J Food Sci Technol
https://doi.org/10.1007/s13197-019-03940-1
ORIGINAL ARTICLE
Effect of enzyme pretreatment on dehulling, cooking time
and protein content of pigeon pea (variety BDN2)
M. N. Dabhi1 • V. P. Sangani1 • P. J. Rathod1
Revised: 6 July 2019 / Accepted: 9 July 2019
Ó Association of Food Scientists & Technologists (India) 2019
Abstract This study was carried out to investigate the
effect of enzymatic pretreatment at different enzyme con-
centration, incubation time, incubation temperature and
tempering water pH on the hulling efficiency, cooking
quality and protein content of pigeon pea (variety BDN2).
Response surface methodology based on a four-factor, five-
level, central composite design was employed to study the
effect of the independent variables and optimize processing
conditions. A quadratic model satisfactorily described the
hulling efficiency, cooking time and protein content with
high value for the coefficient of determination R2 (0.95,
0.92 and 0.97 respectively). It predicted a maximum hul-
ling efficiency of 84.35%, minimum cooking time
13.06 min and maximum protein content 22.60% at
enzyme concentration, and 31.34 mg/100 g dry matter,
incubation time, 8.72 h, incubation temperature, 43.47 °C
and tempering water, pH 5.99. Results were also compared
with hulling efficiency, cooking time and protein content
obtained with traditional oil pretreated method 78.30%,
14.30 min and 18.53% respectively. It revealed that hulling
efficiency and protein content of enzyme pretreated pigeon
pea could be increased 2.44% and 6.77% respectively,
whereas cooking time could be reduced 1.50 min com-
pared to the oil pretreated method.
Keywords Enzyme  Pigeon pea  Hulling efficiency 
Cooking time  Protein content
& M. N. Dabhi
mndabhi@jau.in
1 1999).
Processing and Food Engineering Department, College of
Agricultural Engineering and Technology, Junagadh
Agricultural University, Junagadh, India
Introduction
Pigeon pea is generally dehulled to improve their cooking
and nutritional qualities. Dehulling of pigeon pea also helps
to remove antinutritional compounds such as polyphenols
located in the seed coat. Pre-dehulling treatment for loos-
ening the husk from the cotyledons is one of the important
steps in dehulling of pigeon pea. The pigeon pea is usually
pre-treated to loosen the hulls before it can be separated
using mechanical means. Kernel preconditioning is gen-
erally designed to toughen the hull and loosen the gummy
bond between the hull and the cotyledon and to harden the
cotyledon to reduce damage. Loosening the hulls during
dehulling is traditionally achieved either by wet or dry
methods (Kurien 1981). Pre-treatments may heat treatment
only or soaking in water, chemical solutions, tempering
followed by hot dehulling (Phirke and Bhole 2000; Phirke
et al. 1992; Ramkrishnaiah and Kurien 1983; Srivastva
et al. 1988). The limitations of these treatments were either
a shape deformation or poor cooking quality of dehulled
splits. These treatments are also labour-intensive and time-
consuming.
It was also reported that due to the presence of calac-
tomonus disaccharide, glucoronai acid and glycol protein
bonds husk with cotyledons (Kurien and Parpia 1968).
Even arabinogalactan type polysaccharide was found
responsible, which is gummy and hygroscopic in nature for
binding husk with cotyledons (Swamy et al. 1991). This
binding of husk and cotyledons makes the dehulling of
pigeonpea a difficult process. Therefore, it is important to
dissolve the gum layer from between husk and cotyledon
with pretreatment to result in higher dhal recovery (Saxena
It was reported that pigeon pea is hard-to dehull because
of the presence of mucilage and gum forming a strong bond
123

between the hulls and cotyledons. The chemical nature of
the mucilage and gums present in the interface between the
husk and cotyledons play an important role in the dehulling
of pigeon pea grains (Ramkrishnaiah and Kurien 1983).
These mucilages and gums of pigeon pea seeds are network
of cellulosic microfibrils embedded in a matrix of non-
starch polysaccharides (NSP) and proteins (Cosgrove
1997). Partial hydrolysis of these NSP and/or proteins by
enzymatic pretreatment results in easy dehulling of
legumes (Arora et al. 2007; Verma et al. 1993). It was
reported that, xylanase mediated degradation of cell wall
polysaccharides of horse gram resulted in expansion of the
grain with improved nutritional and functional properties
upon thermal treatment (Sreerama et al. 2008). Further,
improvement in the physical and expansion properties of
pigeon pea and horse gram was also achieved by protease
or sodium bicarbonate pre-treatments (Sreerama et al.
2009a).
Keeping in view of above most favoured variety of
pigeon pea i.e. BDN2 was selected for the study with an
objective to standardize the enzymatic pre-treatments to
increase the dhal recovery and protein content as well as to
reduce the cooking time.
Materials and methods
Selection of variety
Amongst different varieties of pigeon pea being cultivated
in Gujarat, the BDN2 variety was purchased from Juna-
gadh Agricultural University. Care was taken to purchase
all the seeds from a single batch. The seeds were then taken
to the laboratory in jute bags.
Dehusking machine
The laboratory scale dehusking machine based on CIAE
dhal mill design and fabricated at Department (Salve et al.
2008) with overall dimensions of 600 mm 9 620 mm 9
935 mm, capacity of 85 kg/h and power unit of 1 hp
electric motor was used for all the milling studies. The
optimum operating speed and feed rate of the dehusking
machine were 1420 rpm and 64 kg/h, respectively.
Selection of enzymes
The selection of enzymes was made on the basis of the
chemical composition and binding substances present
between husk and cotyledon of pigeon pea grain. The
xylanase enzyme is widely used as bio-bleaching agent for
lignin isolation (Saxena and Srivastava 1998). Cellulase
and pectinase break down cellulose to beta-glucose and
123
J Food Sci Technol
pectin to pectic acid, respectively. Thus, the xylanase,
cellulase and pectinase are the key enzymes which rupture
the binding materials leading to increase the dehulling
efficiency. The xylanase (12.5 l/mg) was procured from
Advanced Enzyme Technologies Ltd., Thane (Maharash-
tra) while cellulase (C 10 l/mg) and pectinase were
obtained from HiMedia Laboratories Pvt. Ltd., Mumbai
(Maharashtra).
Standardization of ratio of enzymes
Preliminary trials were undertaken to arrive at standard
proportions of enzymes, i.e., xylanase: pectinase: cellulase
for maximizing the husk removal. Initially, the proportion
was selected arbitrarily. The effect of selected enzyme
combination on husk removal of pigeon pea grain was
evaluated keeping the enzyme concentration, incubation
time, incubation temperature and tempering water pH
constant based on the technical specifications of the prod-
ucts provided by manufacturer.
Following equations were used to calculate husk
removal and hulling efficiency (Shanta et al. 1978).
Husk Removed during dehusking
Husk removed ðHRÞ; % ¼
Total husk content
 100
ð1Þ
Coefficient of hulling ðChÞ
Weight of unhulled grain after milling ð2Þ
¼1
Weight of unhulled grain used for milling
Wf
Coefficient of wholeness of kernel ðCwkÞ ¼
Wf þ Wb þ Wp
ð3Þ
where Wf, weight of finished product; Wb, weight of bro-
kens; Wp, weight of powder
Hulling efficiency ¼ Ch  Cwk  100 ð4Þ
Enzymatic pre-treatment
The enzyme solution was prepared at the standardized
proportion of all three selected enzymes. In this enzymatic
pre-treatment, the degumming might be due to the action of
different enzymes used for pre-treatment, i.e., xylanase,
pectinase and cellulase.
Dry milling method followed as control
Generally, the dry milling method is followed throughout
the Indian subcontinent for milling of pigeon pea. Hence,
for the comparison of enzymatic pre-treatment, the dry
J Food Sci Technol
milling method was taken as control. The cleaned and size
graded grains were pitted through dehusking roller
machine. Then, mustard oil was used for oil treatment @
0.5 kg oil per 100 kg pigeon pea grains (Saxena and Sri-
vastava, 1998). For 2 kg pigeon pea grains 10 g mustard oil
was mixed and kept in a glass bottle (5 L) for 36 h for
diffusion of oil. After 36 h, the distilled water was sprayed
@ 100 g/2 kg grain, on the grains and heaped for 12 h.
Subsequently, after tempering, the grains were dried in tray
dryer (Khera Instruments Pvt. Ltd., New Delhi) at 60 °C up
to a moisture content of 10% ± 0.5% (w.b.). This sequence
of operation was repeated three to four times.
Milling of sample
Enzyme and oil treated samples of 1 kg weight having
about 10% ± 0.5% moisture content (w.b.) were milled
using laboratory scale dehusking machine. After milling,
all obtained fractions were collected in polyethylene bag.
Each of the samples was milled separately and care was
taken to obtain all the fractions without any loss, using a
cleaning brush.
Dehulled sample separation
The different fractions of the milled product such as whole
dehulled grains, split dehulled grains, partly dehulled and
unhulled grains, broken, husk and powder were separated
by suitable sieves (BS sieves no. 4, 6, 18). A grain was
considered completely dehulled when there was no husk
adhering to it.
Cooking time
Pigeon pea dhal samples obtained through various enzymatic
treatments were cooked in a stainless steel pan having a ratio
of dhal: distilled water as 1:10. For determination of cooking
time, distilled water was heated to boiling point in a 150 mL
beaker and then 15 g dhal was added. During boiling, the
level of water was maintained by regular addition of boiled
water. Boiling was continued and samples were drawn at
1 min interval to check the cooking time by pressing
between the thumb and the forefinger till no hard core is left
as described by Singh et al. (1984). Full cooking time was
recorded as the time when 90% of the dhal became soft
enough to masticate (Williams et al. 1983).
Experimental design
The effects of four independent variables’ viz., enzyme
concentration, incubation time, incubation temperature and
tempering water pH value on hulling efficiency, cooking
time and protein content were studied with variables coded
as X1, X2, X3 and X4 respectively. The levels of parameter
values were carefully chosen based on the literature
available on the enzymatic hydrolysis of pigeon pea grain.
Response variable, i.e., hulling efficney, cooking time and
protein content were determined for optimization of the
process. Response Surface Methodology (RSM) was used
for designing the experiments. A Central Composite
Rotatable Design (CCRD) of 4 variables at 5 levels each
with 6 centre point combinations was used (Khuri and
Cornell 1987). Altogether, 30 combinations (including 6
replications at the centre point and single observation at
other points) were chosen to accord to a central composite
rotatable design.
For data analysis and optimization, the CCRD design
was used to conduct experiments and the Response Surface
Methodology (RSM) was applied to the experimental data
using a commercial statistical package, Design Expert-
version 8.0.0.6 (State-Ease Inc 2009). Analysis of variance
(ANOVA) was calculated for fitting the model represented
by Eq. (5) to examine the statistical significance of the
model terms. Model analysis with respect to lack-of fit test
and R2 (co-efficient of determination) was done for deter-
mining adequacy of model. The coefficient of variance
(CV) was calculated to find the relative dispersion of the
experimental points from the prediction of the model.
Response surfaces were generated and by using the same
software, numerical optimization was done. The most
commonly used model for optimization using response
surface methodology is a second order polynomial equa-
tion (Bas and Boyaci 2007). The model is of the form:
X
3 X
3 X
3
Yk ¼ bk0 þ bki Xi þ bkij Xi Xj þ bkii Xi2
i¼1 i6¼j¼1 i¼1
þ eðk ¼ 0; 1; 2; 3. . .Þ ð5Þ
where Yk is the response; bk0, bki, bkij, bkii and e are the
constant, linear, and quadratic cross-product regression
coefficients, random error respectively and Xis are the
coded independent variables.
Validity test
The optimum conditions obtained through statistical anal-
ysis was verified by conducting the experiment in tripli-
cates. The average value of hulling efficiency, cooking
time and protein content was considered for the validation.
Results and discussion
The observation of different enzyme pretreatment for all
thirty experiments were recorded. The best combination of
enzyme concentration, incubation temperature, incubation
123
 J Food Sci Technol

time and pH was selected with respect to hulling efficiency,
cooking time and protein content. The data obtained were
tabulated in Table 1. It was observed that hulling effi-
ciency, cooking time and protein content were influenced
significantly by enzyme concentration, incubation temper-
ature, incubation time and tempering water pH.
The analysis of variance (ANOVA) was conducted on
experimental data and the significance of enzyme con-
centration, incubation temperature, incubation period and
tempering water pH as well as their interactions on hulling
efficiency, cooing time and protein content were calcu-
lated. The quadratic model was fitted to experimental data
and statistical significance of linear, quadratic and inter-
action effects were calculated for each response.
The regression analysis and ANOVA results for differ-
ent dependent parameters of pigeon pea dhal are presented
in Table 2.
Effect of enzymatic pre-treatment on hulling
efficiency
Results showed that among linear effects of incubation
temperature had significant effect on hulling efficiency
(p \ 0.001) (Table 2). This finding was confirmed by San-
gani et al. (2014a) and Murumkar et al. (2016). Linear effects

Table 1 Effect of enzymatic treatment variables on hulling efficiency, cooking time and protein content
Treat Enzymatic treatment variables Responses
no.
Enzyme concentration (mg/ Incubation Incubation Tempering Hulling Cooking time Protein
100 g dry sample) time (h) temperature (°C) water pH efficiency (%) (min) content (%)

Control 78.30 14.30 18.53

1 42.5 10 50 5.5 80.13 13.36 21.40
2 27.5 10 50 5.5 82.93 13.84 22.04
3 42.5 6 50 5.5 82.29 14.33 22.51
4 27.5 6 50 5.5 82.59 14.82 22.74
5 42.5 10 40 5.5 81.57 12.85 21.34
6 27.5 10 40 5.5 84.02 13.87 21.91
7 42.5 6 40 5.5 83.72 13.81 21.68
8 27.5 6 40 5.5 85.65 13.83 22.63
9 42.5 10 50 4.5 75.87 13.89 19.97
10 27.5 10 50 4.5 78.61 14.86 21.74
11 42.5 6 50 4.5 77.17 14.36 21.45
12 27.5 6 50 4.5 79.33 14.86 21.88
13 42.5 10 40 4.5 79.85 14.36 21.14
14 27.5 10 40 4.5 80.45 15.36 21.56
15 42.5 6 40 4.5 80.80 14.86 21.63
16 27.5 6 40 4.5 81.15 14.39 22.68
17 50.0 8 45 5.0 74.90 13.86 19.43
18 20.0 8 45 5.0 78.15 14.86 21.98
19 35.0 12 45 5.0 79.95 13.37 21.00
20 35.0 4 45 5.0 81.10 15.86 22.44
21 35.0 8 55 5.0 75.40 14.82 21.36
22 35.0 8 35 5.0 78.05 14.33 22.56
23 35.0 8 45 6.0 79.82 13.81 22.06
24 35.0 8 45 4.0 77.35 13.80 22.24
25 35.0 8 45 5.0 83.31 13.35 22.35
26 35.0 8 45 5.0 84.09 12.86 22.39
27 35.0 8 45 5.0 82.17 13.30 22.31
28 35.0 8 45 5.0 84.35 13.35 22.53
29 35.0 8 45 5.0 81.60 13.80 22.56
30 35.0 8 45 5.0 82.15 13.36 21.83

123
J Food Sci Technol

Table 2 Analysis of variance

Source Hulling efficiency (%) Cooking time (min) Protein content (%)
table and regression coefficients
for response surface quadratic b0 (intercept) 83.26*** 13.32*** 22.44***
model of different dependent
parameters of pigeon pea dhal b1 (X1) - 0.61** - 0.24*** - 0.50***
b2 (X2) - 0.51** - 0.29*** - 0.36***
b3 (X3) - 1.00*** 0.11 - 0.16**
b4 (X4) 1.67*** - 0.27*** 0.20***
b12 (X1X2) - 0.27 - 0.16** - 0.023
b13 (X1X3) - 0.30 - 0.057 - 0.025
b14 (X1X4) 0.030 - 3.750E - 003 0.056
b23 (X2X3) 0.069 - 0.12 - 0.076
b24 (X2X4) - 0.090 - 0.18* 0.020
b34 (X3X4) 0.40 0.16 0.20**
b11 (X21) - 0.51* 0.25*** - 0.39***
b22 (X22) - 0.43* 0.24*** - 0.19***
b33 (X23) - 1.30*** 0.31*** - 0.091*
b44 (X24) - 0.51** 0.096 - 0.030
R2 0.9471 0.9155 0.9652
Adj-R2 0.8977 0.8366 0.9327
Pred-R2 0.7153 0.5568 0.8927
Adeq. precision 15.705 10.293 21.148
F-value 19.17 11.60 29.70
Lack of fit NS NS NS
C.V. (%) 0.96 1.92 0.91
X1, enzyme concentration; X2, incubation time; X3, incubation temperature; X4, pH
***Significant at p \ 0.001; **significant at p \ 0.01; *significant at p \ 0.05

of tempering water pH had also significant effect on hulling
efficiency (p \ 0.001). Sangani et al. (2014a) also reported
significant (p \ 0.05) effects of pH on hulling efficiency.
However, linear effects of enzyme concentration was found
significant effect on hulling efficiency (p \ 0.01) (Mu-
rumkar et al. 2016; Sreerama et al. 2009b; Chandini et al.
2016) but Sangani et al. (2014a) reported the non-significant
effect of enzyme concentration on hulling efficiency. Linear
effects of incubation time were found significant effect on
hulling efficiency (p \ 0.01) (Sangani et al. 2014a; Mur-
umkar et al. 2016). Opoku et al. (2003) reported tempering is
necessary for achieving better dehulling results after soaking
and drying or steaming and drying. Interaction effect were
found to be non-significant (Sangani et al. 2014a). Quadratic
effect of enzyme concentration and incubation time had
significant effect on hulling efficiency (p \ 0.05) while that
of incubation temperature had extremely significant effect
on hulling efficiency (p \ 0.001) and tempering water pH
had highly significant effect on hulling efficiency (p \ 0.01)
(Table 2).
The hulling efficiency varied from 74.90 to 85.65%.
(Table 1). The minimum hulling efficiency was found in
treatment number 17 having the combination of enzyme
concentration 50 mg/100 g, incubation temperature 45 °C,
incubation period 8 h and tempering water pH 5.00 while,
maximum hulling efficiency was found in the treatment
number 8 having the combination of enzyme concentration
27.5 mg/100 g, incubation temperature 40 °C, incubation
period 6 h and pH 5.50. This showed that enzyme con-
centration, incubation time, incubation temperature and pH
played prominent role on hulling efficiency.
R2 and CV percent value for hulling efficiency was 0.95
and 0.96% respectively. The response surface equation of
second order was obtained in terms of coded factors to
predict the variation in hulling efficiency due to enzyme
pretreatment on pigeon pea processing with varying levels
of processing parameters under,
Hulling efficiency ð%Þ ¼ 83:26  0:61X1  0:51X2
 1:00X3 þ 1:67X4  0:27X1 X2
 0:30X1 X3 þ 0:03X1 X4
þ 0:069X2 X3  0:09X2 X4
þ 0:40X3 X4  0:51X12  0:43X22
 1:30X32  0:51X42
ð6Þ
where X1, X2, X3 and X4 are the coded factors of enzyme
concentration, incubation time, incubation temperature and
pH, respectively.

123

Three dimensional response surface plot for hulling
efficiency of enzyme pretreated samples were generated
with center point of two parameters central and another two
parameters as variables.
Figure 1 shows that at central value of incubation tem-
perature and tempering water pH. Hulling efficiency was
increased (up to 83.53%) with an increase of enzyme
concentration up to 31.39 mg/100 g sample and incubation
time up to 7.11 h, respectively (Fig. 1a). However, with
further increase in enzyme concentration and incubation
time, the hulling efficiency was decreased. Similarly, at
central value of incubation time and tempering water pH
that there was an increase in hulling efficiency (up to
83.58%) with an increase in enzyme concentration up to
31.26 mg/100 g sample and incubation temperature up to
43.36 °C (Fig. 1b). For central value of incubation period
and incubation temperature there was an increase in hulling
efficiency (up to 84.79%) with an increase in enzyme
concentration up to 30.95 mg/100 g sample and tempering
water pH up to 5.82 (Fig. 1c). Similarly, at central value of
enzyme concentration and tempering water pH there was
an increase in hulling efficiency (up to 83.62%) with an
increase in incubation time up to 6.71 h and incubation
temperature up to 43.05 °C (Fig. 1d). At central value of
enzyme concentration and incubation temperature there
was an increase in hulling efficiency (84.88%) with an
increase in incubation time up to 6.44 h and tempering
water pH up to 5.86 (Fig. 1e). For central value of enzyme
concentration and incubation period there was an increase
in hulling efficiency (84.65%) with an increase in incuba-
tion temperature up to 44.26 °C and tempering water pH up
to 5.79 (Fig. 1f).
This may indicate the existence of optimum levels of
hydrolysis parameters within the selected range. Higher
than optimum enzyme concentration reduces the enzymatic
activity due to saturation of active sites of enzymes with
substrate leading to lower hulling efficiency. Prolonged
exposure of grain to enzymes may have decreased the
hulling efficiency because of hardening effect due to
combined effect of temperature and moisture (Sangani
et al. 2014a; Murumkar et al. 2016). The reduction in
enzymatic activity at above optimum incubation tempera-
ture was due to denaturing of enzyme, resulting in the
reduction in hulling efficiency. It also confirmed the facts
that maximum enzymatic reaction occurred at optimum
enzyme concentration and temperature levels. It could be
observed that with increase in tempering water pH, the
hulling efficiency increased at a particular enzyme con-
centration. The reduction in enzyme activity at above
optimum tempering water pH was due to denaturing of
enzymes, resulting in a decrease in the hulling efficiency.
123
J Food Sci Technol
Effect of enzyme pretreatment parameters
on cooking time
The response surface quadratic model implied the signifi-
cant effect of selected enzymatic pre-treatments on cooking
time of pigeon pea dhal. It was observed that among linear
effects of enzyme concentration, incubation time and
tempering water pH had significant effect on cooking time
(p \ 0.001). However, linear effects of incubation tem-
perature was found non-significant effect on cooking time
(Table 2). Sangani et al. (2014b) confirmed the significant
effect (p \ 0.05) of enzyme concentration and tempering
water pH, and they observed highly significant effect
(p \ 0.01) of incubation time. Interaction effect of enzyme
concentration and incubation time was found significant on
cooking time (p \ 0.01) and interaction of incubation time
and tempering water pH was found significant on cooking
time (p \ 0.05). Other interactions were found to be non-
significant effect on cooking time (Table 2). Sangani et al.
(2014b) observed non-significant effect of all the interac-
tion on cooking time. Quadratic effect of enzyme con-
centration, incubation temperature and incubation time had
significant effect on cooking time (p \ 0.001) (Table 2).
Sangani et al. (2014b) also observed the significant effect
(p \ 0.05) of enzyme concentration and significant effect
(p \ 0.01) of incubation time and incubation temperature.
However, quadratic effect of tempering water pH had non-
significant effect on cooking time (Table 2). Tiwari et al.
(2008) also reported significant effect (p \ 0.05) of con-
ditioning on cooking time. Bhokre and Joshi (2015) also
reported that soaking of cowpea reduces the cooking time.
Coskuner and Karababa (2003) also supported the results
for chickpea. Murumkar et al. (2016) also reported the
reduction of cooking time by enzymatic pretreatment to
pigeon pea. No significant change in the cooking times of
dehulled splits was observed for control and enzyme (xy-
lanase and protease) pre-treated legumes (Sreerama et al.
2009b).
The cooking time varied from 12.86 to 15.86 min.
(Table 1). The minimum cooking time was found in
treatment number 26 having the combination of enzyme
concentration 35%, incubation temperature 45 °C, incu-
bation period 8 h and tempering water pH 5.00 while,
maximum cooking time was found in the treatment number
20 having the combination of enzyme concentration 35%,
incubation temperature 45 °C, incubation period 4 h and
tempering water pH 5.00. This showed that incubation time
played prominent role on cooking time.
R2 and CV percent value for cooking time was 0.92 and
1.92% respectively. The response surface equation of
second order was obtained in terms of coded factors to
predict the variation in cooking time due to enzyme
J Food Sci Technol

Design-Expert® Software
Design-Expert® Software
Hulling efficiency (%)
84.35
Hulling efficiency (%)
75.4 84.35
X1 = A: Enzyme concentration 75.4
X2 = B: Incubation time

Actual Factors X1 = A: Enzyme concentration

C: Incubation temp. = 45.00 84
X2 = C: Incubation temp.

H ulling efficiency ( %)
D: pH = 5.00
Hulling efficiency (%)
83.6
Actual Factors 80.75
81.725 B: Incubation time = 8.00
D: pH = 5.00 77.5
79.85

77.975 74.25

76.1
71
12.00 50.00

20.00 55.00
10.00 42.50

27.50 50.00
8.00 35.00
35.00 45.00
6.00 27.50
B: Incubation time A: Enzyme concentration
42.50 40.00
A: Enzyme concentration C: Incubation temp.
4.00 20.00
50.00 35.00

(a) Effect of enzyme concentration and (b) Effect of enzyme concentration and
incubation time. incubation temperature

Design-Expert® Software
Design-Expert® Software
Hulling efficiency (%)
84.35
Hulling efficiency (%)
84.35 75.4

75.4 X1 = B: Incubation time

X2 = C: Incubation temp.

X1 = A: Enzyme concentration Actual Factors

85 A: Enzyme concentration = 35.00
X2 = D: pH
D: pH = 5.00
H ulling efficiency ( %)

Hulling efficiency (%)

84
Actual Factors 82.25
B: Incubation time = 8.00 81.25

C: Incubation temp. = 45.00 79.5 78.5

76.75 75.75

73
74
55.00 12.00

20.00 6.00
50.00 10.00

27.50 5.50
45.00 8.00
35.00 5.00
40.00 6.00
42.50 4.50 C: Incubation temp. B: Incubation time
A: Enzyme concentration D: pH
35.00 4.00
50.00 4.00

(d) Effect of incubation time and incubation

(c) Effect of enzyme concentration and
temperature
tempering water pH

Design-Expert® Software Design-Expert® Software

Hulling efficiency (%) Hulling efficiency (%)

84.35 84.35

75.4 75.4

X1 = B: Incubation time X1 = C: Incubation temp.

84.9 85
H ulling efficiency ( %)

H ulling efficiency ( %)

X2 = D: pH X2 = D: pH

Actual Factors 82.525 Actual Factors 81

A: Enzyme concentration = 35.00 A: Enzyme concentration = 35.00
C: Incubation temp. = 45.00 80.15 B: Incubation time = 8.00 77

77.775 73

75.4 69

4.00 35.00

6.00 6.00
6.00 40.00
5.50 5.50
8.00 45.00
5.00 5.00

B: Incubation time 10.00

4.50 C: Incubation temp. 50.00 4.50
D: pH D: pH
12.00 4.00 55.00 4.00

(e) Effect of incubation time and (f) Effect of incubation temperature and
tempering water pH tempering water pH

Fig. 1 Response surface and contour plots for hulling efficiency of pigeon pea as a function of enzyme concentration, incubation time,
incubation temperature and tempering water pH. For each plot, the two parameter are at central point

123

pretreatment on pigeon pea processing with varying levels
of processing parameters under,
Cooking time ðminÞ ¼ 13:32  0:24X1  0:29X2 þ 0:11X3
 0:27X4  0:16X1 X2
 
 0:057X1 X3  3:750E003 X1 X4
 0:12X2 X3  0:18X2 X4
þ 0:16X3 X4 þ 0:25X12 þ 0:24X22
þ 0:31X32 þ 0:096X42
ð7Þ
where X1, X2, X3 and X4 are the coded factors of enzyme
concentration, incubation time, incubation temperature and
tempering water pH, respectively.
Three dimensional response surface plot for cooking
time of enzyme pretreated samples were generated with
center point of two parameters central and another two
parameters as variables.
Figure 2 shows that at central value of incubation tem-
perature and tempering water pH. Cooking time was
decreased (up to 13.10 min) with an increase of enzyme
concentration up to 40.58 mg/100 g sample and incubation
time up to 9.63 min, respectively (Fig. 2a). However, with
further increase in enzyme concentration and incubation
time, the cooking time was increased. Similarly, at central
value of incubation time and tempering water pH there was
a decrease in cooking time (up to 13.25 min) with an
increase in enzyme concentration up to 38.52 mg/100 g
sample and incubation temperature up to 44.36 °C
(Fig. 2b). The cooking time was found to be increased with
further increase in enzyme concentration and temperature.
For central value of incubation time and incubation tem-
perature cooking time decreased (up to 13.06 min) with an
increase in enzyme concentration up to 38.67 mg/100 g
sample and tempering water pH up to 5.70 (Fig. 2c). The
cooking time was found to be increased with further
increase in enzyme concentration and tempering water pH.
Whereas for central value of enzyme concentration and
tempering water pH there was decrease in cooking time (up
to 9.17 min) with an increase in incubation time up to
9.17 h and incubation temperature up to 44.65 °C
(Fig. 2d). The cooking time was found to be increased with
further increase in incubation time and incubation tem-
perature. For central value of enzyme concentration and
incubation temperature cooking time decreases (up to
12.73 min) with an increase in incubation time up to
10.64 h and tempering water pH up to 6.0 (Fig. 2e). The
cooking time was found to be increased with further
incubation time and tempering water pH. Similarly, at
central value of enzyme concentration and incubation time
cooking time decreases (up to 13.00 min) with an increase
in incubation temperature up to 41.52 °C and tempering
water pH up to 5.98 (Fig. 2f). The cooking time was found
123
J Food Sci Technol
to be increased with further increase in incubation tem-
perature and tempering water pH.
Effect of enzyme pretreatment parameters
on protein content
During dehulling, outer layers of cotyledons are scarified
resulting in 12% yield loss as powder fraction (Singh and
Jambunathan 1990). The outer portion of cotyledons is a
rich source of protein, sugar, fiber, and ash but poor in
starch. It was observed that linear effects of enzyme con-
centration, incubation time and tempering water pH had
significant effect on protein content (p \ 0.001) at 0.1%
level of significance. However, linear effects of incubation
temperature had significant effect on protein content
(p \ 0.01) at 1% level of significance (Table 2). Chandini
et al., 2016 also reported the effect of higher soaking time
in reduction of crude protein in pigeon pea. This may be
due to the hydrophilic property its crude protein exhibits
which could have leached out while soaking in water.
Murumkar et al., 2016 reported increase of 2.96% protein
content due to enzyme pretreatment to pigeon pea. Inter-
action effect of incubation temperature and tempering
water pH was found significant on protein content
(p \ 0.01) at 1% level. Other interactions were found to be
non-significant effect on protein content. Quadratic effect
of enzyme concentration and incubation time had signifi-
cant effect on protein content (p \ 0.001) at 0.1% level of
significance whereas quadratic effect of incubation tem-
perature had significant effect on protein content
(p \ 0.05) at 5% level of significance. However, quadratic
effect of tempering water pH had non-significant effect on
protein content (Table 2). Tiwari et al. (2008) also reported
pretreatment increases the protein content. The pectinase
preparation (Pectinex) containing high polygalacturonase
activity and a b-glucanase side activity was the most
effective preparation in terms of carbohydrate hydrolysis
and protein release. Enzymatic carbohydrate hydrolysis
correlated with increased protein extractability during
water extraction or saline extraction at tempering water pH
6 (Rommi et al. 2014). Das et al. (2008) reported cellulase
mediated treatment resulted in increase in antioxidant
activity, reducing power, free amino acids, proteins, crude
fiber, ash, oil, and phenolic content in the order of brown
rice [ enzyme treated rice [ milled rice.
The protein content varied from 19.43 to 22.74 min.
(Table 1). The minimum protein content was found in
treatment number 17 having the combination of enzyme
concentration 50%, incubation temperature 45 °C, incu-
bation period 8 h and tempering water pH 5.00 while,
maximum protein content was found in the treatment
number 4 having the combination of enzyme concentration
27.5%, incubation temperature 50 °C, incubation period
J Food Sci Technol

Design-Expert® Software Design-Expert® Software

Cooking time (min) Cooking time (min)

15.26 15.26

12.85 12.85

X1 = A: Enzyme concentration 16.1 X1 = A: Enzyme concentration

X2 = B: Incubation time X2 = C: Incubation temp.

Cooking time (min)

Cooking time (min)

15.35 16.5
Actual Factors Actual Factors
C: Incubation temp. = 45.00 B: Incubation time = 8.00 15.675
D: pH = 5.00 14.6 D: pH = 5.00 14.85
14.025
13.85
13.2
13.1
50.00

35.00
20.00
42.50
12.00 40.00
27.50
10.00 35.00
45.00
35.00
8.00
27.50
A: Enzyme concentration 42.50 A: Enzyme concentration 50.00
6.00
B: Incubation time C: Incubation temp.
20.00 55.00
50.00 4.00

(a) Effect of enzyme concentration and (b) Effect of enzyme concentration and
incubation time. incubation temperature.

Design-Expert® Software Design-Expert® Software

Cooking time (min) Cooking time (min)

15.26 15.26

12.85 12.85

X1 = A: Enzyme concentration X1 = B: Incubation time

X2 = D: pH X2 = C: Incubation temp. 16.8

Cooking time (min)

Actual Factors Actual Factors
15.9
Cooking time (min)

B: Incubation time = 8.00 15.8 A: Enzyme concentration = 35.00

C: Incubation temp. = 45.00 D: pH = 5.00
15.1
15
14.4
14.1
13.7
4.00
13 13.2

50.00 4.50 35.00

12.00
42.50 40.00
5.00 10.00
35.00 45.00
8.00
5.50 D: pH
27.50 50.00
B: Incubation time 6.00
C: Incubation temp.
A: Enzyme concentration 20.00 6.00 4.00 55.00

(c) Effect of enzyme concentration and (d) Effect of incubation time and
tempering water pH incubation temperature
Design-Expert® Software Design-Expert® Software

Cooking time (min) Cooking time (min)

15.26 15.26

12.85 12.85
15.5 16
Cooking time (min)

X1 = B: Incubation time X1 = C: Incubation temp.

Cooking time (min)

X2 = D: pH X2 = D: pH
15.25
14.8
Actual Factors Actual Factors
A: Enzyme concentration = 35.00 A: Enzyme concentration = 35.00 14.5
14.1
C: Incubation temp. = 45.00 B: Incubation time = 8.00
13.75
13.4

13
12.7

55.00
4.00

4.50 4.00 50.00

4.00
6.00 4.50
5.00 45.00
8.00 5.00
D: pH 5.50 40.00
10.00 C: Incubation temp. 5.50

6.00 12.00
B: Incubation time 35.00 6.00 D: pH

(e) Effect of incubation time and (f) Effect of incubation temperature and
tempering water pH tempering water pH

Fig. 2 Response surface and contour plots for cooking time of pigeon pea as a function of enzyme concentration, incubation time, incubation
temperature and tempering water pH. For each plot, the two parameter are at central point

6 h and tempering water pH 5.50. This showed that time and tempering water pH played prominent role on
enzyme concentration, incubation temperature, incubation protein content.

123
 J Food Sci Technol

Design-Expert® Software Design-Expert® Software

Protein Content (%) Protein Content (%)

22.88 22.88

19.87 19.87

X1 = A: Enzyme concentration X1 = A: Enzyme concentration

X2 = B: Incubation time X2 = C: Incubation temp.

Actual Factors Actual Factors

C: Incubation temp. = 45.00 B: Incubation time = 8.00 22.8
Protein Content (%) 22.8

Protein Content (%)

D: pH = 5.00 D: pH = 5.00
21.625 21.85

20.45 20.9
12.00 55.00
19.275 19.95
10.00 50.00
18.1 19

8.00 45.00
20.00 B: Incubation time 20.00
27.50 27.50 C: Incubation temp.
6.00 40.00
35.00 35.00
42.50 42.50
50.00 4.00 50.00 35.00

A: Enzyme concentration A: Enzyme concentration

(a) Effect of enzyme concentration and (b) Effect of enzyme concentration and
incubation time. incubation temperature.

Design-Expert® Software Design-Expert® Software

Protein Content (%) Protein Content (%)

22.88 22.88

19.87 19.87

X1 = A: Enzyme concentration X1 = B: Incubation time 22.8

X2 = D: pH X2 = C: Incubation temp.

Protein Content (%)

Actual Factors Actual Factors 22.075
B: Incubation time = 8.00 22.9 A: Enzyme concentration = 35.00
Protein Content (%)

C: Incubation temp. = 45.00 D: pH = 5.00 21.35

21.95
20.625
21

6.00 19.9
20.05

5.50 4.00
19.1 55.00

6.00
5.00 50.00
20.00
D: pH 8.00
27.50 45.00
4.50
35.00
10.00
42.50 B: Incubation time 40.00 C: Incubation temp.
50.00 4.00
A: Enzyme concentration 12.00 35.00

(c) Effect of enzyme concentration and (d) Effect of incubation time and
tempering water pH incubation temperature

Design-Expert® Software Design-Expert® Software

Protein Content (%) Protein Content (%)

22.88 22.88

19.87 19.87

X1 = B: Incubation time X1 = C: Incubation temp. 22.9

X2 = D: pH X2 = D: pH
Protein Content (%)

22.9 22.275
Actual Factors Actual Factors
A: Enzyme concentration = 35.00 A: Enzyme concentration = 35.00
Protein Content (%)

C: Incubation temp. = 45.00 22.25 B: Incubation time = 8.00 21.65

21.6 21.025

20.95 20.4
6.00
20.3
35.00
5.50 6.00

4.00 40.00
5.50
5.00
6.00
45.00 5.00
8.00 D: pH
4.50
10.00 C: Incubation temp. 50.00 4.50
D: pH
12.00 4.00
B: Incubation time 55.00 4.00

(f) Effect of incubation temperature and

(e) Effect of incubation time and
tempering water pH
tempering water pH

Fig. 3 Response surface for protein content of pigeon pea as a function of enzyme concentration, incubation time, incubation temperature and
tempering water pH. For each plot, the two parameter are at central point

R2 and CV percent value for protein content was 0.96 pretreatment on pigeon pea processing with varying levels
and 0.91% respectively. The response surface equation of of processing parameters under,
second order was obtained in terms of coded factors to
predict the variation in protein content due to enzyme

123
J Food Sci Technol
Table 3 Constraints, criteria
Constraint
and output for numerical
optimization of pigeon pea dhal Variables
production
Enzyme concentration (mg/100 g sample)
Incubation time (h)
Incubation temperature (°C)
Tempering water pH
Constraint
Responses
Hulling efficiency (%)
Cooking time (min)
Protein content (%)
Protein Content ð%Þ ¼ 22:44  0:50X1  0:36X2  0:16X3
þ 0:20X4  0:023X1 X2
 0:025X1 X3 þ 0:056X1 X4
 0:076X2 X3 þ 0:02X2 X4
þ 0:20X3 X4  0:39X12  0:19X22
 0:091X32  0:03X42
where X1, X2, X3 and X4 are the coded factors of enzyme
concentration, incubation time, incubation temperature and
tempering water pH, respectively.
Three dimensional response surface plot for protein
content of enzyme pretreated samples were generated with
center point of two parameters central and another two
parameters as variables.
Figure 3 shows that at constant central value of incu-
bation temperature and tempering water pH. Protein con-
tent was increased (up to 22.75%) with an increase of
enzyme concentration up to 30.45 mg/100 g sample and
incubation time up to 6.14 h respectively (Fig. 3a). Simi-
larly, for central value of incubation period and tempering
water pH there was an increase in protein content up to
22.65% with an increase in enzyme concentration up to
30.31 mg/100 g sample and incubation temperature up to
40.87 °C (Fig. 3b). The protein content was expected to be
increased at this combination of enzyme concentration and
temperature. For central value of incubation period and
incubation temperature protein content increased up to
22.81% an increase in enzyme concentration up to
30.88 mg/100 g sample and tempering water pH up to 5.99
(Fig. 3c). Whereas central value of enzyme concentration
and tempering water pH there was an increase in protein
content up to 22.63% with an increase in incubation time
up to 6.33 h and incubation temperature up to 42.13 °C
(Fig. 3d). For central value of enzyme concentration and
incubation temperature protein content with an increased
Goal Importance Optimum value
Minimize 3 31.34
In the range 3 8.72
In the range 3 43.47
In the range 3 5.99
Goal Importance Predicted value Experimental value Deviation
Maximize 5 84.35 80.74 3.61
Minimize 5 13.06 12.80 0.26
Maximize 5 22.60 25.30 2.7
up to 22.85% in incubation time up to 6.30 h and tem-
pering water pH up to 5.98 (Fig. 3e). Similarly, for central
value of enzyme concentration and incubation period
highest protein content (22.87%) could be obtained for the
interaction of incubation temperature of 51.39 °C and
tempering water pH of 5.99 (Fig. 3f). It was also observed
that above optimum value of variable parameters there is
ð8Þ
decrease in protein content. The increase in protein content
up to optimum value of variable parameters is directly
related to increase in hulling efficiency. It was reported that
outer layer of cotyledon have more protein (Singh and
Jambunathan 1990). This enzymatic process produces less
powder and more recovery of dhal hence outer layer of
cotyledon remains intact.
Optimization of process variables
The optimum condition for the production of pigeon pea
dhal was determined by the numerical optimization tech-
nique, using Design Expert version 8.0.0.6 (Trial version;
State-Ease Inc., Minneapolis, MN, USA). The main criteria
applied for constraints optimization in the study were:
(a) enzyme concentration: minimum, (b) hulling efficiency:
maximum, (c) cooking time: minimum, (d) protein content
of dhal: maximum. The constraints, criteria and output for
numerical optimization of pigeon pea dhal production are
given in Table 3. Under these constraints, the optimum
treatment conditions were found to be, 31.34 mg/100 g
enzyme concentration, 8.72 h incubation time, 43.47 °C
incubation temperature and 5.99 tempering water pH.
It may be mentioned that the optimum values of dif-
ferent variables for enzymatic pretreatment were within the
range considered in the study.
The hulling efficiency, cooking time and protein content
of oil treated (control) sample was found 78.30%,
14.30 min and 18.53% respectively while the observed
123

value of that of enzymatic treatment sample at optimum
condition was 80.74%, 12.80 min and 25.30%. Hence,
there was an increase in hulling efficiency of 2.44%,
reduction in cooking time 1.50 min and increase in protein
content 6.77% over oil treated sample.
Conclusion
For enzymatic pretreatment, the enzyme solution having
xylanase, pectinase and cellulase enzymes increases the
recovery of dhal in terms of hulling efficiency, reduces
energy requirement for cooking in terms of cooking time
and increases most useful biochemical content in terms of
protein content as compared to traditional oil treatment.
The requirement of frequent touching through emery
coated roller is not required in this process, hence the
power requirement for dhal making will reduce. This will
be more beneficial to pigeon pea processing industries to
economize their processing as well as to consumer for
better protein content.
Acknowledgements Authors acknowledge the financial support
received from Department of Agriculture, Cooperation and Farmers
Welfare, Ministry of Agriculture and Farmers Welfare, Government
of India, New Delhi under National Food Security Mission Project.
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