.
Prema*
Biotechnology
Division,
National
Institute
for
Interdisciplinary
Science
and
Technology
(Formerly
Regional
Research
Laboratory),
CSIR,
Trivandrum
695019,
Kerala,
India
Received
6
June
2007;
received
in
revised
form
20
June
2007;
accepted
18
July
2007
Abstract
The
effectiveness
of
using
crude
extracellular
oc-galactosidase
from
Streptomyces
griseoloalbus
for
the
treatment
of
horse
gram
and
green
gram
flours
was
investigated
by
comparing
with
traditional
treatments
such
as
soaking
and
cooking.
The
enzymatic
treatment
was
most
effective
and
the
raffinose
content
in
horse
gram
flour
was
reduced
by
97.5%
and
stachyose
content
by
93.2%.
The
reduction
in
the
raffinose
content
of
green
gram
flour
was
96.3%
and
that
for
stachyose
was
91.8%.
The
information
obtained
from
the
present
investigation
is
advantageous
for
the
large-scale
production
of
horse
gram
flour
and
green
gram
flour
free
of
flatulence-causing
oligosaccharides.
©
2007
Elsevier
Ltd.
All
rights
reserved.
Keywords:
Horse
gram
flour;
Green
gram
flour;
Flatulence;
ot
-
Galactosidase
1.
Introduction
Legumes
are
widely
grown
throughout
the
world
and
their
dietary
and
economic
importance
is
globally
appreci-
ated
and
recognized.
Legumes
not
only
add
variety
to
human
diet,
but
also
serve
as
an
economical
source
of
pro-
teins,
dietary
fibres
and
a
variety
of
micronutrients
and
phytochemicals,
for
a
large
human
population
in
develop-
ing
countries,
e.g.,
India,
where
most
of
the
population
is
vegetarian.
Recently
research
efforts
are
being
directed
towards
identifying
and
evaluating
under-exploited
legume
food
sources
such
as
protein
crops
for
the
future,
so
as
to
meet
the
nutritional
demands
of
an
increasing
population.
Horse
gram
(Dolichos
Olorus)
and
green
gram
or
mung
bean
(Vigna
radiata
L.)
are
among
the
most
important
food
legumes
grown
and
consumed
in
India.
In
addition
to
proteins,
horse
gram
is
a
rich
source
of
iron
and
molyb-
*
Corresponding
author.
Tel.:
+91
471
2515325;
fax:
+91
471
2491712.
E-mail
address:
(P.
Prema).
0308-8146/$
-
see
front
matter
©
2007
Elsevier
Ltd.
All
rights
reserved.
doi:10.1016/j.foodchem.2007.07.058
denum.
Green
gram
is
an
excellent
source
of
carbohy-
drates,
proteins
and
minerals
and
its
protein
quality
is
similar
to
or
better
than
other
legumes,
such
as
chickpea,
black
gram,
peas,
pigeonpea
(food,
Mehta,
&
Singh,
1986).
Horse
gram
and
green
gram
are
consumed
as
whole
seeds
or
sprouts
by
a
large
population
in
rural
areas
of
southern
India.
Like
other
legumes,
the
utilization
of
horse
gram
and
green
gram
for
human
nutrition
is
constrained
by
the
presence
of
raffinose
family
oligosaccharides
(RFO)
which
have
a
tendency
to
induce
flatulence.
The
production
of
flatulence
is
regarded
as
being
due
to
the
lack
of
ability
of
the
human
intestinal
tract
to
synthesize
the
enzyme
oc-
galactosidase,
which
is
necessary
to
hydrolyze
oligosaccha-
rides
containing
oc-galactosidic
linkages.
The
predominant
RFO,
raffinose
and
stachyose,
are
relatively
large
mole-
cules
and
are
hence
not
resorbed
by
the
intestinal
wall.
The
intact
oligosaccharides
therefore
enter
the
lower
intes-
tine
where
they
are
metabolized
by
the
microflora
into
carbon
dioxide,
hydrogen
and,
to
a
lesser
extent,
methane.
It
is
the
production
of
these
gases
which
leads
to
the
1176
G.S.
Anisha,
P.
Prema/Food
Chemistry
106
(2008)
1175-1179
characteristic
features
of
flatulence,
namely
nausea,
cramps,
diarrhea,
abdominal
rumbling,
and
the
social
dis-
comfort
associated
with
the
ejection
of
rectal
gas
(Cristof-
aro,
Mattu,
&
Wuhrmann,
1974).
The
problem
area
in
the
manufacture
of
protein
foods
is
therefore
the
breakdown
of
RFO
which
are
present
in
the
leguminous
seeds.
Many
methods
are
being
practised
for
the
processing
of
legume
seeds,
such
as
soaking,
cooking
and
sprouting
(Mulimani
&
Devendra,
1998;
Viana
et
al.,
2005),
which
may
bring
about
changes
in
the
levels
of
RFO.
The
newly
released
high-yielding
cultivars
may
not
only
have
different
grain
quality
characteristics,
but
also
may
behave
differ-
ently
from
existing
cultivars
after
processing
and
cooking.
Of
all
the
techniques
proposed,
the
enzymatic
processing
by
oc-galactosidase
has
proved
most
effective
(Mansour
&
Khalil,
1998;
Viana
et
al.,
2005).
oc-Galactosidase
or
melibi-
ase
(a-D-galactoside
galactohydrolase,
EC
3.2.1.22)
is
an
exo-glycosidase
that
cleaves,
the
terminal
non-reducing
oc-
D-galactose
residues
from
oc-D-galactosides,
including
gal-
actose
oligosaccharides,
such
as
melibiose,
raffinose
and
stachyose,
and
branched
polysaccharides,
such
as
galacto-
mannans
and
galacto
(gluco)
mannans
(Naumoff,
2004).
Currently,
there
is
a
lot
of
interest
in
the
scientific
commu-
nity
around
the
world,
in
exploiting
novel
microorganisms
for
the
production
of
industrially
important
enzymes
and
actinomycetes
which
have
immense
potential
as
source
of
exo-enzymes
are
yet
to
be
harnessed
as
source
of
oc-galacto-
sidase
for
commercial
application.
We
have
previously
identified
the
filamentous
actinobacterium,
Streptomyces
griseoloalbus
as
a
novel
source
of
oc-galactosidase
(Anisha
&
Prema,
2006)
and
the
potential
of
this
enzyme
in
soymilk
hydrolysis
has
also
been
demonstrated
(Anisha
&
Prema,
2007).
The
present
study
was
aimed
at
evaluating
the
suit-
ability
of
oc-galactosidase
from
this
novel
source,
for
the
reduction
of
RFO
in
horse
gram
and
green
gram
flours.
The
effect
of
enzymatic
treatment
was
compared
with
tra-
ditional
techniques,
such
as
soaking
and
cooking.
2.
Materials
and
methods
2.1.
Microorganism
Streptomyces
griseoloalbus
MTCC
7447
used
in
the
study
was
isolated
in
our
laboratory
from
a
soil
sample
col-
lected
from
mangrove
regions
along
the
West
Coast
of
India.
The
organism
was
maintained
at
4
°C
on
starch
casein
agar
(SCA)
slants
and
was
sub-cultured
fortnightly.
2.2.
Production
and
extraction
of
crude
a-galactosidase
Solid-state
fermentation
was
carried
out
for
the
produc-
tion
of
oc-galactosidase
from
S.
griseoloalbus.
Inoculum
was
prepared
by
transferring
a
loopful
of
culture
from
fresh
SCA
slants
into
sterile
medium
(100
ml
in
250
ml
Erlen-
meyer
flask)
composed
of
(g/l):
locust
bean
gum,
10;
yeast
extract,
3;
(NH
4
)
2
HPO
4
,
3.03;
KH
2
PO
4
,
1;
MgSO
4
7H
2
0,
0.49;
and
1
ml
of
trace
elements
solution.
The
trace
ele-
ments
solution
was
composed
of
(g/l):
FeSO
4
7H
2
0,
0.1;
MnC1
2
4H
2
0,
0.1
and
ZnSO
4
7H
2
0,
0.1.
The
flasks
were
incubated
at
30
°C
on
a
rotary
shaker
at
175
rpm.
A
48
h
old
culture
containing
3
x
10
6
CFU/ml
was
used
as
the
inoculum.
For
solid-state
fermentation,
10
g
of
soybean
flour,
taken
in
a
250
ml
Erlenmeyer
flask,
was
moistened
with
mineral
salt
solution
(g/1:
KH
2
PO
4
,
1;
MgSO
4
,
0.4;
pH
7.0),
thoroughly
mixed
and
autoclaved
at
121
°C
for
30
min.
The
cooled
medium
was
inoculated
with
2.25
x
10
7
CFU
of
inoculum
and
incubated
at
30
°C
for
5
days.
The
final
moisture
content
of
the
medium
was
40%.
Enzyme
extraction
was
carried
out
by
mixing
the
fer-
mented
matter
with
distilled
water
(1:5,
w/v)
on
a
rotary
shaker
at
200
rpm
for
1
h.
The
thoroughly
agitated
fer-
mented
matter
was
then
filtered
through
muslin
cloth
and
the
filtrate
obtained
was
centrifuged
at
10000
rpm
and
4
°C
for
20
min.
The
resultant
supernatant
was
used
as
the
enzyme
preparation.
2.3.
Enzyme
assay
The
activity
of
oc-galactosidase
was
routinely
determined
according
to
the
method
of
Dey
and
Pridham
(1969)
using
p-nitrophenyl
oc-D-galactopyranoside
(pNPG),
with
minor
modifications.
The
pNPG
hydrolyzing
activity
was
esti-
mated
by
incubating
100
IA
of
enzyme
with
50
IA
of
2
mM
pNPG
and
850
µI
of
0.1
M
McIlvaine
buffer
(Cit-
rate—Na
2
HPO
4
,
pH
7.0)
at
55
°C
for
10
min.
The
reaction
was
terminated
by
the
addition
of
2
ml
of
1
M
sodium
car-
bonate.
The
p-nitrophenol
released
was
estimated
spectro-
photometrically
by
absorbance
at
400
nm.
One
unit
(U)
of
enzyme
activity
was
expressed
as
the
amount
of
enzyme
that
liberated
1
µmol
of
p-nitrophenol/min
under
the
assay
conditions.
2.4.
Processing
of
legume
seeds
2.4.1.
Soaking
Dry
whole
seeds
of
horse
gram
and
green
gram,
pur-
chased
from
the
local
market,
were
soaked
in
distilled
water
(1:10,
w/v)
for
12
h
at
room
temperature.
After
12
h,
the
water
was
drained
off
and
the
soaked
seeds
were
washed
three
times
with
distilled
water.
2.4.2.
Cooking
Whole
horse
gram
and
green
gram
seeds
were
cooked
in
distilled
water
(1:10,
w/v)
on
a
hot
plate
for
60
min.
After
cooking,
the
seeds
were
rinsed
three
times
with
distilled
water.
2.4.3.
a-Galactosidase
treatment
Five
grammes
of
horse
gram
and
green
gram
seed
flour,
which
passes
through
a
500
um
sieve,
were
treated
with
1
ml
of
oc-galactosidase
(40
U)
diluted
to
50
ml
with
0.1
M
McIlvaine
buffer
(pH
7.0),
in
a
rotary
shaker
at
120
rpm
and
55
°C
for
2
h.
After
incubation,
the
treated
seed
flour
samples
were
filtered
through
a
Whatman
No.
G.S.
Anisha,
P.
Prema
I
Food
Chemistry
106
(2008)
1175-1179
1177
1
filter
paper,
dried
and
the
oligosaccharide
content
was
determined.
For
control,
the
volume
of
enzyme
was
replaced
with
an
equal
volume
of
buffer.
2.4.4.
Determination
of
oligosaccharide
content
The
raffinose
oligosaccharides
were
extracted
by
treating
5
g
of
seed
flour
sample
with
50
ml
of
70%
ethanol
(v/v)
in
a
rotary
shaker
at
120
rpm
for
12
h.
The
alcoholic
extract
obtained
after
filtration
through
Whatman
No.
1
filter
paper
was
concentrated
under
vacuum
at
40
°C
in
a
rotary
evaporator.
The
concentrated
sugar
syrup
was
made
up
to
10
ml
with
distilled
water.
Ten
microlitres
each
of
the
sugar
extract
were
applied
to
silica
gel
G
plates
(20
x
20
cm)
and
developed
by
ascending
chromatography,
using
n-propa-
nol:ethyl
acetate:water
(6:1:3,
v/v)
as
the
solvent
system
(Tanaka,
Thananunkul,
Lee,
&
Chichester,
1975).
The
sugar
spots
were
located
by
keeping
the
plates
in
an
oven
at
140
°C
for
5
min
after
spraying
with
1%
a-naphthol
in
absolute
ethanol
containing
10%
of
ortho-phosphoric
acid
(Albon
&
Gross,
1950).
For
quantitative
determination,
the
area
(2
x
2
cm)
corresponding
to
each
oligosaccharide
spot
was
scraped
from
unsprayed
duplicate
plates
and
eluted
with
3
ml
distilled
water
for
12
h.
The
mixture
was
centrifuged
to
remove
silica
gel
and
1
ml
of
the
supernatant
was
used
for
the
estimation
of
oligosaccharides
by
the
method
of
Tanaka
et
al.
(1975).
2.5.
Analytical
procedures
Total
soluble
sugars
in
the
concentrated
sugar
syrup
were
estimated
by
the
phenol-sulphuric
acid
method
(Dubois,
Gilles,
Hamilton,
Rebers,
&
Smith,
1956).
The
reducing
sugars
were
estimated
by
the
method
of
Nelson
(1944).
2.6.
Statistical
analysis
All
experiments
were
carried
out
in
triplicate
to
check
the
reproducibility
of
results.
The
data
presented
here
are
the
averages
of
triplicate
determinations
and
the
standard
deviations
for
all
the
values
were
<±5%.
3.
Results
and
discussion
The
levels
of
RFO
in
raw
horse
gram
and
green
gram
flour
samples
are
presented
in
Table
1.
The
results showed
that
green
gram
contained
more
RFO
than
did
horse
gram
Table
1
Oligosaccharide
content
of
raw
horse
gram
and
green
gram
Seed
flour
sample
Total
soluble
Raffinose
Stachyose
sugars
(g/kg
DMa)
(g/kg
DMa)
(g/kg
DMa)
and
the
concentration
of
stachyose
was
highest
in
both
horse
gram
and
green
gram.
The
relative
levels
of
raffinose
and
stachyose
obtained
in
our
study
were
in
accordance
with
those
presented
by
other
workers
(Adsule,
Kadam,
&
Salunkhe,
1986;
Rathbone,
1980).
3.1.
Effect
of
soaking
The
reduction
of
RFO
in
dry
whole
seeds
of
horse
gram
and
green
gram
by
various
treatments
is
given
in
Figs.
1
and
2,
respectively.
Soaking
of
dry
whole
seeds
of
horse
gram
in
distilled
water
for
12
h
resulted
in
mean
reductions
of
raffinose
content
by
23.8%
and
stachyose
content
by
12.3%.
For
green
gram
flour
samples,
the
reduction
of
raf-
finose
content
was
19%
and
that
of
stachyose
was
10%.
The
reduction
of
raffinose
content
was
higher
than
that
of
stachyose
content
in
both
the
cases.
The
sucrose
content
of
both
the
seed
flour
samples
decreased
after
soaking
(Table
2).
Mulimani,
Thippeswamy,
and
Ramalingam
(1997)
have
reported
that
soaking
of
whole
soybean
seeds
led
to
a
mean
decrease
of
80.3%
for
raffinose
and
44.8%
for
stachyose.
Reduction
in
raffinose
and
stachyose
con-
tents
of
red
gram
flour
by
54.6%
and
55.4%,
respectively,
was
reported
by
Mulimani
and
Devendra
(1998).
Reduc-
tion
of
RFO
in
cow
pea
seeds
by
soaking
was
reported
by
Somiari
and
Balogh
(1993).
Leaching
could
be
one
of
the
reasons
for
the
reduction
of
the
raffinose
family
of
25
g
20
15
c
10
0
cc
5
0
41•1•114.
Soaked
Cooked
Enzyme
treated
Fig.
1.
Raffinose
and
stachyose
0
contents
of
raw,
soaked,
cooked
and
enzyme-treated
horse
gram
flour.
30
§
10
-
0
cc
5-
Raw
Horse
gram
Green
gram
28.9
±
0.2
59.2
±
0.25
6.8
±
0.15
19.4
±
0.17
16.5
±
0.11
27.5
±
0.26
0
,71
The
data
are
means
and
standard
errors
of
three
independent
samples
with
triplicate
determinations.
a
Dry
matter.
Raw
Soaked
Cooked
Enzyme
treated
Fig.
2.
Raffinose
E
and
stachyose
0
contents
of
raw,
soaked,
cooked
and
enzyme-treated
green
gram
flour.
1178
G.S.
Anisha,
P.
Prema
I
Food
Chemistry
106
(2008)
II
75-11
79
Table
2
Sucrose
content
of
horse
gram
and
green
gram
before
and
after
various
treatments
Treatments
Sucrose
(g/kg
DM
a
)
Horse
gram
Green
gram
Raw
2.3
±
0.03
6.5
±
0.04
Soaked
0.7
±
0.02
2.0
±
0.03
Cooked
5.1
±
0.08
11.2
±
0.17
ot-Galactosidase-treated
8.9
±
0.06
22.4
±
0.1
The
data
are
means
and
standard
errors
of
three
independent
samples
with
triplicate
determinations.
a
Dry
matter.
sugars
during
soaking
(Price,
Lewis,
Wyatt,
&
Fenwick,
1988).
Upadhyay
and
Garcia
(1988)
have
demonstrated
that
the
differential
solubilities
of
individual
sugars
and
their
diffusion
rates
are
the
two
factors
that
influence
the
sugar
losses
during
soaking.
The
extent
of
reduction
in
level
of
oligosaccharides
can
be
enhanced
by
increasing
the
soaking
time
and
employing
different
soaking
media
(Pugalenthi,
Siddhuraju,
&
Vadivel,
2006).
However,
the
presence
of
off-odour
in
flours
obtained
from
the
legume
seeds
after
soaking
would
affect
the
acceptability
of
such
products
(Somiari
&
Balogh,
1993).
3.2.
Effect
of
cooking
Cooking
brought
about
a
greater reduction
in
the
raffi-
nose
family
sugars
than
did
soaking
(Figs.
1
and
2).
Cook-
ing
of
horse
gram
and
green
gram
seeds
for
60
min
resulted
in
mean
decreases
of
49.6%
and
46.3%,
respectively,
for
raffinose
and
24.3%
and
20.1%,
respectively,
for
stachyose.
Mulimani
et
al.
(1997)
reported
52.3%
removal
of
raffinose
and
20.7%
removal
of
stachyose
from
soybean
seeds
after
cooking.
Somiari
and
Balogh
(1993)
reported
that
cooking
of
cow
pea
for
50
min
reduced
the
raffinose
content
by
44%
and
the
stachyose
by
28.6%.
Onigbinde
and
Akinyele
(1983)
have
proposed
that
decrease
in
the
levels
of
raffinose
and
stachyose
during
cooking
might
be
attributed
to
heat
hydrolysis
to
disaccharides
and
monosaccharides
or
the
formation
of
other
compounds.
In
contrast,
Rao
and
Belavady
(1978)
reported
an
increase
in
the
level
of
oligo-
saccharides
after
cooking
of
pulses.
The
sucrose
content
of
both
horse
gram
and
green
gram
increased
after
cooking
(Table
2).
This
might
be
due
to
the
breakdown
of
storage
polysaccharides
to
sucrose,
as
reported
by
Onigbinde
and
Akinyele
(1983).
Though
cooking
resulted
in
a
decrease
in
the
level
of
RFO,
it
affected
the
colour,
texture
and
aroma
of
the
seed
flours.
It
is
also
reported
that
legumes,
such
as
horse
gram,
require
prolonged
cooking
to
obtain
products
of
accept-
able
nature
(Kadam
&
Salunkhe,
1985).
Price
et
al.
(1988)
have
reported
that
treatments
such
as
soaking
and
cooking
could
change
the
physicochemical
properties
of
legumes.
Moreover,
soaking
and
cooking
alone
will
not
be
sufficient
to
bring
about
any
significant
reduction
in
the
flatulence-inducing
activity
of
legumes
(Price
et
al.,
1988).
3.3.
Effect
of
crude
a-galactosidase
treatment
Horse
gram
flour,
when
treated
with
oc-galactosidase,
resulted
in
a
reduction
of
raffinose
content
by
97.5%
and
stachyose
content
by
93.2%
(Figs.
1
and
2).
The
enzyme
treatment
of
green
gram
samples
resulted
in
96.3%
and
91.8%
reductions
of
raffinose
and
stachyose,
respectively.
On
the
other
hand,
no
reduction
of
RFO
was
observed
in
the
control.
The
sucrose
content
of
the
enzyme-treated
seed
flour
samples
was
higher
than
that
of
the
soaked
and
cooked
samples
(Table
2).
The
reduction
in
RFO
by
crude
oc-galactosidase
treatment
was
due
to
the
conversion
of
oligosaccharides
into
di
and
monosaccharides
by
the
hydrolysis
of
oc-galactosidic
linkages
between
the
sugar
molecules.
The
increase
in
sucrose
content
could
be
due
to
the
formation
of
sucrose
through
raffinose
hydrolysis.
The
crude
oc-galactosidase
extracts
from S.
griseoloalbus
markedly
reduced
the
levels
of
raffinose
and
stachyose
in
horse
gram
and
green
gram
flours.
There
are
several
reports
available
in
the
literature
of
the
use
of
oc-galactosidase
from
fungal
and
plant
sources
for
the
removal
of
the
RFO
from
soymilk
and
legume
flours.
Somiari
and
Balogh
(1993)
have
used
crude
preparations
of
oc-galactosidase
from
Aspergillus
niger
for
the
removal
of
raffinose
and
stachyose
present
in
cowpea
flours.
Man-
sour
and
Khalil
(1998)
have
reported
100%
reduction
of
raffinose
oligosaccharide
content
in
chickpea
flours
by
crude
fungal
oc-galactosidase
treatment.
Mulimani
et
al.
(1997)
have
used
crude
oc-galactosidase
from
germinating
guar
seeds
for
the
hydrolysis
of
galactooligosaccharides
in
soybean
flour
and
have
reported
90.4%
reduction
of
raf-
finose
and
91.9%
reduction
of
stachyose.
4.
Conclusions
The
crude
a-galactosidase
extracts
from
S.
griseoloalbus
were
clearly
more
effective
for
reducing
the
levels
of
raffi-
nose
and
stachyose
in
legume
seed
flours
than
were
the
con-
ventional
methods,
soaking
and
cooking.
Crude
enzyme
treatments
would
seem
to
have
the
greatest
potential
as
the
technique
for
controlling
flatulence-inducing
activity
of
horse
gram,
green
gram
and
other
legume
seed
flours.
This
is
the
first
report
documenting
the
suitability
of
using
Streptomycete-a-galactosidase
for
the
treatment
of
legume
flours.
Although
the
results
suggest
that
oc-galactosidase
from S.
griseoloalbus
has
a
great
potential
in
the
treatment
of
legume
flours, safety,
palatability,
functionality
and
storage
properties
of
enzyme-treated
flours
have
to
be
determined
before
they
can
be
commercialized.
Adoption
of
effective
processing
methods
may
further
enhance
the
utilization
of
horse
gram
and
green
gram
as
potential
sources
of
proteins,
especially
among
the
economically
weaker
section
of
people
in
developing
countries.
G.S.
Anisha,
P.
Prema
I
Food
Chemistry
106
(2008)
II
75-11
79
1179
Acknowledgements
The
authors
would
like
to
thank
the
Director,
National
Institute
for
Interdisciplinary
Science
and
Technology,
Tri-
vandrum
for
providing
all
the
facilities
to
carry
out
this
work.
AGS
is
grateful
to
the
Council
of
Scientific
and
Industrial
Research
(CSIR)
for
the
award
of
research
fel-
lowship
given
to
her.
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