In-vitro digestibility and microscopic appearance of germinated legume starches and their effect on dietary protein utilization


Shekib, Laila, A.

Food Chemistry 50(1): 59-63

1994


The effect of germination on the in-vitro digestibility of some legume starches and their effect on dietary protein were studied. Starch was extracted from faba bean (Vicia faba L.), kidney bean (Phaseolus vulgaris L.) and chick pea (Cicer artinum) before and after germination for 3 days. Germination increased the starch digestibility of the above three legumes (33.2%-32.3%, 39.5-47.2% and 44.7149.4%, respectively). The microscopic appearance of the isolated starch was conducted using a scanning electron microscopic technique. The size of the starch granules increased with slight damage in the starch isolated from germinated seeds in comparison to the ungerminated. The effect of the isolated legume starches on the utilization of casein as the sole protein in rat diets was evaluated using protein efficiency ratio (PER), digestibility coefficient (DC) and net protein utilization (NPU) as parameters. The utilization of casein significantly increased when the diets were supplemented with isolated starches from germinated legume seeds compared to ungerminated legume starches. Cooked starch from germinated chick pea had PER, DC and NPU values similar to those of a corn starch diet, being 2.4, 93.4 and 74.1%, respectively. This study found a good correlation between the nature of the starch granules and their in-vitro digestibilities and the utilization of the protein in the rat diets.

Food
Chemistry
50
(1994)
59-63
In-vitro
digestibility
and
microscopic
appearance
of
germinated
legume
starches
and
their
effect
on
dietary
protein
utilization
Laila
A.
Shekib
Food
Science
and
Technology
Department,
Faculty
of
Agriculture,
University
of
Alexandria,
Egypt
(Received
14
April
1993;
revised
version
received
and
accepted
24
June
1993)
The
effect
of
germination
on
the
in-vitro
digestibility
of
some
legume
starches
and
their
effect
on
dietary
protein
were
studied.
Starch
was
extracted
from
faba
bean
(Vicia
faba
L.),
kidney
bean
(Phaseolus
vulgaris
L.)
and
chick
pea
(Cicer
artinum)
before
and
after
germination
for
3
days.
Germination
increased
the
starch
digestibility
of
the
above
three
legumes
(33.2%-32.3%,
39.5-47-2%
and
4471-
49.4%,
respectively).
The
microscopic
appearance
of
the
isolated
starch
was
con-
ducted
using
a
scanning
electron
microscopic
technique.
The
size
of
the
starch
granules
increased
with
slight
damage
in
the
starch
isolated
from
germinated
seeds
in
comparison
to
the
ungerminated.
The
effect
of
the
isolated
legume
starches
on
the
utilization
of
casein
as
the
sole
protein
in
rat
diets
was
evaluated
using
protein
efficiency
ratio
(PER),
digestibility
coefficient
(DC)
and
net
protein
utilization
(NPU)
as
parameters.
The
utilization
of
casein
significantly
increased
when
the
diets
were
supplemented
with
isolated
starches
from
germinated
legume
seeds
compared
to
ungerminated
legume
starches.
Cooked
starch
from
germinated
chick
pea
had
PER,
DC
and
NPU
values
similar
to
those
of
a
corn
starch
diet,
being
2.4,
93.4
and
74.1%,
respectively.
This
study
found
a
good
correlation
between
the
nature
of
the
starch
granules
and
their
in-vitro
digestibilities
and
the
utilization
of
the
protein
in
the
rat
diets.
INTRODUCTION
Food
legumes
form
an
important
source
of
protein,
vitamins,
carbohydrates
and
minerals
in
diets
of
large
population
groups
in
Egypt
and
some
other
developing
countries
(Aykroyd
&
Doughty,
1964).
Legume
starches
have
been
found
to
be
less
digestible
than
cereal
or
cassava
starches
(Hoover
&
Sosulski,
1985).
They
showed
that,
during
a
6
h
period,
corn
starch
was
hydrolysed
(75%)
by
porcine
pancreatic
a-amylase
whereas
the
corresponding
values
for
legume
starches
belonging
to
the
species
Phaseolus
vulgaris
ranged
from
26
to
35%.
On
the
other
hand,
the
blood
glucose
responses
of
a
wide
spectrum
of
starchy
foods
were
found
to
correlate
with
the
in-vitro
digestion
rates
of
their
starches.
(Tappy
et
al.,
1986).
The
microscopic
structure
of
broad
bean
has
been
studied
by
El-Shimi
et
al.
(1980)
using
scanning
electron
microscopy
(SEM).
They
found
that
the
size
of
the
starch
granules
increased
after
soaking
and
germina-
tion.
Moreover,
the
utilization
of
legume
starches
may
have
some
effect
on
the
utilization
of
legume
protein.
Gervani
&
Theophilus
(1981)
reported
that
the
source
Food
Chemistry
0308-8146/94/$07.00
©
1994
Elsevier
Science
Limited,
England.
Printed
in
Great
Britain
of
carbohydrates
affects
the
utilization
of
the
protein.
They
found
that
black
gram
and
green
gram
starches
promoted
rat
growth
similarly
to
corn
starch,
but
the
growth
of
rats
fed
on
Red
gram
and
Bengal
gram
starches
was
significantly
less
(P
<
0-05).
El-Shemi
et
al.
(1992)
reported
that
the
nutritional
value
of
legume
seeds
can
be
improved
by
germination.
Germination
induces
biochemical
changes
which
produce
some
changes
in
the
physical
properties
and
functionality
of
seed
components
(Wursch
et
al.,
1986).
This
study
was
undertaken
to
explore
the
effect
of
germination
on
the
in-vitro
digestibility
of
some
legume
starches
and
the
effects
of
their
isolated
legume
starches
on
the
utilization
of
the
protein
in
the
rat
diets.
Also,
the
structure
of
raw
and
germinated
starches
was
investigated
using
an
SEM
technique
to
study
the
correlation
between
legume
starch
nature
and
its
nutritional
value.
MATERIALS
AND
METHODS
Materials
Faba
bean
(
Vicia
faba
L.),
chick
pea
(Cicer
artinum)
and
kidney
bean
(Phaseolus
vulgaris
L.)
were
purchased
59
60
Laila
A.
Shekib
from
a
local
market
in
Alexandria,
Egypt.
The
legume
seeds
were
kept
tightly
in
polyethylene
bags
at
4°C
until
used.
Methods
Germination
After
soaking
the
beans
in
distilled
water
for
12
h
at
room
temperature,
the
beans
were
washed
and
placed
in
stainless
steel
screen
baskets,
which
were
covered
with
a
cheese
cloth.
During
the
germination
period,
the
seeds
were
rinsed
with
tap
water
every
12
h.
Samples
were
collected
after
72
h
of
germination
and
air-dried.
Ungerminated
and
germinated
seeds
were
decorticated
and
ground
to
pass
through
a
60
mesh
sieve.
Extraction
of
legume
starch
Starch
was
extracted
from
the
raw
and
germinated
beans
by
method
B
described
by
Schoch
&
Maywald
(1968).
In
the
case
of
faba
beans
and
kidney
beans,
0.2%
NaOH
was
used
instead
of
distilled
water
for
the
isola-
tion
of
starch.
The
extracted
starch
was
air-dried
and
powdered
before
use.
The
isolated
starches
contained
98.8%
starch
based
on
dry
matter,
0.28%
nitrogen
and
0.06%
ash.
The
starch
was
determined
by
the
anthrone
method
as
described
by
(McCready
et
al.,
1950).
In
vitro
digestibility
of
legume
starch
Ground
samples
(100
mg)
were
suspended
in
9
ml
of
sodium
phosphate
buffer
(0.2
M,
pH
7.0)
and
incubated
at
37°C
for
15
mins.
Further,
1
ml
of
a-amylase
solution
in
0.2
N4
sodium
phosphate
buffer
(containing
200
units
of
enzyme
activity
and
preincubated
at
37°C
for
15
mins)
was
added.
At
the
end
of
30
minutes
the
reaction
was
stopped
by
heating
in
a
boiling
water
bath
for
3
min
(Deshpande
&
Salunkhe,
1982).
The
liberated
sugars
(expressed
as
maltose)
were
estimated
by
the
method
described
in
AACC
(1975).
Scanning
electron
microscopy
(SEM)
The
starch
specimen
was
mounted
on
circular
stubs
using
double
sided
tape
and
coated
with
gold
to
a
thickness
of
4
nm.
The
specimen
was
then
examined
on
a
Joel-JSM
252
II,
microscope,
(Japan).
Selected
areas
were
photographed
on
panatomic
X
film.
Biological
methods
The
protein
efficiency
ratio
(PER),
digestibility
coefficient
(DC)
and
net
protein
utilization
(NPU)
were
used
as
a
parameters
to
assess
the
effects
of
legume
starches
on
protein
utilization
in
the
diet.
Eight
groups
of
six
weanling
male
albino
rats,
3
to
4
weeks
old
(weight
average
50
g),
obtained
from
the
High
Institute
of
Public
Health,
University
of
Alexandria,
were
used.
The
composition
of
the
diet
is
given
in
Table
1.
Rats
were
fed
ad
libitum
daily
for
4
weeks.
Uneaten
and
scattered
feeds
were
estimated
daily
and
rats
were
weighed
daily.
The
PER
was
calculated
according
to
the
AOAC
(1980).
At
the
end
of
the
feed
period,
faeces
were
collected
daily
and
composited
for
5
days.
Food
Table
1.
Composition
of
casein
diet
supplemented
with
isolated
starches
from
some
legume
seeds
Legume
starches
(%)
Faba
bean
Kidney
bean
Chick-
pea
Casein
Casein
10
10
10
10
Corn
starch
75
Legume
starch
75
75
75
Corn
oil
10
10
10
10
Salt
mixture'
4
4 4
4
Vitamin
mixture"
1
1
1
1
From
INC
Pharmaceuticals,
OH,
USA.
The
composition
of
the
salt
mixture
is
(%):
54.3
calcium
car-
bonate,
2.5
magnesium
carbonate,
1.6
magnesium
sulphate.
7H
2
0,
6.9
sodium
chloride,
11.2
potassium
chloride,
21.2
potassium
phosphate
(monobasic),
205
ferric
phosphate,
0.008
potassium
iodide,
0.035
manganese,
0.01
sodium
fluoride,
0.017
aluminium
sulphate
and
0.9
copper
sulphate.
The
composition
of
the
vitamin
mixture
is
(mg/kg):
800
thiamin
HCI,
1600
riboflavin
HCI,
800
pyridoxin
HC1,
5000
Ca
pantothenate,
8000
niacinamide,
200
folic
acid,
40
biotin,
100
menadione,
2000
a-tocopheryl
succinate,
30
cyanocobal-
amin,
20
vitamin
A,
32.5
IU
vitamin
D/g
mixture
and
961.43
g
starch.
intake
and
body
weight
were
also
recorded
to
calculate
the
DC,
as
described
by
Mitchell
(1924).
Correction
of
faecal
nitrogen
excretion
was
made
by
calculating
the
faecal
nitrogen
on
rats
receiving
a
nitrogen-free
diet.
NPU
was
determined
by
the
method
of
Miller
&
Bender
(1955).
RESULTS
AND
DISCUSSION
In-vitro
starch
digestibility
values
are
presented
in
Table
2.
Starches
isolated
from
germinated
seeds
were
significantly
(P
<
0.01)
more
digestible
than
those
from
ungerminated
samples.
These
values
were
33.2,
32.3
and
39.5%
for
ungerminated
faba
bean,
kidney
bean
and
chickpea,
respectively,
and
became
47.2,
44.7
and
49.4%
after
germination
for
3
days.
El-Faki
et
al.
(1984)
found
a
modest
effect
of
germination
on
the
in-vitro
digestibility
of
legume
starch.
Moreover,
Nnanna
&
Phillips
(1990)
reported
that
in-vivo
digestibility
of
legume
starch
increased
by
germination.
As
indicated
by
Table
2,
cooking
of
the
isolated
starches
increased
their
digestibility
significantly
(P
<
0.01)
for
both
the
ungerminated
and
germinated
samples.
It
can
be
seen
that
the
cooked
germinated
starch
had
the
highest
starch
digestibility
values
(84.3-90.3%).
Cooking
was
found
to
increase
the
digestibility
of
starches
from
germinated
beans.
The
digestibilities
of
starches
from
cooked,
germinated
chickpea,
cowpea
and
green
gram
were
found
to
be
two
to
four
times
higher
than
that
seen
with
uncooked
germinated
seeds
(Kumar
&
Venkataraman,
1976).
The
corresponding
value
for
black
gram
and
chick
pea
was
1.3
(Jood
et
al.,
1988).
Swelling,
gelatinization
and
destruction
of
the
crystalline
structure
of
starch
granules
are
known
to
increase
the
action
of
amylase
enzymes
on
them.
Ingredient
(g)
Effect
of
germinated
legume
starches
on
dietary
protein
utilization
61
I(
,
.„
Taft
1
N...kb
(A)
(B)
ct
,
o4
(C)
(D)
(E)
(F)
Fig.
1.
SEM
micrographs
of
starches
isolated
from
ungerminated
and
germinated
legume
seeds.
(original
magnification
x
700).
(A)
ungerminated
faba
bean;
(B)
germinated
faba
bean
(3
days);
(C)
ungerminated
kidney
bean;
(D)
germinated
kidney
bean
(3
days);
(E)
ungerminated
chick
pea;
(F)
germinated
chick
pea
(3
days).
62
Laila
A.
Shekib
Table
2.
In
vitro
starch
digestibility
of
ungerminated
and
Table
3.
Effect
of
isolated
legume
starches
on
PER,
DC
and
germinated
isolated
legume
starches
in
various
forms
NPU
of
casein
as
a
protein
source
in
rat
diets
Treatment
Faba
bean
(%)
Kidney
bean
(%)
Chick-
pea
(%)
Corn
starch
(%)
Isolated
starch
A
33.2
32.3
39.5
B
65.1
62.1
67.2
C
47.2
44.7
49.4
D
84.3
82.1
90.3
Cooked
corn
starch
90.3
A:
isolated
starch
from
ungerminated
seeds.
B:
cooked
isolated
starch
from
ungerminated
seeds.
C:
isolated
starch
from
germinated
seeds.
D:
cooked
isolated
starch
from
germinated
seeds.
Cooking:
the
samples
was
steamed
(115°C
for
15
min).
SEM
micrographs
of
starch
granules
isolated
from
ungerminated
and
germinated
faba
bean,
kidney
bean
and
chickpea
are
shown
in
Fig.
1.
It
is
clear
that
the
size
of
the
starch
granules
increased
after
3
days
of
germination.
The
lengths
of
ungerminated
legume
starch
granules
ranged
from
0.9
to
1.8
mm.
After
germination
the
granules
averaged
1
to
2.9
mm
for
the
three
legume
seeds.
However,
faba
bean
starch
showed
the
highest
swelling
during
the
germination
period
(its
starch
length
ranged
from
1.1
to
1.8
mm
and
increased
from
1.7
to
2.9
mm
after
germination).
Some
of
the
legume
starch
granules
were
broken
while
others
maintained
their
integrity.
Germinated
kidney
starches
were
more
rigid
than
germinated
chickpea
starches,
which
were
more
fragile
and
some
of
the
starch
granules
lost
their
structure.
EI-Shimi
et
al.
(1980)
stated
that
broad
bean
starch
granules
appeared
to
be
very
fragile
after
8
days
of
germination.
From
the
results
of
Table
2
and
Fig.
1,
germinated
seeds
which
had
large
and
somewhat
broken
starch
granules
had
higher
digestibility
values
than
ungerminated
ones.
So
it
can
be
concluded
that
the
digestibility
of
the
legume
starch
is
correlated
to
the
nature
of
the
starch.
El-Faki
et
al.
(1984)
stated
that
the
nature
of
starch
may
decide
the
type
and
degree
of
amylolysis
in
the
in-vitro
system.
Further,
Wursch
et
al.
(1986)
reported
that
disruption
of
the
cells,
especially
before
cooking,
improves
the
susceptibility
of
starch
to
a-amylase
digestion.
Contradicting
views
have
been
expressed
by
other
investigators
on
the
relationship
between
granule
size
and
rate
of
enzymolysis,
some
indicating
that
large
granules
are
less
readily
attacked
(Kulp
&
Mattern,
1973)
and
some
not
finding
any
association
(Leach
&
Schoch,
1961).
The
effect
of
ungerminated
starches
on
the
utilization
of
casein
as
a
sole
source
of
protein
in
rate
diets
is
given
in
Table
3.
All
the
ungerminated
legume
starches
under
study
were
inferior
to
corn
starch
in
promoting
growth.
The
adjusted
PER
value
for
the
rats
fed
on
diets
supple-
mented
with
corn
starch
was
2.5%,
while
it
was
1.87,
1.99
and
2.15%
for
faba
bean,
kidney
bean
and
chick-
pea
starch
diets,
respectively.
The
protein
DC
values
of
the
diets
containing
ungerminated
legume
starches
were
lower
than
that
of
the
corn
starch
diet.
Chickpea
had
Source
of
starch'
Actual
PER
Adjusted"
PER
DC
(%)
NPU
Corn
starch
2.21
±
0.04
2.50
96.5
75.9
Faba
bean
Ungerminated
1.65
±
0.09
1.87
69.4
60.3
±
4
21
Germinated
1.98
±
0.05
2.23
88.5
69.4
±
3.09
Kidney
bean
Ungerminated
1-76
±
0.06
1.99
66.3
61.1
±
6.10
Germinated
1.90
±
0.08
2.15
83.1
65.2
±
5.07
Chick
pea
Ungerminated
1.94
±
0.11
2.15
79.9
67.2
±
4.04
Germinated
2.15
±
0.08
2.43
93.4
74.1
±
5.06
"
Steamed
(115°C
for
15
mins).
"
Adjusted
PER
Actual
PER
x
2.5
-
PER
of
corn
starch
diet
the
highest
DC
value
among
the
three
legumes
under
study,
being
79-9%.
NPU
values
for
the
legume
starch
diets
confirmed
the
above
results
where
NPU
values
for
the
diets
containing
ungerminated
legume
starches
were
lower
than
those
containing
corn
starch.
The
results
of
Table
3
also
show
that
the
germination
process
significantly
improves
(P
<
0.05)
the
utilization
of
the
protein
in
the
diets.
Adjusted
PER,
DC
and
NPU
values
increased
by
19.3%,
20.3
and
15.1%,
12.9,
22.7
and
18.3%,
and
13.0;
17.5
and
10.3%
for
faba
bean,
kidney
bean
and
chick
pea,
respectively.
Booher
et
al.
(1951)
reported
that
apparent
digestibility
of
protein
varies
according
to
the
component
of
the
diet.
Also,
Yoshida
&
Morimoto
(1955)
pointed
out
that
PER
and
DC
of
diets
supplemented
with
potato
starch
were
less
than
that
of
corn
starch.
Gervani
&
Theophilus
(1981)
postulated
that
the
influence
of
starches
on
protein
utilization
may
be
through
their
interference
with
digestibility.
They
also
reported
that
the
PER
of
red
gram
and
Bengal
gram
protein
can
be
improved
if
the
digestibility
of
the
respective
carbo-
hydrates
is
improved.
The
results
of
this
study
confirm
their
findings
as
re-enforced
by
the
following:
Table
2
shows
that
germination
significantly
improved
the
in-
vitro
digestibility
of
legume
starches.
Also,
Table
3
shows
that
the
diets
supplemented
with
starches
isolated
from
germinated
seeds,
which
had
high
starch
digestibility
values,
supported
the
utilization
of
the
protein
almost
as
well
as
corn
starch
(especially
chick
pea).
REFERENCES
AACC
(1975).
Cereal
Laboratory
Methods,
American
Association
of
Cereal
Chemists,
St.
Paul,
MN.
AOAC
(1980).
Official
Methods
of
Analysis,
Association
of
Official
Analytical
Chemists,
Washington,
DC.
Aykroyd,
W.
B.
&
Doughty,
J.
(1964).
Legumes
in
human
nutrition.
FAO,
Nutrition
Studies
No.
19,
Rome.
Booher,
L.
E.,
Behman,
I.
MC.
&
Means,
E.
(1951).
Biological
utilization
of
unmodified
and
modified
food
starches.
J.
Nutr.,
45,
75-95.
Deshpande,
S.
S.
&
Salunkhe,
D.
K.
(1982).
Effect
of
Effect
of
germinated
legume
starches
on
dietary
protein
utilization
63
dehulling
on
phytic
acid,
polyphenol,
and
enzyme
in-
hibitors
of
dry
beans
(Phaseolus
vulgaris).
J.
Food
Sci.,
47,
1846-9.
El-Faki,
H.
A.,
Venkatraman,
L.
V.
&
Desikachar,
H.
S.
R.
(1984).
Effect
of
processing
on
the
in-vitro
digestibility
of
proteins
and
carbohydrates
in
some
Indian
legumes.
Plant
Foods
Human
Nutr.,
34,
127-31.
El-Shimi,
N.
M.,
Luh,
B.
S.
&
Shehata,
E.
(1980).
Changes
in
microstructure
of
starch
granules
and
sugars
of
germinated
broad
beans.
J.
Food
Sci.,
45,
1652-7.
El-Shimi,
N.
M.,
Shekib,
L.
A.
&
Kotet,
S.
M.
(1992).
Changes
in
nitrogenous
constituents,
phytate
and
nutritive
value
during
the
germination
of
black
pea
(Vigna
unguiculate)
and
kidney
beans
(Phaseolus
vulgaris)
2nd
Alexandria
Conf.
Food
Sci.
Technol.,
Faculty
of
Agriculture,
Alexandria
University,
p.
173-9.
Gervani,
P.
&
Theophilus,
F.
(1981).
Influence
of
legume
starches
on
protein
utilization
and
availability
of
lysine
and
methionine
to
albino
rats.
J.
Food
Sci.,
46,
817-8.
Hoover,
R.
&
Sosulski,
F.
W.
(1985).
Studies
on
the
functional
characteristics
and
digestibility
of
starches
from
Phaseolus
vulgaris.
Staerke,
37,
181-91.
Jood,
S.,
Chauhan,
B.
M.
&
Kapoor,
A.
C.
(1988).
Contents
and
digestibility
of
carbohydrates
of
chickpea
and
black
gram
as
affected
by
domestic
processing
and
cooking.
Food
Chem.,
30,
113-27.
Kulp,
K.
&
Mattern,
P.
J.
(1973).
Properties
of
starches
derived
from
wheat
of
varied
maturity.
Cereal
Chem.,
50,
496-504.
Kumar,
K.
G.
&
Venkataraman,
L.
V.
(1976).
Studies
on
In-vitro
digestibility
of
starch
in
some
legumes
before
and
after
germination.
Nutr.
Rep.
Int.,
13,
115-24.
Leach,
H.
W.
&
Schoch,
T.
J.
(1961).
Structure
of
the
starch
granule.
II-Action
of
various
amylases
on
granular
starches.
Cereal
Chem.,
38,
34-40.
McCready,
R.
M.,
Griggolz,
J.,
Silviera,
V.
&
Ownes,
H.
S.
(1950).
Determination
of
starch
and
amylose
in
vegetables.
Application
to
pea.
Anal.
Chem.,
22,
1156-60.
Miller,
D.
S.
&
Bender,
A.
E.
(1955).
The
determination
of
utilization
of
protein
by
shortened
method.
Br.
J.
Nutr.,
9,
392-8.
Mitchell,
H. H.
(1924).
A
method
of
determining
the
biological
value
of
protein.
J.
Biol.
Chem.,
58,
873-81.
Nnanna,
I.
A.
&
Phillips,
R.
D.
(1990).
Protein
and
starch
digestibility
and
flatulence
potential
of
germinated
cowpeas
(Vigna
unguiculata).
J.
Food
Sci.,
55,
151-3.
Schoch,
T.
&
Maywald,
E.
C.
(1968).
Preparation
and
properties
of
various
legume
starches.
Cereal
Chem.,
45,
564-73.
Tappy,
L.,
Wursch,
P.,
Randin,
J.
P.,
Felber,
J.
P.
&
Jequier,
E.
(1986).
Metabolic
effect
of
pre-cooked
instant
preparation
of
bean
and
potato
in
normal
and
diabetic
subjects.
Am.
J.
Clin.
Nutr.,
43,
30-5.
Wursch,
P.,
Vedovo,
S.
D.
&
Koellreutter,
B.
(1986).
Cell
structure
and
starch
nature
as
key
determinants
of
the
digestion
rate
of
starch
in
legumes.
Am.
J.
Clin.
Nutr.,
43,
25-9.
Yoshida,
M.
&
Morimoto,
H.
(1955).
Utilization
of
sweet
potato
starch
by
rats
and
its
effect
on
the
digestion
of
dietary
protein.
J.
Nutr.,
57,
565-8.