The impact of germination and dehulling on nutrients antinutrients in vitro iron and calcium bioavailability and in vitro starch and protein digestibility of some legume seed

Abstract

The content of nutrients (protein, starch, ash, calcium, iron, phosphorous and thiamin) and antinutritional components (dietary fiber fractions, phytic acid and tannin), and in vitro bioavailability of calcium and iron and in vitro digestibility of protein and starch were determined in control, germinated and dehulled green gram, cowpea, lentil and chickpea. Germination caused significant (P<0.05) increase in protein, thiamin, in vitro iron and calcium bioavailability and in vitro starch and protein digestibility contents of all the legume samples. Further increase in mentioned parameters was observed after dehulling the germinated legumes. Phytic acid and tannin were reduced by 18–21% and 20–38%, respectively, on germination and more reduction was observed in dehulled over germinated samples. There were negative correlations between nutrients bioavailability and digestibility with antinutritional factors.

Full text

LWT 40 (2007) 1292–1299
The impact of germination and dehulling on nutrients, antinutrients, in
vitro iron and calcium bioavailability and in vitro starch and protein
digestibility of some legume seeds
Reihaneh Ahmadzadeh Ghavidel

, Jamuna Prakash
Department of Studies in Food Science and Nutrition, University of Mysore, Manasagangotri, Mysore 570 006, India
Received 3 October 2005; received in revised form 29 July 2006; accepted 8 August 2006
Abstract
The content of nutrients (protein, starch, ash, calcium, iron, phosphorous and thiamin) and antinutritional components (dietary fiber
fractions, phytic acid and tannin), and in vitro bioavailability of calcium and iron and in vitro digestibility of protein and starch were
determined in control, germinated and dehulled green gram, cowpea, lentil and chickpea. Germination caused significant (Po0.05)
increase in protein, thiamin, in vitro iron and calcium bioavailability and in vitro starch and protein digestibility contents of all the
legume samples. Further increase in mentioned parameters was observed after dehulling the germinated legumes. Phytic acid and tannin
were reduced by 18–21% and 20–38%, respectively, on germination and more reduction was observed in dehulled over germinated
samples. There were negative correlations between nutrients bioavailability and digestibility with antinutritional factors.
r2006 Published by Elsevier Ltd. on behalf of Swiss Society of Food Science and Technology.
Keywords: Germination; Dehulling; Antinutrients; In vitro mineral availability; In vitro starch and protein digestibility; Legumes
1. Introduction
Legumes play an important role in the agriculture and
diet of many developing countries and are a major source
of dietary nutrients for many people. However, their role
appears to be limited because of several factors including
low protein and starch digestibility (Kataria, Chauhan, &
Punia, 1989;Negi, Boora, & Khetarpaul, 2001),
poor mineral bioavailability (Kamchan, Puwastien,
Sirichakwal, & Kongkachuichai, 2004;Rao & Prabha-
vathi, 1982) and high antinutritional factors (Das,
Chaturvedi, & Nagar, 1999;Ramulu & Udayasekhara,
1997;Savelkoul, Vanderpoel, & Tamminga, 1992).
It has been reported that protein and thiamin (Sattar,
Durrani, Mahmood, Ahmad, & Khan, 1989), mineral
bioavailability (Ghanem & Hussein, 1999;Rao & Prabha-
vathi, 1982) and protein and starch digestibility (Kataria,
Chauhan, & Punia, 1992;Preet & Punia, 2000) increased,
whereas phytic acid (Ayet et al., 1997;Kataria et al., 1989;
Sattar et al., 1989) and tannin (Ayet et al., 1997;Savelkoul
et al., 1992) decreased during germination of legumes.
Most researchers have studied the effect of soaking and
germination on nutritional quality of legumes, but
information on effect of a combination of processes such
as soaking, germination and dehulling on improvement of
nutritional quality of legumes is scarce. Therefore, the aims
of this work were (a) to study the effect of soaking and
germination individually and in combination with dehul-
ling on proximate composition; antinutrients such as
dietary fiber fractions, phytic acid and tannin; minerals,
viz. iron, calcium and phosphorous; in vitro iron and
calcium bioavailability and in vitro starch and protein
digestibility, and (b) to analyse data statistically to
establish regression equations for predicting in vitro iron
and calcium bioavailability as well as in vitro starch and
protein digestibility by substituting antinutritional factors
in equations.
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www.elsevier.com/locate/lwt
0023-6438/$30.00 r2006 Published by Elsevier Ltd. on behalf of Swiss Society of Food Science and Technology.
doi:10.1016/j.lwt.2006.08.002

Corresponding author. Tel.: +91 821 5288 220; fax: +91 821 2412 589.
E-mail address: reahmadzadeh@yahoo.com (R.A. Ghavidel).

2. Materials and methods
2.1. Sample preparation
Green gram (Phaseolus aureus), cowpea (Vigna catjang),
lentil (Lens culinaris) and chickpea (Cicer arietinum) were
obtained from local market. Legume seeds were cleaned,
washed and soaked in 4–5 volumes of water (22–25 1C) for
12 h under ambient laboratory conditions. At the end of
the period, the water was drained and the seed samples
were allowed to germinate under a wet muslin cloth for
24 h and then dried in a cabinet dryer (Magumps, Mumbai,
India) at 50751C for 16–18 h. A portion of germinated
samples was dehulled in a dehusker (Versatile dhal mill,
designed and developed by Central Food Technological
Research Institute, Mysore, India). Ungerminated seeds
served as control. All the three samples, (1) control
(ungerminated), (2) germinated and (3) dehulled (after
germination) were milled to flour in a plate mill (Bhavani
Industries, Bangalore, India). The processing of samples
was done in one batch and processed samples were stored
in airtight containers for further analysis.
2.2. Chemicals
The chemicals required for the study were obtained from
SD Fine, Qualigen Laboratories Pvt. Ltd., Mumbai, India.
The enzymes used were obtained from Himedia Company,
Mumbai, India. Glucose oxidase peroxidase kit (Code No:
B0112/Lot No: 5354) was procured from Span Diagnostics
Ltd., Surat, India.
2.3. Chemical analysis
Moisture, fat, ash (minerals) and tannin contents were
estimated by standard AOAC methods (AOAC, 1990). The
nitrogen content was estimated by Kjeldhal method, based
on the assumption that plant proteins contain 16 g/100 g
nitrogen, protein content was calculated using the formula,
protein ¼nitrogen 6.25. Thiamin was analysed by
oxidation to thiochrome, which fluoresces in UV light
(Raghuramulu, Nair, & Kalyansundaram, 1983). Insoluble
and soluble dietary fiber was analysed by separation of non-
starch polysaccharides by enzymatic gravimetric method
(Asp, Johansson, Hallmer, & Siljestro
¨m, 1983). Phytic acid
was extracted and determined by estimation of phosphorous
according to the precipitate analysis method of Thompson
and Erdman (1982). The conversion factor 3.55 for
phosphorous to phytic acid was used (Sen & Bhattacharyya,
2003). The samples were ashed in a muffle furnace and ash
solution was prepared by dry ashing. Iron was estimated
colorimetrically by a-a- dipyridyl method (AOAC, 1990).
An in vitro method for the determination of bioavailability
of nonheme iron from foods was investigated. Sample was
extracted with pepsin–HCl at pH 1.35 and subsequently the
pH was adjusted to pH 7.5 and filtered. Ionizable iron was
determined in the filtrate by the a-a- dipyridyl method.
Percent iron bioavailability is predicted using the following
regression equation; Y¼0.4827+0.4707X,whereYis the
percent available iron and Xis the percent ionizable iron
(Rao & Prabhavathi, 1978). Calcium was analysed by
precipitation as calcium oxalate and subsequent titration by
potassium permanganate (AOAC, 1990). In vitro bioavail-
ability of calcium was determined by a simulated gastro-
intestinal digestion using pepsin for the gastric stage
followed by pepsin and bile salts for the intestinal stage
(Luten et al., 1996). The content of calcium diffused through
a semi permeable membrane was determined by precipita-
tion and titration method (AOAC, 1990). Phosphorous was
estimated colorimetrically by Taussky and Shorr (1953)
method. The enzymatic method of Batey and Ryde (1982)
was used for total starch analysis. In vitro starch digestibility
was determined by modification of method of Kumar and
Venkataramann (1976) by using enzymatic glucose oxidase
peroxidase kit instead of dinitrosalicylic reagent. Glucose
was used as a standard and the degree of hydrolysis was
expressed as mg of glucose liberated from the samples after
correction for blank values and percent in vitro starch
digestibility was calculated on the basis of total starch
content using the following equation; glucose released
(g) 0.9 100/g of total starch. In vitro protein digestibility
was estimated by enzymatic method of Akeson and
Stahmann (1964).
2.4. Statistical analysis
The analysis was carried out in four replicates for all
determinations. The mean and standard deviation of
means were calculated. The data were analysed by one-
way analysis of variance (ANOVA). A multiple compar-
ison procedure of the treatment means was performed by
Duncan’s new multiple range test (Duncan, 1955). The
correlation coefficients were computed and regression
equations were made. Significance of the differences was
defined as Po0.05.
3. Results and discussion
The results in Table 1 showed that moisture in control
samples ranged from 8.5 to 11.7 g/100 g. There was a
reduction in moisture on germination in all samples and an
increase of it after dehulling. Fat content of control seeds
ranged from 0.89 g/100 g in lentil to 5.45 g/100 g in
chickpea. On germination, there was a statistically sig-
nificant (Po0.05) decrease of fat content, which could be
due to total solid loss during soaking prior to germination
(Wang, Lewis, Brennan, & Westby, 1997) or use of fat as
an energy source in sprouting process. The results are
comparable with findings of Venderstoep (1981) for
germinated green gram and lentil. Protein and thiamin
levels of control samples were within previously reported
ranges (Gopalan, Sastri, & Balasubramanian, 1989;Sa-
vage, 1988). Protein and thiamin contents after germina-
tion increased significantly (Po0.05) by 6.1–9.7% and
ARTICLE IN PRESS
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Table 1
Effect of germination and dehulling on moisture, fat, protein, thiamin, and ash contents of legume flours (on dry weight basis/100 g)
a
Sample Moisture (g) Fat (g) Protein (g) Thiamin (mg) Ash (g)
Green gram
Raw 10.970.1
a
1.2970.02
b
27.770.3
c
0.5670.02
c
4.0370.09
a
Germinated 8.070.2
c
1.2170.01
c
29.170.1
b
0.7170.03
b
3.8870.04
b
Dehulled 10.070.1
b
1.770.01
a
29.970.6
a
0.8570.04
a
3.7970.01
b
SE (df ¼9) 0.084 0.007 0.208 0.016 0.028
Cowpea
Raw 8.570.4
a
1.1670.04
b
25.770.1
c
0.6470.02
c
3.6270.01
a
Germinated 6.170.1
c
1.0770.02
c
27.270.2
b
0.6970.03
b
3.5370.01
b
Dehulled 7.670.3
b
1.4870.01
a
28.470.3
a
0.8570.02
a
3.4770.06
b
SE (df ¼9) 0.174 0.012 0.146 0.011 0.018
Lentil
Raw 11.770.1
a
0.8970.06
b
26.570.5
c
0.5170.03
c
2.4470.02
a
Germinated 10.070.2
c
0.7870.02
c
28.570.2
b
0.6870.04
b
2.2870.14
b
Dehulled 11.070.3
b
1.270.09
a
29.670.2
a
0.8170.03
a
2.1370.13
b
SE (df ¼9) 0.025 0.031 0.187 0.016 0.055
Chickpea
Raw 9.970.3
a
5.4570.02
b
22.170.5
c
0.3470.009
c
3.1470.17
a
Germinated 7.170.2
b
5.1870.02
c
24.270.2
b
0.4270.01
b
2.9470.01
b
Dehulled 6.570.3
b
5.770.04
a
27.270.3
a
0.5170.009
a
2.8770.02
b
SE (df ¼9) 0.154 0.013 0.195 0.005 0.048
SE ¼standard error of means.
df ¼degree of freedom.
All mean scores bearing different superscripts in columns in each sample are significantly different on application of Duncan’s new multiple range test (Po
0.05).
a
Values are expressed as mean7standard deviation (n¼4).
Table 2
Effect of germination and dehulling on antinutritional factors in legume flours (g/100 g on dry weight basis)
a
Sample Soluble dietary fiber Insoluble dietary fiber Total dietary fiber Phytic acid Tannin
Green gram
Raw 3.4270.06
b
16.5870.07
a
20.070.09
b
0.6170.02
a
0.6670.02
a
Germinated 4.7170.07
a
15.870.02
b
20.5170.07
a
0.570.03
b
0.5970.01
b
Dehulled 2.5770.06
c
12.670.06
c
15.1670.02
c
0.2970.03
c
0.3670.01
c
SE (df ¼9) 0.027 0.026 0.041 0.014 0.004
Cowpea
Raw 2.1970.08
b
25.1870.34
b
27.3870.26
b
0.670.01
a
0.4770.01
a
Germinated 2.370.02
a
25.870.11
a
28.170.13
a
0.4870.02
b
0.3470.01
b
Dehulled 1.3470.01
c
21.4870.06
c
22.8270.07
c
0.2970.01
c
0.2570.01
c
SE (df ¼9) 0.024 0.161 0.186 0.007 0.006
Lentil
Raw 0.8570.01
b
15.6270.03
a
16.4770.02
b
0.1970.01
a
0.7570.01
a
Germinated 1.4870.06
a
15.5270.08
b
17.070.02
a
0.1570.02
b
0.6170.01
b
Dehulled 0.3470.01
c
11.3970.08
c
11.7370.06
c
0.170.01
c
0.3670.01
c
SE (df ¼9) 0.018 0.033 0.019 0.004 0.005
Chickpea
Raw 1.2770.02
b
25.9170.04
a
27.1870.06
b
0.4870.02
a
0.5370.01
a
Germinated 2.4170.08
a
25.5670.08
b
27.9870.03
a
0.3870.02
b
0.4470.01
b
Dehulled 0.5470.01
c
19.6970.02
c
20.2370.02
c
0.2470.01
c
0.370.01
c
SE (df ¼9) 0.022 0.027 0.019 0.008 0.006
SE ¼standard error of means.
df ¼degree of freedom.
All mean scores bearing different superscripts in columns in each sample are significantly different on application of Duncan’s new multiple range test (Po
0.05).
a
Values are expressed as mean7standard deviation (n¼4).
R.A. Ghavidel, J. Prakash / LWT 40 (2007) 1292–12991294

ARTICLE IN PRESS
Table 3
Effect of germination and dehulling on mineral content and % bioavailability of iron and calcium of legume flours (on dry weight basis/100g)
a
Sample Iron (mg) Bioavailable iron (%) Calcium (mg) Bioavailable calcium (%) Phosphorous (mg)
Green gram
Raw 11.1470.36
a
10.970.1
c
13674.2
a
15.770.7
c
41777.0
a
Germinated 10.1570.22
b
18.370.2
b
11473.7
b
24.770.6
b
38477.7
c
Dehulled 9.0570.36
c
35.770.3
a
7173.6
c
40.570.8
a
40171.1
b
SE (df ¼9) 0.159 0.122 1.944 0.397 3.028
Cowpea
Raw 6.570.24
a
11.270.3
c
8771.8
a
22.670.5
c
39974.5
a
Germinated 5.8770.15
b
19.770.2
b
7574.1
b
38.270.3
b
34174.3
c
Dehulled 5.5270.12
c
37.670.1
a
5472.1
c
54.870.4
a
37872.0
b
SE (df ¼9) 0.089 0.102 1.448 0.243 1.912
Lentil
Raw 8.5270.24
a
10.270.1
c
7772.4
a
29.370.4
c
467712.4
a
Germinated 6.7370.18
b
18.570.2
b
6373.7
b
46.570.4
b
42075.7
c
Dehulled 5.5570.18
c
40.470.2
a
5073.2
c
59.670.5
a
44877.6
b
SE (df ¼9) 0.104 0.078 1.581 0.256 4.54
Chickpea
Raw 4.6870.24
a
11.370.2
c
22273.7
a
19.370.7
c
36675.5
a
Germinated 3.7770.11
b
18.670.2
b
17673.5
b
32.970.5
b
32575.5
c
Dehulled 2.9470.22
c
38.670.3
a
6375.6
c
48.170.7
a
34475.5
b
SE (df ¼9) 0.098 0.117 2.208 0.365 2.772
SE ¼standard error of means.
df ¼degree of freedom.
All mean scores bearing different superscripts in columns in each sample are significantly different on application of Duncan’s new multiple range test (Po
0.05).
a
Values are expressed as mean+standard deviation (n¼4).
Table 4
Effect of germination and dehulling on in vitro starch and protein digestibility in legume flours (on dry weight basis)
a
Sample In vitro starch digestibility
Total starch (g%) Glucose released (g%) In vitro starch digestibility (%) In vitro protein digestibility (%)
Green gram
Raw 46.770.8
a
9.870.3
c
18.970.6
c
61.071.0
c
Germinated 42.370.9
c
16.270.2
b
34.470.6
b
72.770.8
b
Dehulled 44.870.7
b
27.571.5
a
55.273.3
a
77.770.8
a
SE (df ¼9) 0.433 0.469 0.994 0.469
Cowpea
Raw 38.171.0
a
9.170.3
c
21.470.7
c
63.870.6
c
Germinated 35.270.5
c
13.970.2
b
35.670.7
b
72.971.0
b
Dehulled 37.070.9
b
24.370.5
a
59.171.4
a
77.271.0
a
SE (df ¼9) 0.425 0.201 0.518 0.450
Lentil
Raw 38.270.9
a
10.870.4
c
25.571.1
c
65.671.1
c
Germinated 34.370.5
c
14.970.2
b
39.170.7
b
75.171.4
b
Dehulled 37.370.5
b
26.870.3
a
64.770.7
a
78.870.8
a
SE (df ¼9) 0.333 0.174 0.445 0.582
Chickpea
Raw 42.470.4
a
10.370.2
c
21.870.5
c
64.271.8
c
Germinated 38.570.6
c
15.670.4
b
36.570.9
b
73.470.7
b
Dehulled 41.370.4
b
27.270.2
a
59.370.5
a
77.671.0
a
SE (df ¼9) 0.253 0.162 0.364 0.649
SE ¼standard error of means.
df ¼degree of freedom.
All mean scores bearing different superscripts in columns in each sample are significantly different on application of Duncan’s new multiple range test (Po
0.05).
a
Values are expressed as mean7standard deviation (n¼4).
R.A. Ghavidel, J. Prakash / LWT 40 (2007) 1292–1299 1295

8.0–33.0%, respectively, which could be due to biosynthesis
during germination (Sattar et al., 1989;Venderstoep,
1981). Fat, protein and thiamin levels improved
significantly after dehulling due to removal of hull
portion and concentration of endosperm. The highest ash
content was recorded in green gram (4.03 g/100 g) and the
lowest in lentil (2.44 g/100 g). These results were in
agreement with those reported earlier by several workers
(Gopalan et al., 1989;Savage, 1988;Venderstoep, 1981).
Leaching out of solid matter during pre germination
soaking process could be the reason for significant
reduction of mineral matter on germination. The further
insignificant decrease of ash after dehulling could be
contributed to removal of hull portion, which may have
some amounts of minerals.
Table 2 presents the antinutritional factors of legume
flours. Out of four legumes analysed, control lentil samples
had the lowest percent of fiber fractions and cowpea had
the highest. On germination, soluble and total dietary fiber
fractions increased and insoluble dietary fiber fraction
reduced significantly (Po0.05). There were marked reduc-
tions of all fiber fractions on dehulling of all legume
samples studied. These data agree with the findings of
Ramulu and Udayasekhara (1997) for dehulled green
gram, pigeonpea, lentil and chickpea. Control samples
contained considerable amounts of phytic acid
(0.19–0.61 g/100 g). The 18–21% significant (Po0.05)
decrease in phytic acid in germinated samples were
comparable to the results reported for other germinated
legumes including African oil bean (Enujiugha, Badejo,
Iyiola, & Oluwamukomi, 2003), black gram and pea (Das
et al., 1999) and pearl millet (Kumar & Chauhan, 1993).
Decrease in phytic acid content during germination could
be due to increase in phytase activity as reported by several
authors in faba bean (Eskin & Wiebe, 1983), broad bean,
chickpea and lentil (Egli, Davidsson, Juillerat, Barclay, &
Hurrell, 2002) and several other legumes (Kyriakidis,
Panayotou, Stavropoulou, & Athanasopoulos, 1998).
Tannin levels in control samples ranged from 0.47 g/100 g
in cowpea to 0.75 g/100 g in lentil. Germination signifi-
cantly (Po0.05) reduced the tannin contents of all studied
legumes as previously observed by Savelkoul et al. (1992) in
pigoenpea, chickpea, black gram and green gram. After
dehulling, there was little phytic acid and tannin detectable
in cotyledons, indicating that most of the phytic acid and
tannin are present in seed coat. Rao and Prabhavathi
(1982) also reported similar results for some decorticated
legumes.
ARTICLE IN PRESS
Table 5
Correlation coefficients and the regression equations for the association between % of iron and calcium bioavailability and antinutritional factorsin
legume flours
YXRegression equation Correlation coefficient
a
Green gram
Bioavailable iron Phytic acid Y¼58.13178.209X0.999
Bioavailable iron Tannin Y¼65.0680.92X0.997
Bioavailable iron Total dietary fiber Y¼95.94X0.928
Bioavailable calcium Phytic acid Y¼63.07577.92X0.999
Bioavailable calcium Tannin Y¼69.61179.452X0.990
Bioavailable calcium Total dietary fiber Y¼98.1173.834X0.898
Cowpea
Bioavailable iron Phytic acid Y¼62.09885.982X0.997
Bioavailable iron Tannin Y¼63.866116.131X0.953
Bioavailable iron Total dietary fiber Y¼133.6084.244X0.902
Bioavailable calcium Phytic acid Y¼85.269102.35X0.993
Bioavailable calcium Tannin Y¼89.594144.523X0.993
Bioavailable calcium Total dietary fiber Y¼156.8244.532X0.806
Lentil
Bioavailable iron Phytic acid Y¼72.864339.754X0.982
Bioavailable iron Tannin Y¼68.11478.629X0.996
Bioavailable iron Total dietary fiber Y¼98.835.031X0.936
Bioavailable calcium Phytic acid Y¼93.922332.675X0.990
Bioavailable calcium Tannin Y¼87.85974.527X0.972
Bioavailable calcium Total dietary fiber Y¼105.7344.022X0.770
Chickpea
Bioavailable iron Phytic acid Y¼65.227115.619X0.986
Bioavailable iron Tannin Y¼73.969120.794X0.990
Bioavailable iron Total dietary fiber Y¼100.9623.109X0.937
Bioavailable calcium Phytic acid Y¼77.252119.508X0.998
Bioavailable calcium Tannin Y¼85.933124.018X0.995
Bioavailable calcium Total dietary fiber Y¼104.3142.82X0.833
a
Significant (Po0.05).
R.A. Ghavidel, J. Prakash / LWT 40 (2007) 1292–12991296

A significant (Po0.05) decrease found in iron, calcium
and phosphorous contents on germination in present study
(Table 3) is well documented by other authors (Das et al.,
1999;Enujiugha et al., 2003;Giri, Pravatham, & Santhini,
1981). This is easily observable in the lower ash contents
obtained in the germinated samples (Table 1). This
reduction could be due to leaching of solid matter in
soaking water. Further decline in iron and calcium levels
after dehulling was observed, which may be contributed to
presence of these minerals in hull portion. But there was a
significant (Po0.05) improvement in phosphorous con-
tents in dehulled samples compared to germinated samples.
On germination, the percent bioavailable iron increased
significantly (Po0.05) by 64.6%, 67.8%, 75.8% and 81.3%
in chickpea, green gram, cowpea and lentil, respectively,
over the control samples (Table 3). These data are in
agreement with previous reports for germinated green
gram, horse gram and red gram (Giri et al., 1981). The
presence of tannin and phytic acid in seed coat as inhibitors
was demonstrated to reduce iron absorption (Davies &
Nightingale, 1975;Rao & Prabhavathi, 1982) by chelating
the iron ion (McDonald, Mila, & Scalbert, 1996). The
statistical analysis confirmed that, the percent biovailable
iron correlated significantly (Po0.05) and negatively to
phytic acid, tannin and total dietary fiber contents (Table
5). The process of germination and dehulling are associated
with a significant (Po0.05) enhancement in the bioavail-
ability of calcium (Table 3). Ghanem and Hussein (1999)
also found an appreciable improvement in calcium
bioavailability of germinated faba bean. Increase of
calcium bioavailability after germination and dehulling of
legume samples could be contributed to simultaneous
reduction of phytic acid, tannin and dietary fiber. Several
reports show the negative correlation of phytic acid and
dietary fiber contents of foods with percent of calcium
bioavailability (Allen, 1982;Cheryan, 1980;Ghanem &
Hussein, 1999;Kamchan et al., 2004). While there is no
doubt that unabsorbable complexes of calcium with uronic
acid in hemicellulose fraction of dietary fiber and with
phytic acid reduce the bioavailability of calcium (Allen,
1982;James, Branch, & Southgate, 1978). High negative
correlation coefficient values of bioavailable calcium vs.
phytic acid, tannin and total dietary fiber in all samples
presented in Table 5 support the findings in this regard.
Increase in a-amylase activity during germination
(Nnanna & Phillips, 1988;Sumathi, Malleshi, & Rao,
1995;Uriyo, 2001) could be a possible explanation of the
total starch loss and appreciable improvement in percent
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Table 6
Correlation coefficients and the regression equations for the association between % in vitro starch and protein digestibility and antinutritional factors in
legume flours
YXRegression equation Correlation coefficient
a
Green gram
In vitro starch digestibility Phytic acid Y¼88.215111.532X0.995
In vitro starch digestibility Tannin Y¼97.019113.39X0.977
In vitro starch digestibility Total dietary fiber Y¼135.1675.335X0.864
In vitro protein digestibility Phytic acid Y¼93.06748.43X0.918
In vitro protein digestibility Tannin Y¼95.80747.219X0.864
In vitro protein digestibility Total dietary fiber Y¼106.5131.942X0.669
Cowpea
In vitro starch digestibility Phytic acid Y¼94.322121.801X0.999
In vitro starch digestibility Tannin Y¼97.693166.962X0.969
In vitro starch digestibility Total dietary fiber Y¼190.3265.809X0.873
In vitro protein digestibility Phytic acid Y¼90.18641.357X0.944
In vitro protein digestibility Tannin Y¼93.04861.553X0.995
In vitro protein digestibility Total dietary fiber Y¼112.2911.57X0.657
Lentil
In vitro starch digestibility Phytic acid Y¼107.441438.689X0.993
In vitro starch digestibility Tannin Y¼100.854100.734X0.999
In vitro starch digestibility Total dietary fiber Y¼136.5966.205X0.904
In vitro protein digestibility Phytic acid Y¼94.24143.689X0.951
In vitro protein digestibility Tannin Y¼91.28931.609X0.917
In vitro protein digestibility Total dietary fiber Y¼96.1391.524X0.649
Chickpea
In vitro starch digestibility Phytic acid Y¼96.647156.674X0.999
In vitro starch digestibility Tannin Y¼108.214163.027X1.0
In vitro starch digestibility Total dietary fiber Y¼137.3093.904X0.880
In vitro protein digestibility Phytic acid Y¼91.59754.173X0.952
In vitro protein digestibility Tannin Y¼95.35855.806X0.943
In vitro protein digestibility Total dietary fiber Y¼99.011.085X0.675
a
Significant (Po0.05).
R.A. Ghavidel, J. Prakash / LWT 40 (2007) 1292–1299 1297

glucose released (Table 4). Total starch and glucose
contents enhanced significantly (Po0.05) after dehulling.
Germination significantly (Po0.05) increased the in vitro
starch and protein digestibility of all the samples as
compared to control samples by 53–82% and 14–18%,
respectively (Table 4), which is supported by the findings of
Kataria et al. (1989),Negi et al. (2001) and Archana,
Sehga, and Kawatra (2001). Dehulling brought about
further enhancement in starch and protein digestibility as
reported also by Preet and Punia (2000) for dehulled
cowpea. Dehulled samples had the highest levels of in vitro
starch and protein digestibility that may be attributed to
lowered levels of antinutrients. Findings about interaction
of starch with fiber, phytic acid and tannin (Flores,
Castanon, & McNab, 1994;Reddy, Sathe, & Salunkhe,
1982;Thorne, Thompson, & Jenkins, 1983), suppression of
pepsin activity by dietary fiber and consequent reduction of
in vitro protein digestibility (Horie, Sugase, & Horie, 1995;
Mongeau, Sarwar, Peace, & Brassard, 1989), negative
correlation between phytic acid and tannin with in vitro
digestibility of protein support this study observations
(Agarwal & Chitnis, 1995;Kumar & Chauhan, 1993).
Significant negative correlations (Po0.05) were observed
between in vitro starch and protein digestibility and phytic
acid, tannin and total dietary fiber, with least correlation in
case of fiber (Tables 5 and 6).
4. Conclusion
Germination improved protein, thiamin, in vitro iron
and calcium bioavailability and in vitro starch and protein
digestibility contents of all the legumes samples in this
study, significantly (Po0.05). On dehulling the germinated
legumes, further enhancement in mentioned parameters
was observed. Phytic acid and tannin reduced by 47–52%
and 43–52%, respectively, in dehulled samples over
control. Significant (Po0.05) negative correlations were
found between antinutritional factors and nutrients bioa-
vailability and digestibility. Germination combined with
dehulling process improved quality of legumes by enhan-
cing the bioavailability and digestibility of nutrients and
reducing the antinutrients.
References
Agarwal, P., & Chitnis, U. (1995). Effect of treatments on phytate
phosphorous, iron bioavailability, tannins and in vitro protein
digestibility of grain sorghum. Journal of Food Science and Technology,
32, 453–458.
Akeson, W. R., & Stahmann, M. A. (1964). A pepsin—Pancreatin digest
index of protein quality. Journal of Nutrition,83, 257–261.
Allen, L. H. (1982). Calcium bioavailability and absorption: A review.
American Journal of Clinical Nutrition,35, 783–808.
AOAC. (1990). Official methods of analysis (15th ed.). Washington, DC:
Association of Official Analytical Chemists.
Archana, Sehga, S., & Kawatra, A. (2001). In vitro protein and starch
digestibility of pearl millet (Pennisetum gluacum L.) as affected by
processing techniques. Nahrung,45(1), 25–27.
Asp, N. G., Johansson, C. G., Hallmer, H., & Siljestro
¨m, M. (1983).
Rapid enzymatic assay of insoluble and soluble dietary fiber. Journal of
Agricultural Food Chemistry,31, 476–482.
Ayet, G., Burbano, C., Cuadrado, C., Pedrosa, M. M., Robredo, L. M.,
Muzquiz, M., et al. (1997). Effect of germination, under different
environmental conditions, on saponins, phytic acid and tannins in
lentils (Lens culinaris). Journal of the Science of Food and Agriculture,
74, 273–279.
Batey, I. L., & Ryde, N. (1982). Starch analysis using thermo stable alpha-
amylases. Starch,34, 125–128.
Cheryan, M. (1980). Phytic acid interactions in food systems. CRC
Critical Review in Food Science and Nutrition,13, 297–332.
Das, J., Chaturvedi, Y., & Nagar, R. (1999). Effect of germination on
inorganic, labile and phytate phosphorous of some legumes. Journal of
Food Science and Technology,36(6), 532–534.
Davies, N. T., & Nightingale, R. (1975). The effects of phytate on
intestinal absorption and secretion of zinc and whole-body retention of
zinc, copper, iron and manganese in rats. British Journal of Nutrition,
34(2), 243–258.
Duncan, D. B. (1955). Multiple range and multiple F-test. Biometrics,11,
1–42.
Egli, I., Davidsson, l., Juillerat, M. A., Barclay, D., & Hurrell, R. F.
(2002). The influence of soaking and germination on the phytase
activity and phytic acid content of grains and seeds potentially useful
for complementary feeding. Journal of Food Science,67(9), 3484–3488.
Enujiugha, V. N., Badejo, A. A., Iyiola, S. O., & Oluwamukomi, M. O.
(2003). Effect of germination on the nutritional and functional
properties of African oil bean (Pentaclethra macrophylla Benth) seed
flour. Food, Agriculture and Environment,1, 72–75.
Eskin, N. A. M., & Wiebe, S. (1983). Changes in phytase activity and
phytate during germination of two faba bean cultivars. Journal of Food
Science,48, 270–271.
Flores, M. P., Castanon, J. I., & McNab, J. M. (1994). Effect of tannins on
starch digestibility and TMEn of triticale and semipurified starches
from triticale and field bean. British Poultry Science,35(2), 281–286.
Giri, J., Pravatham, R., & Santhini, K. (1981). Effect of germination on
the levels of pectin, phytins and minerals in the selected legumes. Indian
Journal of Nutrition and Dietetics,18, 87–91.
Gopalan, C., Sastri, B. V. R., & Balasubramanian, S. C. (1989). Nutritive
value of Indian foods. Hyderabad, India: National Institute of
Nutrition, Indian Council for Medical Research.
Horie, Y., Sugase, K., & Horie, K. (1995). Physiological differences of
soluble and insoluble dietary fiber fractions of brown algae and
mushrooms in pepsin activity in vitro and protein digestibility. Asian
Pacific Journal of Clinical Nutrition,4, 251–255.
Hussein, L., & Ghanem, K. Z. (1999). Calcium bioavailability from
selected Egyptian foods with emphasis on the impact of germination
and fermentation. International Journal of Food Sciences and Nutrition,
50, 351–356.
James, W. P. T., Branch, W. J., & Southgate, D. A. T. (1978). Calcium
binding by dietary fiber. Lancet,1, 638–639.
Kamchan, A., Puwastien, P., Sirichakwal, P. P., & Kongkachuichai, R.
(2004). In vitro calcium bioavailability of vegetables, legumes and
seeds. Journal of Food Composition and Analysis,17, 311–320.
Kataria, A., Chauhan, B. M., & Punia, D. (1989). Antinutrients and
protein digestibility (in vitro) of mungbean as affected by domestic
processing and cooking. Food Chemistry,32, 9–17.
Kataria, A., Chauhan, B. M., & Punia, D. (1992). Digestibility of proteins
and starch (in vitro) of amphidiploids (black gram mung bean) as
affected by domestic processing and cooking. Plant Foods for Human
Nutrition,42(2), 117–125.
Kumar, A., & Chauhan, B. M. (1993). Effect of phytic acid on protein
digestibility (in vitro) and HCl-extractability of minerals in pearl millet
sprouts. Cereal Chemistry,70(5), 504–506.
Kumar, G. K., & Venkataramann, L. V. (1976). Studies on the in vitro
digestibility of starch in some legumes before and after germination.
Nutrition Report International,13, 115–124.
ARTICLE IN PRESS
R.A. Ghavidel, J. Prakash / LWT 40 (2007) 1292–12991298

Kyriakidis, N. B., Panayotou, M. G., Stavropoulou, A., & Athanaso-
poulos, P. (1998). Increase in phytase activity and decrease in phytate
during germination of four common legumes. Biotechnology Letters,
20(5), 475–478.
Luten, J., Crew, H., Flynn, A., Dael, P. V., Kastenmayer, P., Hurell, R.,
et al. (1996). Interlaboratory trial on the determination of the in vitro
dialysability from food. Journal of the Science of Food and Agriculture,
72, 415–424.
McDonald, M., Mila, I., & Scalbert, A. (1996). Precipitation of metal ions
by plant polyphenols: Optimal conditions and origin of precipitation.
Journal of Agricultural Food Chemistry,44(2), 599–606.
Mongeau, R., Sarwar, G., Peace, R. W., & Brassard, R. (1989).
Relationship between dietary fiber levels and protein digestibility in
selected foods as determined in rats. Plant Foods for Human Nutrition,
39(1), 45–51.
Negi, A., Boora, P., & Khetarpaul, N. (2001). Starch and protein
digestibility of newly released moth bean cultivars: Effect of soaking,
germination and pressure-cooking. Nahrung,45(4), 251–254.
Nnanna, I. A., & Phillips, R. D. (1988). Changes in oligosaccharide
content, enzyme activities and dry matter during controlled germina-
tion of cowpeas (Vigna unguiculata). Journal of Food Science,53(6),
1782–1786.
Preet, K., & Punia, D. (2000). Antinutrients and digestibility (in vitro) of
soaked, dehulled and germinated cowpea. Nutrition and Health,14(2),
109–117.
Raghuramulu, N., Nair, M. K., & Kalyansundaram, S. (1983). A manual
of laboratory techniques. Jami-Osmania, Hyderabad, India: National
Institute of Nutrition, Indian Council for Medical Research.
Ramulu, P., & Udayasekhara, P. R. (1997). Effect of processing on dietary
fiber content of cereals and pulses. Plant Foods for Human Nutrition,
50, 249–257.
Rao, B. S. N., & Prabhavathi, T. (1978). An in vitro method for predicting
the bioavailability of iron from foods. American Journal of Clinical
Nutrition,31, 169–175.
Rao, B. S. N., & Prabhavathi, T. (1982). Tannin content of foods
commonly consumed in India and its influence on ionisable iron.
Journal of the Science of Food and Agriculture,33, 89–96.
Reddy, N. R., Sathe, S. K., & Salunkhe, D. K. (1982). Phytates in legumes
and cereals. Advances in Food Research,28, 1–92.
Sattar, A., Durrani, S. K., Mahmood, F., Ahmad, A., & Khan, I. (1989).
Effect of soaking and germination temperatures on selected nutrients
and antinutrients in mungbean. Food Chemistry,34, 111–120.
Savage, G. P. (1988). The composition and nutritive value of lentils (Lens
culinaris). Nutrition Abstract Review,58(5), 319–343.
Savelkoul, F. H. M. G., Vanderpoel, A. F. B., & Tamminga, S. (1992).
The presence and inactivation of trypsin inhibitors, tannins, lectins and
amylase inhibitors in legume seeds during germination: A review. Plant
Foods for Human Nutrition,42, 71–85.
Sen, M., & Bhattacharyya, D. (2003). Nutritional effects of a mustard seed
protein on growing rats. International Journal of Food Science and
Technology,38, 225–232.
Sumathi, A., Malleshi, N. G., & Rao, S. V. (1995). Elaboration of amylase
activity and changes in paste viscosity of some common Indian
legumes during germination. Plant Foods for Human Nutrition,47,
341–347.
Taussky, H. H., & Shorr, E. (1953). A micro-colorimetry method for
determination of inorganic phosphorous. Journal of Biological
Chemistry,202, 675–685.
Thompson, D. B., & Erdman, J. W. (1982). Phytic acid determination in
soyabeans. Journal of Food Science,47, 513–517.
Thorne, M. J., Thompson, L. U., & Jenkins, D. J. (1983). Factors
affecting starch digestibility and the glycemic response with special
reference to legumes. American Journal of Clinical Nutrition,38,
481–488.
Uriyo, M. G. (2001). Changes in enzyme activities during germination of
cowpeas (Vigna unguiculata, cv. California blackeye). Food Chemistry,
73, 7–10.
Venderstoep, J. (1981). Effect of germination on the nutritive value of
legumes. Food Technology,43, 83–85.
Wang, N., Lewis, M. J., Brennan, J. G., & Westby, A. (1997). Effect of
processing methods on nutrients and antinutritional factors in cowpea.
Food Chemistry,58, 59–68.
ARTICLE IN PRESS
R.A. Ghavidel, J. Prakash / LWT 40 (2007) 1292–1299 1299