Sequential Acid, Alkaline, and Enzymatic Modifications of Chickpea and Lentil Flours
Impacted Batter Physical Properties
Mohammed Saleh,1,† Ghadeer Mehyar,1 Ayed Amr,1 and George Ondier2
ABSTRACT
The effect of sequential acid, alkaline, and enzymatic treatment of chick-
pea and lentil flours on batter rheological properties was investigated.
Substitution of wheat with disrupted chickpea and lentil flours significantly
(P < 0.05) increased water-holding capacity from 66.8% in wheat flour to
more than 70.0% based on the disruption treatment, indicating an improved
adhesion of coated batter. Flow behavior index of batter treatments of
partially replaced wheat flour with various ratios of disrupted chickpea and
lentil flours ranged from 0.88 to 1.36 and was significantly (P < 0.05) lower
than the flour (i.e., 2.15) and nondisrupted control (i.e., 1.28–1.38 for
Batter application to various food products is widely used to
enhance food functional properties including texture, flavor, ap-
pearance, and acceptability. A thin, uniform, and continuous layer
of batters can, for example, contribute to reducing oil absorption of
food products during deep frying (Pinthus et al. 1993; Fiszman and
Salvador 2003).
Batter is mainly a liquid mixture composed of water, flour, starch,
flavoring, and seasonings into which food products are usually
dipped prior to frying. Several types of flour or combinations of
flours including rice flour, wheat–corn, and corn–rice are also used
in batter making and are reported to have a significant impact on
batter rheological properties (Shih and Daigle 1999; Mukprasirt
et al. 2000; Dogan et al. 2005; Xue and Ngadi 2007). Hydrocolloids,
for example, were effective in improving batter performance and
reducing oil absorption in fried coated foods (Xue and Ngadi 2007).
However, batter is a highly complex system in which chemical
composition, nature, and interaction of ingredients vary widely, thus
determining the final product quality (Hsia et al. 1992; Shih and
Daigle 1999; Dogan et al. 2005). Proteins, for example, were
reported to modify the coated product texture when added in the
range of 10–25% (Loewe 1993, 2011). Proteins were reported to
enhance binding and coated product texture and to improve water-
absorption capacity of flour, thus increasing the system’s viscosity
(Hoseney 1994; Shih and Daigle 1999; Mukprasirt et al. 2000).
Pulses including beans, chickpeas, lentils, lupines, and peas are
gaining interest as healthy, popular food choices because of their
high protein, fiber, starch, vitamin, and mineral contents and their
low-fat and gluten-free status (Dzudie et al. 2002; Mbofung et al.
2002; Torres et al. 2007; Utrilla-Coello et al. 2007). The use of
legumes in cereal products, however, appears to be restricted be-
cause of factors including low protein and starch digestibility and
the content of significant amounts of antinutritional factors that
might be distributed ubiquitously within plant foods including
vegetables, cereals, legumes, and fruits, especially when used as
whole grain (Khokhar and Apenten 2014). Chickpeas, for instance,
contain trypsin inhibitors, phytic acid, and tannins that can reduce
the availability of amino acids and affect growth (Adebowale et al.
† Corresponding
author. Phone: +962-6-5355000, ext. 22426.
E-mail: misaleh@ju.edu.jo
1 Department of Nutrition and Food Technology, Faculty of Agriculture, The
University of Jordan, Amman, Jordan.
2 Taylor Laboratories Inc., 12010 Taylor Road, Houston, TX 77041, U.S.A.
http://dx.doi.org/10.1094/CCHEM-07-14-0168-R
© 2015 AACC International, Inc.
Cereal Chem. 92(2):161–170
chickpea and 1.22–1.28 for lentil) flours. Consistency coefficients of
disrupted chickpea and lentil flours were significantly (P < 0.05) greater
when replacing wheat control, indicating a best fit for the shear-thickening
model. Flour disruption decreased the treatment’s pasting properties, except
the setback, providing support for the significant role of proteins in dic-
tating the pasting characteristics of batter flour treatments. Results of this
study suggested a potential use for treated chickpea and lentil flours in
enhancing batter rheological properties including adhesion and water-holding
capacity.
2005; Wang et al. 2010). Likewise, the presence of phenolic com-
pounds and their oxidized products in lentils promotes essential amino
acid–phenolic complex formation, thus lowering their digestibility
(Shahidi et al. 1992). Additionally, the presence of polyphenols and
phytate in legumes can interfere with the bioavailability of minerals
including iron (Towo et al. 2006).
Modifications of chickpea and lentil flours, therefore, are es-
sential to either eliminate or disrupt their undesirable components to
improve their functionality and allow them to be better utilized as
ingredients in cereal products. Legume processing methods such as
germination, soaking, dehulling, cooking, microwave heating, and
fermentation are examples of modification processes used to reduce
antinutritional factors and to upgrade their nutritional quality (El-
Adawy 2002; Alajaji and El-Adawy 2006). Reyes-Moreno et al.
(2004) for instance, studied the effect of solid fermentation of chick-
peas on the physicochemical properties of tempeh, a traditionally
fermented product of Southeast Asia. Fermentation treatments were
able to reduce phytic acid, tannins, trypsin, and phenols by 71.18,
73.22, 89.78, and 67.84%, respectively, when compared with non-
treated chickpeas. Abu-Salem and Abou-Arab (2011) also reported
a decrease in antinutritional factors with a complete inactivation of
trypsin inhibitor activity after boiling for 90 min as well as autoclaving
at 121°C for 10 min. Clemente et al. (1998) reported an increase in
protein digestibility and a decrease in methionine, cysteine, tyrosine,
and leucine during chickpea cooking.
Although temperature treatments have been indicated to improve
the functionality of legume proteins, there is no indication on im-
proving cereal product functional properties as a result of severe
protein modifications. On the contrary, reports indicated a detrimen-
tal effect of high-temperature-treated legumes on cereal product
quality and processing conditions (Sabanis et al. 2006). Addition-
ally, the interactions between starch and modified proteins during
processing can further impact flour rheological properties, thus
playing a significant role in determining coated product quality and
performance during processing (Shih and Daigle 1999; Dogan et al.
2005; Wang et al. 2006; Renzetti and Arendt 2009; Ribotta et al.
2012). Furthermore, viscosity was reported to dictate characteristics
of batter that include adherence, appearance, texture, and the han-
dling property of the coated product (Mukprasirt et al. 2000). The
flow behaviors and pasting viscoelastic properties of a batter may
provide information that characterizes the influence of temperature,
water content, and various ingredients on a coating’s structural be-
havior during processing. Pasting of flour can further affect batter
viscosity through providing a film barrier that can inhibit oil pen-
etration into the food substrate, providing the finished product with
its crispness (Gibney et al. 1999). Therefore, this study aims to
Vol. 92, No. 2, 2015 161
investigate the effect of partial substitution of wheat flour with
chickpea and lentil flours, whether disrupted or not, on batter rhe-
ological and pasting properties.
MATERIALS AND METHODS
Materials and Experimental Design. Commercial all-purpose
wheat flour (Al-Arabia, U.S.A.) and whole chickpeas and lentils
(Gardenia, Lebanos Food Trading, U.K.) were purchased from
a local market. A cyclone sample mill (UDY, U.S.A.) fitted with
a 100-mesh sieve was used for grinding chickpea and lentil sam-
ples to produce flours. Treatments of fractional substitution of all-
purpose wheat flour with disrupted and nondisrupted chickpea and
lentil flours were used in this study. Treatments of fractional sub-
stitution with nondisrupted chickpea and lentil flours and 100%
Fig. 1. SDS-PAGE of disrupted chickpea (C) and lentil (T) flours. S =
standard; C1 and T1 = acid disruption + enzyme A; C2 and T2 = acid
disruption + enzyme B; C3 and T3 = acid disruption with no enzyme
treatment; C4 and T4 = alkaline disruption + enzyme C; C5 and T5 = acid +
alkaline + enzyme B; C6 and T6 = acid + alkaline + enzyme D; C7 =
alkaline disruption with no enzyme; and T7 = alkaline disruption + enzyme
D. Enzymes A, B, C, and D represent proteases from Rhizopus spp. (EC
3.4.23.21), papaya (EC 3.4.22.2), Aspergillus mellus (EC 232.642.4), and
Aspergillus saitoi (EC 3.4.23.18), respectively.
162 CEREAL CHEMISTRY
wheat flour were included in this study as the control samples. One
acid and one alkaline level (HCl, pH 2; NaOH, pH 10) (pH meter
40675/0001 and pH 211 microprocessor pH meter, electrode type
no. P11/BNC/BANTE, Hanna Instruments, U.S.A.) and four differ-
ent enzymes were also used to disrupt chickpea and lentil flours.
Flour enzymatic disruption treatments included protease from
Rhizopus spp. (EC 3.4.23.21, rhizopuspepsin, P0107-5G, 071M1218,
Japan) with an optimum pH of 3.0 and temperature of 37°C, protease
from Aspergillus saitoi (EC 3.4.23.18, aspergillopepsin I, P2143-
25G, 1001396035, Japan) with an optimum pH of 2.8 and temper-
ature of 37°C, protease from A. melleus (EC 232-642-4, P4032-25G,
1001511077, Japan) with an optimum pH of 7.5 and temperature
of 37°C, and papaya proteinase (EC 3.4.22.2, papain, P3250-25G,
1001465678, Japan) with an optimum pH of 6.2 and temperature
of 55°C.
Sequential acid, alkaline, and enzymatic treatments were per-
formed to disrupt legume flours as indicated by sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE), presented
in Figure 1. For treatment formulation, flours of disrupted and
nontreated chickpeas and lentils were used to replace all-purpose
wheat flour in proportions of 5, 10, 15, and 20%. A control sample
of 0% replacement (i.e., 100% wheat flour) was also included in the
sample set. Replacement of more than 20% of the wheat flour by
treated or nontreated chickpea or lentil flours was not included in
this study because greater replacement of legumes was reported to
be detrimental to batter functionality, including batter textural
characteristics (unpublished data). Treatments were mixed thor-
oughly with a KitchenAid household mixer (model KSM150PSER,
KitchenAid, U.S.A.) at speed 4 for 2 min before performing the
experiment.
Acid Hydrolysis of Chickpea and Lentil Flours. Chickpea
and lentil flours were acid treated according to the method described
by Lee et al. (2001). In brief, chickpea and lentil flours were sus-
pended in distilled water in a ratio of 5:1 water/flour and held in
a water bath (model Wb 14, DIN176-1K1, Memmert, Germany) at
40°C for 30 min. HCl was used to hydrolyze flour by adjusting the
pH of the solution to achieve the concentration in the reaction
medium of 0.1N HCl (pH 2). The suspension was then incubated at
45°C with continuous agitation for 3 h. Acid hydrolysis of flour was
then terminated by cooling the solution for 30 min to room tem-
perature (approximately 23.2°C) and adjusting pH to 6.0–6.2 with
0.1N NaOH. Samples were then dried at 40°C in a drying oven
(Precision, U.S.A.) to a moisture content of 12% (wb) before being
used for alkaline treatments.
Alkaline Treatment of Chickpea and Lentil Flours. Al-
kaline treatments (pH 10) were carried out on the acid-treated
chickpea and lentil flours. In brief, acid-treated chickpea and lentil
flours were suspended in distilled water at a 5:1 water/flour ratio
and held at 40°C for 30 min with a temperature-controlled water
bath. NaOH solution was then added to achieve a concentration in
the reaction medium of 0.1N NaOH (pH 10). The suspension was
then incubated at 45°C with continuous agitation for 2 h. Alkaline
treatments were terminated by cooling the solution for 30 min at
room temperature (approximately 23.2°C) and adjusting the pH to
7.0 with 0.1N HCl. Samples were then dried at 40°C in a drying
oven to a moisture content of 12% (wb).
Enzymatic Hydrolysis of Chickpea and Lentil Flours.
Enzymatic treatments of the chickpea and lentil flours involved com-
mercial sources of the following enzymes: protease from Rhizopus spp.
(EC 3.4.23.21, rhizopuspepsin), protease from A. saitoi (EC 3.4.23.18,
aspergillopepsin I), protease from A. melleus (EC 232-642-4), and
papaya proteinase (EC 3.4.22.2, papain). Prior to enzymatic treat-
ment, a suspension of the acid- and alkaline-treated flour was pre-
pared by adding 250 mL of distilled water per 50 g of flour.
Enzymatic hydrolysis was then carried out depending on the opti-
mum pH (2.8–7) and temperature (37–50°C) for each enzyme by
using a temperature-controlled water bath. Enzymatic amounts and
durations were varied based on the optimum activity for each
 enzyme as described by the manufacturer: 1.2 and 0.5 g of pro- was terminated by adjusting the pH of that treatment solution to
tease from Rhizopus spp., 1.25 and 2.25 g of papaya proteinase, a pH that was different than the optimum for that enzyme. More
0.53 and 0.75 g of protease from A. melleus, and 0.5 and 0.60 g of specifically, protease from A. melleus and papaya proteinase
protease from A. saitoi were used per 250 g of lentil and chickpea having optimum activity at pH 7.5 and 6.2, respectively, and
flour samples, respectively. Enzymatic reaction for each treatment temperature of 37°C was terminated by adjusting the pH of the

Enzyme A- Acid Treatment Enzyme B- Acid Treatment Enzyme C- Acid Treatment Enzyme D- Acid Treatment
1.50 1.50 1.50 1.50

1.25 1.25 1.25 1.25

Viscosity (mPa.s)

Viscosity (mPa.s)

Viscosity (mPa.s)
Viscosity (mPa.s)
1.00 1.00 1.00 1.00
5% 15% 15%
20% 10%
0.75 10% 0.75 0.75
15% 0.75 10%
15% 5% 5%
10%
20% 5%
0.50 0.50 0.50 0.50

0.25 0.25 0.25 0.25

20 20%
0.00 0.00 0.00 0.00
0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0 0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0 0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0 0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0
-1 -1 -1 -1
Shear Rate (s ) Shear Rate (s ) Shear Rate (s ) Shear Rate (s )

Enzyme A- Acid-Alkaline Treatment Enzyme B-Acid-Alkaline Treatment Enzyme C-Acid-Alkaline Treatment Enzyme D-Acid-Alkaline Treatment
1.50 1.50 1.50 1.50

1.25 1.25 1.25 1.25

5%

Viscosity (mPa.s)
Viscosity (mPa.s)

Viscosity (mPa.s)

Viscosity (mPa.s)
1.00 1.00 1.00 1.00
10% 5%
5%
0.75 0.75 0.75 0.75
15% 15% 15%
10% 5% 10% 20%
20% 10%
20%
0.50 0.50 0.50 0.50
15%

0.25 0.25 0.25 0.25

20%

0.00 0.00 0.00 0.00

0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0 0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0 0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0 0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0
-1 -1 -1 -1
Shear Rate (s ) Shear Rate (s ) Shear Rate (s ) Shear Rate (s )

Enzyme A-Alkaline Treatment Enzyme B-Alkaline Treatment Enzyme C-Alkaline Treatment Enzyme D-Alkaline Treatment
1.50 1.50 1.50 1.50

1.25 1.25 1.25 1.25

5%
Viscosity (mPa.s)

Viscosity (mPa.s)
Viscosity (mPa.s)
Viscosity (mPa.s)

1.00 1.00 1.00 1.00

15% 15% 15%
10% 15%
10%
0.75 0.75 0.75 0.75
5% 10% 20%
5% 20%
0.50 0.50 0.50 5%
10% 0.50
20%

0.25 0.25 0.25 0.25

20%
0.00 0.00 0.00 0.00
0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0 0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0 0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0 0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0
-1 -1 -1 -1
Shear Rate (s ) Shear Rate (s ) Shear Rate (s ) Shear Rate (s )

_
Fig. 2. Viscosity (mPa·s) versus shear rate (s 1)
of batter made with wheat and disrupted chickpea flour (5, 10, 15, and 20%) fractions. Enzymes A, B, C,
and D represent proteases from Rhizopus spp. (EC 3.4.23.21), papaya (EC 3.4.22.2), Aspergillus mellus (EC 232.642.4), and Aspergillus saitoi (EC
3.4.23.18), respectively.

Enzyme A- Acid Treatment Enzyme B- Acid Treatment Enzyme C- Acid Treatment Enzyme D- Acid Treatment
1.50 1.50 1.50 1.50

1.25 1.25 1.25 1.25

5% 5%
Viscosity (mPa.s)

Viscosity (mPa.s)

Viscosity (mPa.s)

Viscosity (mPa.s)

1.00 1.00 1.00 1.00

15% 5% 10%
10% 5%
0.75 0.75 0.75 0.75
20% 10% 10% 15%
0.50 0.50 15% 0.50 0.50
15%
0.25 0.25 0.25 0.25
20%
20% 20%
0.00 0.00 0.00 0.00
0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0 0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0 0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0 0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0
-1 -1
Shear Rate (s ) Shear Rate (s ) -1
Shear Rate (s ) -1
Shear Rate (s )

Enzyme A- Acid-Alkaline Treatment Enzyme B- Acid-Alkaline Treatment Enzyme C- Acid-Alkaline Treatment Enzyme D- Acid-Alkaline Treatment
1.50 1.50 1.50 1.50

1.25 1.25 1.25 1.25

Viscosity (mPa.s)

Viscosity (mPa.s)

Viscosity (mPa.s)

Viscosity (mPa.s)

1.00 1.00 1.00 1.00 10%

0.75 20%
10% 0.75 0.75 0.75
5% 20% 15% 20% 5%
20%
10% 5% 10% 5%
0.50 0.50 0.50 0.50

0.25 0.25 0.25 0.25

15% 15%
15%
0.00 0.00 0.00 0.00
0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0 0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0 0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0 0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0
-1 -1
Shear Rate (s ) Shear Rate (s ) -1
Shear Rate (s )
-1
Shear Rate (s )

Enzyme A-Alkaline Treatment Enzyme B-Alkaline Treatment Enzyme C-Alkaline Treatment Enzyme D-Alkaline Treatment
1.50 1.50 1.50 1.50

1.25 1.25 1.25 1.25

5%
Viscosity (mPa.s)
Viscosity (mPa.s)

Viscosity (mPa.s)
Viscosity (mPa.s)

1.00 1.00 1.00 1.00

5% 20% 10%
10% 20%
0.75 20% 0.75 0.75 0.75
10% 5% 20%
10% 5% 15%
15% 15%
0.50 0.50 0.50 0.50

0.25 0.25 0.25 0.25

15%
0.00 0.00 0.00 0.00
0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0 0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0 0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0 0.0 6.0 12.0 18.0 24.0 30.0 36.0 42.0 48.0 54.0 60.0 66.0
-1 -1
Shear Rate (s ) Shear Rate (s ) -1
Shear Rate (s )
-1
Shear Rate (s )

_1
Fig. 3. Viscosity (mPa·s) versus shear rate (s ) of batter made with wheat and disrupted lentil flour (5, 10, 15, and 20%) fractions. Enzymes A, B, C, and
D represent proteases from Rhizopus spp. (EC 3.4.23.21), papaya (EC 3.4.22.2), Aspergillus mellus (EC 232.642.4), and Aspergillus saitoi (EC
3.4.23.18), respectively.

Vol. 92, No. 2, 2015 163

 Non Treated Chickpea and Wheat Control Treatments solution with HCl to pH 2 for 10 min. Similarly, protease from
1.50 Rhizopus spp. and protease from A. saitoi having optimum activity
at pH 2.8 and 3.0 and temperatures of 37 and 55°C, respectively,
1.25
Wheat Control were terminated by adjusting the pH of the solution with NaCl to
pH 11 for 10 min. Samples were then dried at 40°C in a drying
Viscosity (mPa.s)

1.00
10%
oven to a moisture content of 12% (wb) and designated as the
0.75 5%
sequentially modified chickpea and lentil flour samples. Acid and
15%
alkaline modifications of chickpea and lentil flours with no en-
0.50 zymatic modifications were also included in the sample set.
SDS-PAGE. Protein profiling of samples was performed with
0.25
SDS-PAGE as described by Laemmli (1970). In brief, freeze-dried
20%
0.00
protein extracts (10 mg) of chickpea and lentil treatments were
0 6 12 18 24 30 36 42 48 54 60 66 dissolved in 1 mL of sample buffer (distilled water, 50mM Tris, pH
Shear Rate (s )
-1 6.8, 2% SDS, 10% glycerol, 0.1% bromophenol blue, and 5%
b-mercaptoethanol) before being centrifuged 3,800 rpm (14,000 × g)
for 10 min. The supernatants of samples were then collected, heated
at 98°C for 10 min, and cooled.
Non Treated Lentils and Wheat Control Treatments
1.50
Equal amounts (20 µg) of the collected supernatant samples were
each loaded into SDS-PAGE wells. Electrophoresis was performed
1.25 on discontinuous 4% stacking and 12% resolving gels (Bio-Rad,
Wheat Control
U.S.A.). Gel was then fixed and stained with 0.2% Coomassie bril-
Viscosity (mPa.s)

1.00
5%
liant blue R-250 in methanol/acetic acid/water (5:4:1, v/v/v) and
15%
destained before drying and molecular weight calculations. The
0.75
10% standard protein marker contained myosin (200,000), b-galactosidase
0.50
(116,250), phosphorylase b (97,400), serum albumin (66,200), ov-
albumin (45,000), carbonic anhydrase (31,000), trypsin inhibitor
0.25 (21,500), lysozyme (14,400), and aprotinin (6,500).
20% Water-Holding Capacity (WHC) of Flour Treatments.
0.00 WHC of each treatment (i.e., wheat flour substituted with ratios of
0 6 12 18 24 30 36 42 48 54 60 66
-1
disrupted and nondisrupted chickpea and lentil flours) was determined
Shear Rate (s ) following the method described by Abu-Salem and Abou-Arab (2011)
_ with a modified centrifuge speed. In brief, flour treatments were
Fig. 4. Viscosity (mPa·s) versus shear rate (s 1) of batter made with wheat
and nondisrupted chickpea and lentil flours (i.e., 5, 10, 15, and 20%) and dispersed in distilled water and the dispersions allowed to stand for
a wheat control sample. 1 h at room temperature (23.2°C) before centrifuging at 3,800 rpm

TABLE I
Water-Holding Capacity (%) of Wheat Flour Partially Substituted with Various Percentages of Disrupted Chickpea and Lentil Floursy
Chickpea Lentil
Enzymatic
Chemical Treatment Treatmentz 5% 10% 15% 20% LSD 5% 10% 15% 20% LSD
Acid None ND ND 76.0b ND ND ND ND 70.5c ND ND
Acid A 72.4b 74.6c 77.2b 81.4b 0.82 72.4b 73.7a 76.5a 81.0a 0.84
Acid B 71.6c 75.5b 76.6b 79.9c 0.72 73.7a 72.4b 74.7b 77.7b 0.76
Acid C 74.4a 76.3a 80.0a 84.7a 0.95 71.3c 72.1b 74.8b 75.8c 0.62
Acid D 70.1d 73.3d 76.1b 80.5c 0.69 70.4d 73.5a 75.7ab 80.8a 0.78
None None 71.1c 73.6d 75.3b 78.2d 0.89 67.6e 70.5c 73.8b 76.5c 1.5
Flour control 66.8e 66.8e 66.8c 66.8e ND 66.8e 66.8d 66.8d 66.8d ND
Acid + alkaline None ND ND 76.1cd ND ND ND ND 71.2d ND ND
Acid + alkaline A 74.0b 78.4b 77.1c 83.4c 0.92 73.4c 75.6c 82.0a 82.3b 0.89
Acid + alkaline B 69.7d 72.9d 77.9c 84.2c 0.72 74.7b 79.0a 82.2a 78.5d 0.77
Acid + alkaline C 71.3c 76.2c 79.2b 85.0b 0.72 69.1d 68.4e 79.1b 83.9a 0.85
Acid + alkaline D 78.4a 80.8a 83.0a 88.2a 0.80 76.9a 77.0b 79.7b 81.5c 0.89
None None 71.1c 73.6d 75.3d 78.2d 1.02 67.6e 70.5d 73.8c 76.5d 1.20
Flour control 66.8e 66.8e 66.8e 66.8e ND 66.8e 66.8f 66.8e 66.8e ND
Alkaline None ND ND 70.2d ND ND ND ND 68.7d ND ND
Alkaline A 73.7b 75.7b 79.1a 82.9a 0.93 69.5c 72.8c 72.7c 75.3b 0.96
Alkaline B 74.8a 77.4a 78.8a 82.6a 0.88 70.4c 76.0a 79.0a 82.8a 0.94
Alkaline C 71.9c 74.8c 76.8b 80.1b 1.20 74.1a 76.0a 79.0a 82.7a 1.20
Alkaline D 70.7d 73.1d 75.9c 77.5d 1.10 71.8b 74.5b 74.5b 75.7b 0.71
None None 71.1c 73.6d 75.3c 78.2c 0.98 67.6d 70.5d 73.8c 76.5c 0.69
Flour control 66.8e 66.8e 66.8e 66.8e ND 66.8e 66.8e 66.8e 66.8d ND
y Means of water-holding capacity of batter treatments of the same chemical treatment and disrupted chickpea or lentil flour fraction replacements but different
enzymatic treatments (column) with different letters are significantly (P < 0.05) different according to least squares differences (LSD). For similar disrupted,
nondisrupted, and wheat flour samples, means of water-holding capacity of various fraction replacements (same row) are separated according to the provided
LSD values. ND = no data.
z A = protease enzymes from Rhizopus spp. (EC 3.4.23.21); B = papaya (EC 3.4.22.2); C = Aspergillus mellus (EC 232.642.4); and D = Aspergillus saitoi
(EC 3.4.23.18).

164 CEREAL CHEMISTRY

for 30 min (5810R, Eppendorf, Germany). Sediment weights Batter Preparation and Rheological Measurements. Treated
were recorded and used to calculate WHC with the following and nontreated chickpea and lentil flours were used to substitute for
equation: wheat in percentage replacements of 0 (i.e., wheat flour control), 5,
10, 15, and 20%. Batter dough was made with a water/flour ratio of
weight of sediment 70:30. Homogenization of batter was performed with a homogenizer
WHC  ð%Þ = × 100 (1)
weight of dry solids (X 120, Ingenieurbüro CAT, Germany) before rheological property

TABLE II
Flow Behavior Index of Wheat Flour Partially Substituted with Various Percentages of Disrupted Chickpea and Lentil Floursy
Chickpea Lentil
Enzymatic
Chemical Treatment Treatmentz 5% 10% 15% 20% LSD 5% 10% 15% 20% LSD
Acid None ND ND 1.27b ND ND ND ND 1.35b ND ND
Acid A 1.15c 1.13c 1.04d 1.13d 0.035 1.21c 1.36b 1.22c 1.20c 0.065
Acid B 1.14c 1.13c 1.09cd 1.07e 0.121 1.14d 1.14d 1.08d 1.10d 0.723
Acid C 1.07d 1.02d 1.06d 1.11d 0.031 1.11d 1.29c 1.09d 1.07de 0.067
Acid D 1.17c 1.16c 1.11c 1.16c 0.025 1.14d 1.15d 1.22c 1.01e 0.066
None None 1.38b 1.32b 1.28b 1.31b 0.054 1.28b 1.27c 1.22c 1.25b 0.078
Flour control 2.15a 2.15a 2.15a 2.15a ND 2.15a 2.15a 2.15a 2.15a ND
Acid + alkaline None ND ND 1.23c ND ND ND ND 1.35b ND ND
Acid + alkaline A 1.24c 1.14d 1.05e 1.06d 0.058 0.88e 1.34b 1.08e 1.14c 0.034
Acid + alkaline B 1.15d 1.08e 1.10d 1.06d 0.026 1.12c 1.08de 1.06e 1.12c 0.051
Acid + alkaline C 1.03e 1.19c 1.10d 1.08cd 0.052 1.07d 1.05e 1.15d 1.08d 0.065
Acid + alkaline D 1.16d 1.17c 1.15d 1.10c 0.035 1.08d 1.12d ND 1.18b 0.033
None None 1.38b 1.32b 1.28c 1.31b 0.047 1.28b 1.27c 1.22c 1.25b 0.078
Flour control 2.15a 2.15a 2.15a 2.15a ND 2.15a 2.15a 2.15a 2.15a ND
Alkaline None ND ND 1.37c ND ND ND ND 1.35b ND ND
Alkaline A 1.30c 1.15d 1.17e 1.09d 0.036 1.11c 1.15c 1.14d 1.17d 0.056
Alkaline B 1.14d 1.09d 1.11f 1.14c 0.055 1.15c 1.08d 1.07f 1.15de 0.055
Alkaline C 1.13d 1.15d 1.09f 1.13c 0.054 1.12c 1.06d 1.11e 1.10e 0.033
Alkaline D 1.28c 1.24c 1.16e 1.15c 0.025 1.14c 1.16c 1.13d 1.32b 0.052
None None 1.38b 1.32b 1.28d 1.31b 0.025 1.28b 1.27b 1.22c 1.25c 0.078
Flour control 2.15a 2.15a 2.15a 2.15a ND 2.15a 2.15a 2.15a 2.15a ND
y Means of flow behavior index (n) of batter dough having the same chemical treatment and disrupted chickpea or lentil flour fraction but different enzymatic treatments
(column) with different letters are significantly (P < 0.05) different according to least squares differences (LSD). For similar disrupted, nondisrupted, and wheat flour
samples, means of flow behavior index (n) of various fraction replacements (same row) are separated according to the provided LSD values. ND = no data.
z A = protease enzymes from Rhizopus spp. (EC 3.4.23.21); B = papaya (EC 3.4.22.2); C = Aspergillus mellus (EC 232.642.4); and D = Aspergillus saitoi
(EC 3.4.23.18).

TABLE III
Consistency Coefficient (mPa·sn) of Wheat Flour Partially Substituted with Various Percentages of Disrupted Chickpea and Lentil Floursy
Chickpea Lentil
Enzymatic
Chemical Treatment Treatmentz 5% 10% 15% 20% LSD 5% 10% 15% 20% LSD
Acid None ND ND 0.10c ND ND ND ND 0.12b ND ND
Acid A 0.14a 0.12b 0.16a 0.16a 0.025 0.08c 0.05d 0.11bc 0.09c 0.034
Acid B 0.12ab 0.08c 0.08c 0.09b 0.075 0.12b 0.09c 0.08d 0.05d 0.041
Acid C 0.12ab 0.18a 0.09c 0.11b 0.029 0.10b 0.04d 0.09cd 0.09c 0.019
Acid D 0.11b 0.12b 0.13b 0.11b 0.021 0.12b 0.15b 0.13b 0.17b 0.022
None None 0.05d 0.05d 0.06d 0.06c 0.025 0.25a 0.26a 0.26a 0.32a 0.031
Flour control 0.008c 0.008e 0.008e 0.008d ND 0.008d 0.008e 0.008e 0.008e ND
Acid + alkaline None ND ND 0.14a ND ND ND ND 0.14b ND ND
Acid + alkaline A 0.09c 0.11a 0.10b 0.12b 0.011 0.22a 0.08d 0.09c 0.11b 0.012
Acid + alkaline B 0.10c 0.10ab 0.08bc 0.07c 0.012 0.12b 0.14b 0.11b 0.06d 0.025
Acid + alkaline C 0.12b 0.07b 0.10b 0.12b 0.021 0.12b 0.11c 0.11b 0.09c 0.028
Acid + alkaline D 0.18a 0.11a 0.15a 0.22a 0.045 0.12b 0.13b ND 0.11b 0.010
None None 0.05e 0.05c 0.06c 0.06c 0.024 0.25a 0.26a 0.26a 0.32a 0.021
Flour control 0.008f 0.008d 0.008d 0.008d ND 0.008c 0.008e 0.008d 0.008e ND
Alkaline None ND ND 0.13a ND ND ND ND 0.09e ND ND
Alkaline A 0.06c 0.12a 0.09b 0.15b 0.018 0.13c 0.13b 0.11d 0.10b 0.010
Alkaline B 0.11a 0.11a 0.07c 0.07d 0.022 0.10d 0.08e 0.12c 0.10b 0.018
Alkaline C 0.09ab 0.07bc 0.08bc 0.08c 0.021 0.08e 0.10d 0.10e 0.10b 0.010
Alkaline D 0.08b 0.09ab 0.14a 0.16a 0.010 0.14b 0.12c 0.13b 0.06c 0.012
None None 0.05d 0.05c 0.06c 0.06d 0.026 0.25a 0.26a 0.26a 0.32a 0.018
Flour control 0.008e 0.008d 0.008d 0.008e ND 0.008e 0.008e 0.008f 0.008d ND
y Means of consistency coefficient (mPa·sn) of batter dough having the same chemical treatment and disrupted chickpea or lentil flour fraction but different
enzymatic treatments (column) with different letters are significantly (P < 0.05) different according to least squares differences (LSD). For similar disrupted,
nondisrupted, and wheat flour samples, means of consistency coefficient (mPa·sn) of various fraction replacements (same row) are separated according to the
provided LSD values. ND = no data.
z A = protease enzymes from Rhizopus spp. (EC 3.4.23.21); B = papaya (EC 3.4.22.2); C = Aspergillus mellus (EC 232.642.4); and D = Aspergillus saitoi
(EC 3.4.23.18).

Vol. 92, No. 2, 2015 165

 TABLE IV
Pasting Viscosities (cP) of Wheat Flour Substituted with Various Percentages (5, 10, 15, and 20%)
of Chemically and Enzymatically Disrupted Chickpea Floursy
Peak Viscosity Trough
Chemical Enzymatic
Treatment Treatmentz 5% 10% 15% 20% 5% 10% 15% 20%
None None 1,632.0cA 1,559.7dB 1,491.0eC 1,416.7dD 1,023.3dA 997.7cB 963.0eC 958.3dC
Acid A 1,565.0dA 1,478.0eB 1,495.0eC 1,363.7eD 1,019.7dA 977.7cB 952.3efC 917.7eD
Acid B 1,646.3cA 1,606.7cB 1,557.7dC 1,502.7cD 1,057.0cA 1,042.3bB 1,035.3dBC 1,025.7cC
Acid C 1,547.0dA 1,429.3fB 1,381.3fC 1,358.3eD 1,020.0dA 959.0dB 932.0fC 910.7eD
Acid D 1,810.7bA 1,757.0bB 1,694.0cC 1,623.0bD 1,179.7bA 1,166.0bB 1,148.3cC 1,133.0bC
Acid None ND ND 1,825.6b ND ND ND 1,265.4b ND
Flour control 1,873.2aA 1,873.2aA 1,873.2aA 1,873.2aA 1,520.3aA 1,520.3aA 1,520.3aA 1,520.3aA
None None 1,632.0cA 1,559.7cB 1,491.0dC 1,416.7bD 1,023.3cA 997.7dB 963.0cC 958.3cC
Acid + alkaline A 1,585.7dA 1,495.0dB 1,407.7eC 1,330.7cD 993.3dA 975.7dB 987.0cAB 920.3cC
Acid + alkaline B 1,638.7cA 1,580.3cB 1,488.7dC 1,411.0bD 955.0eA 919.3eB 908.7dBC 893.3dC
Acid + alkaline C 1,505.3eA 1,406.0eB 1,292.7eC 1,228.7cD 1,033.7cA 1,012.0cB 969.3cC 948.3cD
Acid + alkaline D 1,673.0bA 1,613.0bB 1,526.7cC 1,424.7dD 1,103.7bA 1,133.7bB 1,146.7bBC 1,126.3bC
Acid + alkaline None ND ND 1,765.6b ND ND ND 1,465.5b ND
Flour control 1,873.2aA 1,873.2aA 1,873.2aA 1,873.2aA 1,520.3aA 1,520.3aA 1,520.3aA 1,520.3aA
None None 1,632.0dA 1,559.7eB 1,491.0fC 1,416.7eD 1,023.3cdA 997.6cdB 963.0deC 958.3cC
Alkaline A 1,609.7dA 1,564.7eB 1,650.0cC 1,408.7eD 1,046.3cA 1,017.0cB 991.0dC 948.0cD
Alkaline B 1,745.0bA 1,639.0cB 1,617.0dC 1,528.3cD 1,033.0cA 984.7dB 944.3eC 895.0dD
Alkaline C 1,673.0cA 1,608.7dB 1,526.3eC 1,479.3dD 999.7dA 944.0eB 891.3fC 863.0dD
Alkaline D 1,730.7bA 1,693.0bB 1,650.3cC 1,575.3bD 1,177.3bB 1,195.7bA 1,171.3cB 1,159.3bC
Alkaline None ND ND 2,925.5a ND ND ND 1,968.8a ND
Flour control 1,873.2aA 1,873.2aA 1,873.2bA 1,873.2aA 1,520.3aA 1,520.3aA 1,520.3bA 1,520.3aA
(continued on next page)
y Means of pasting properties (peak, trough, breakdown, final, and setback) of batter flour having the same chemical treatment and fraction replacement of
disrupted, nondisrupted chickpea flour, and wheat flour with different enzymatic treatments (column) with different lowercase letters are significantly (P < 0.05)
different according to least squares differences (LSD). Means of pasting properties of similar disruption treatment and various fraction replacement (same row)
with different capital letters are significantly (P < 0.05) different according to LSD. cP = centipoise.
z A = protease enzymes from Rhizopus spp. (EC 3.4.23.21); B = papaya (EC 3.4.22.2); C = Aspergillus mellus (EC 232.642.4); and D = Aspergillus saitoi
(EC 3.4.23.18).

measurements. Batter dough was held at room temperature (23.2°C)
for 2 h before rheological measurement. Apparent viscosity of batter
dough was measured during a shear rate of 6–60 s_1 at 23.2°C.
A rotational viscometer (SNB-AI digital viscometer, Shandong,
China) was used for viscosity measurements in which samples
were kept constant in a holding cup during the entire rheological
measurement. Flow behaviors of batters described in terms of con-
sistency coefficient and flow behavior index were evaluated in
this study following the Herschel–Bulkley model (equation 2)
and were used to describe the experimental data for flow curves of
all samples:
t = to +   m g : n
2.5 min and then cooled down to 50°C at a cooling rate of 12°C/min,
and typical RVA parameters were extracted. Parameters recorded
were peak viscosity, trough viscosity, final viscosity, breakdown,
and setback.
Statistical Analysis. All measurements were performed in du-
plicate, and mean values were reported. Analysis of variance
(ANOVA) with JMP software (release 10, SAS Institute, U.S.A.)
was performed to determine any significant differences among
the treatment parameters associated with the rheological prop-
erties. Least significant difference at a 5% level of probability
was determined to separate differences in the properties among
treatments.
(2)
RESULTS AND DISCUSSION

where t is the shear stress (mPa), to is the yield stress (mPa), m is the
consistency coefficient (mPa·sn), g_is the shear rate (s_1), and n is the
flow behavior index (dimensionless). The Herschel–Bulkley model
was used to describe the rheological behavior of batter functional
properties. Flow behavior index (n) is typically used to characterize
fluid and semifluid behavior with an n value of 1 describing New-
tonian, <1 describing shear-thinning, and >1 describing shear-
thickening fluid behavior. In addition, the Herschel–Bulkley model
was used in this study because it can describe shear-thinning or
shear-thickening behavior depending on the value of the flow be-
havior index.
Pasting and Viscosity Measurements. Pasting profile and
viscosities (i.e., peak, trough, setback, breakdown, and final) and
pasting temperature of flour treatments were assessed and recorded
with a Rapid Visco Analyzer (RVA-4, Foss North America, U.S.A.)
following AACC International Approved Method 61-02.01 as de-
scribed by Perdon et al. (2001). Approximately 3 g of flour sample
was mixed with 25 mL of distilled water; the slurry was then mixed
at 50°C for 1 min at 160 rpm before being heated from 50 to 95°C at
a heating rate of 12°C/min. The hot paste was then held at 95°C for
SDS-PAGE results of proteins extracted from disrupted chickpea
and lentil flours (sequential acid, alkaline, and enzymatic treatments
as well as the controls) are presented in Figure 1. Results demon-
strated that treatments were effective in modifying chickpea and
lentil proteins, resulting in increased low-molecular-weight proteins
in almost all of the treated samples.
Flow behavior of batter dough containing various proportions of
disrupted chickpea and lentil flours was evaluated by testing shear
stress versus shear rate at a flour/water ratio of 70% water/30%
treatment at 23.2°C. Results of flow behavior for chickpea and lentil
treatments are presented in Figures 2 and 3, respectively, and that
for the control treatments representing the nondisrupted chickpea
and lentil flours and the wheat flour control are presented in
Figure 4. Results clearly demonstrated that apparent viscosity val-
ues of all batters decreased with increasing shear rate and when
wheat flour was substituted with greater proportions of disrupted
legume flours. The increase in shear stress with the increase in
shear rate revealed a shear-thickening characteristic of the batter
treatments. However, because batter behavior acquired a yield
stress, having a flow behavior index of >1, further fitting of batter

166 CEREAL CHEMISTRY

 TABLE IV
(continued from previous page)
Breakdown Final Viscosity Setback
5% 10% 15% 20% 5% 10% 15% 20% 5% 10% 15% 20%
608.7bA 562.0bB 528.0bC 458.3bD 2,235.3c 2,169.7c 2,052.3e 1,999.3c 1,212.0d 1,172.0c 1,089.3e 1,041.0c
545.3cA 500.3cB 486.7cC 446.0abD 2,160.3d 2,067.0d 2,000.3d 1,909.3d 1,140.7c 1,089.3d 1,048.0d 991.7e
589.3bA 564.3bB 522.3bC 477.0aD 2,567.7b 2,768.0b 2,916.7b 2,986.3b 1,510.7b 1,725.7b 1,881.3b 1,960.7b
527.0cA 470.3dB 449.3dC 427.7bD 2,164.0d 2,033.0d 1,987.3c 1,937.7e 1,144.0c 1,074.0d 1,055.3d 1,027.0d
631.0aA 591.0aB 545.7aC 490.0aD 3,216.7a 3,319.0a 3,381.7a 3,357.5a 2,037.0a 2,153.0a 2,233.3a 2,224.5a
ND ND 560.2a ND ND ND 2,565.2c ND ND ND 1,299.8c ND
352.9dA 352.9eA 352.9eA 352.9cA 1,950.5e 1,950.5e 1,950.5f 1,950.5e 430.2e 430.2e 430.2f 430.2f
608.7bA 562.0cB 528.0dC 458.3cD 2,235.3c 2,169.7e 2,052.3d 1,999.3d 1,212.0bc 1,172.0d 1,089.3e 1,041.0d
616.3bB 589.0bC 663.0bA 488.3cD 2,193.0c 2,145.3d 2,160.3c 1,980.0d 1,199.7c 1,169.7d 1,173.3d 1,059.7d
790.0aA 719.7aB 708.3aB 635.0aC 2,211.7c 2,209.0c 2,231.3b 2,275.3b 1,256.7b 1,289.7b 1,322.7b 1,382.0b
639.3bA 596.7bB 557.0cC 531.0bD 2,288.7b 2,241.0b 2,191.0c 2,169.3c 1,255.0b 1,229.0c 1,221.7c 1,221.0c
627.0bA 559.3cB 503.7eC 449.0cD 2,764.7a 3,073.7a 3,219.3a 3,175.0a 1,661.0a 1,940.0a 2,072.7a 2,048.7a
ND ND 300.1g ND ND ND 2,012.3e ND ND ND 546.8f ND
352.9cA 352.9dA 352.9fA 352.9dA 1,950.5d 1,950.5f 1,950.5f 1,950.5e 430.2d 430.2e 430.2g 430.2e
608.7aA 562.0aB 528.0bC 458.3aD 2,235.3b 2,169.7c 2,052.3d 1,999.3c 1,212.0b 1,172.0c 1,089.3e 1,041.0d
558.3bA 537.7bB 499.3cC 459.0aD 2,236.0b 2,227.3b 2,196.0c 2,127.3b 1,189.7b 1,210.3b 1,205.0c 1,179.3b
534.3cA 480.3cB 434.7dC 410.3bC 2,188.3d 2,128.7d 2,077.7d 2,024.0c 1,155.3c 1,144.0d 1,133.3d 1,129.0c
508.7dA 455.7dB 413.7eC 385.7cD 2,123.3c 2,012.0e 1,918.0e 1,856.0d 1,123.7d 1,068.0e 1,026.7f 993.0e
622.3aA 563.0aB 517.7bC 470.3aD 3,198.7a 3,333.0a 3,377.3a 3,326.3a 2,021.3a 2,137.3a 2,206.0a 2,167.0a
ND ND 956.7a ND ND ND 3,256.0b ND ND ND 1,287.2b ND
352.9eA 352.9eA 352.9fA 352.9dA 1,950.5e 1,950.5f 1,950.5e 1,950.5e 430.2e 430.2f 430.2g 430.2f

dough with the Herschel–Bulkley model was considered. The
Herschel–Bulkley model characteristics will be discussed later.
Wheat flour had viscosity values of 1.418, 1.125, 0.716, and
0.511 mPa·s when sheared at 6, 12, 30, and 60 rpm, respectively,
which was greater than that of samples containing legume flours,
whether disrupted or not (i.e., Figs. 2–4). The shear-thinning
behavior of batter was further studied as exhibited in the middle
region, in which the apparent viscosity changed with shear rate
under the Herschel–Bulkley model. Results were attributed to
changes in chemical structure and composition of batter, in which
the greater the amount of disrupted flour substituting for wheat
flour (i.e., greater amount of disrupted chickpea and lentil flours),
the lower the viscoelastic properties.
Table I presents the WHC of wheat flour substituted with various
percentages of disrupted and nondisrupted legume flours. Results
presented a significant increase in WHC with the increase in sub-
stitution ratio, regardless of enzyme or chemical disruption treat-
ment, compared with wheat flour control (i.e., having a WHC of
66.8). For example, substituting wheat flour with 5, 10, 15, and 20%
of chickpea flour disrupted by acid and enzyme C had WHC values
of 74.4, 76.3, 80.0, and 84.7%, respectively. Substituting wheat
flour with 5, 10, 15, and 20% of nontreated chickpea flour had
WHC values of 71.1, 73.6, 75.3, and 78.2%, respectively. In the
same manner, substituting wheat flour with 5, 10, 15, and 20% of
lentil flour disrupted by alkaline and enzyme B had WHC values of
70.4,76.0, 79.0, and 82.8%, respectively, with substituting with
nontreated lentil flour having WHC values of 67.6, 70.5, 74.8, and
76.5%, respectively. Similar trends were reported for acid + alkaline +
enzyme and alkaline + enzyme flour disruption treatment across
samples. Table I also shows an increase in WHC with the increase
in fractions of legume flour (i.e., whether disrupted or not), although
to a lower extent than when using disrupted legume flours (except
when using enzyme D). Results are in agreement with the findings of
Chaikham and Apichartsrangkoon (2012), Xu et al. (2007), and Xue
and Ngadi (2007) that viscoelastic properties of gluten are affected
by the structural properties of the gliadin and glutenin subfractions
and the interactions between them. The observed nonlinear shear
viscoelastic property of gliadin and glutenin was also indicated to be
dependent on their concentration.
These findings provide support that changes in WHC in fact
could be attributed to the increased percentage of legumes in the
substituted samples as well as to the disruption in legume flours.
These results were also agreement with those of Kohajdova et al.
(2011), Thushan Sanjeewa et al. (2010), Modi et al. (2004), Dzudie
et al. (2002), and Elhardallou and Walker (1993), in which legumes
were reported to be added to various food products including meats
to increase the WHC and yield as well as decrease cooking losses.
The resulting viscosity data were fitted to the Herschel–Bulkley
model as described in equation 2. Flow behavior index (n) and
consistency coefficient (m) of batter substituted with various ratios
of disrupted legume flours are presented in Tables II and III, re-
spectively. Flow behavior index (n) for batter treatments was >1,
indicating best fit of batter dough with the Herschel–Bulkley model
having a correlation of determination (R2) range from 0.87 to 0.99,
indicating the suitability of the fitted model to describe batter vis-
coelastic properties. ANOVA analyses indicated a significant de-
crease in flow behavior index and an overall increase in consistency
coefficient with the disruption treatments across treatments when
compared with the nondisrupted flour or the wheat control (Tables II
and III). For all chemical and enzymatic treatments, flow behavior
index (n) varied from 1.00 to 1.36, whereas the consistency co-
efficient (m) ranged from 0.04 to 0.26 mPa·sn. Flow behavior index
and consistency coefficient of wheat flour were n = 2.15 and m =
0.008 mPa·sn, respectively. These results are in accordance with
WHC results presented in Table I, with legumes having greater
WHC compared with wheat flour. Our results are in agreement with
those of Changala et al. (1989) that lowering WHC decreased the
consistency coefficient and increased flow behavior index for both
native and fermented black gram flour dispersions. Batter visco-
elastic behavior is a function of several variables including its
chemical composition, water availability, and temperature. For in-
stance, free water was reported to play a critical role in the visco-
elastic properties because starch granules are usually not soluble in
cold water; thus, higher resistance is normally caused by low water
availability (Marco et al. 2007; Ketjarut et al. 2010). Similarly,
results were in accordance with the findings of Marco and Rosell
(2008), who reported a decrease in viscoelastic properties of batter
as a result of increasing water absorption, produced by the addition
of protein isolate. Our results further suggested that the batters con-
taining disrupted legume flours exhibited more pronounced New-
tonian behavior because flow behavior indexes of treated samples
were more closely approaching an n value of 1.

Vol. 92, No. 2, 2015 167

 TABLE V
Pasting Viscosities (cP) of Wheat Flour Substituted with Various Percentages (5, 10, 15, and 20%)
of Chemically and Enzymatically Disrupted Lentil Floursy
Peak Viscosity Trough
Enzymatic
Chemical Treatment Treatmentz 5% 10% 15% 20% 5% 10% 15% 20%
None None 1,754.0cA 1,654.3bB 1,604.0cC 1,486.0cD 1,145.3bA 1,092.0bB 1,055.7bC 946.0bD
Acid A 1,679.3dA 1,617.7cdB 1,513.7eD 1,519.7bC 985.7eA 911.0eB 851.0fC 789.7dD
Acid B 1,805.0bA 1,639.7bcB 1,586.7dC 1,513.7bD 1,045.3dA 1,016.3cB 977.0eC 930.7bD
Acid C 1,728.7cA 1,610.3dB 1,425.0fC 1,358.0dD 1,005.3eA 953.7dB 891.0gC 851.7cD
Acid D 1,651.7dA 1,656.0bB 1,595.7dC 1,526.0bD 1,067.3dA 1,072.7bB 1,016.7dC 977.0bD
Acid None ND ND 1,756.5b ND ND ND 1,251.5c ND
Flour control 1,873.2aA 1,873.2aA 1,873.2aA 1,873.2aA 1,520.3aA 1,520.3aA 1,520.3aA 1,520.3aA
None None 1,754.0cA 1,654.3cB 1,604.0cC 1,486.0cD 1,145.3bA 1,092.0bB 1,055.6cC 946.0cD
Acid + alkaline A 1,604.7dA 1,554.7dB 1,490.3dC 1,407.0dD 1,033.0dA 1,003.3cB 967.0eC 864.0dD
Acid + alkaline B 1,567.3eA 1,465.0eB 1,379.0eC 1,305.3eD 1,061.0cdA 1,060.0bA 1,031.3dC 993.7bD
Acid + alkaline C 1,508.3fA 1,399.7fB 1,305.0fC 1,248.7fD 1,063.7cA 1,008.7cB 944.7eC 926.7cD
Acid + alkaline D 1,799.7bA 1,758.7bB 1,689.0bC 1,629.7bD 1,072.3cA 1,085.7bB 1,056.7cC 1,027.3bD
Acid + alkaline None ND ND 1,702.3b ND ND ND 1,202.3b ND
Flour control 1,873.2aA 1,873.2aA 1,873.2aA 1,873.2aA 1,520.3aA 1,520.3aA 1,520.3aA 1,520.3aA
None None 1,754.0bA 1,654.3bB 1,604.0cC 1,486.0bD 1,145.3bA 1,092.0bB 1,055.6cC 946.0bD
Alkaline A 1,635.0cA 1,555.3cB 1,472.3eC 1,370.3dD 1,025.7dA 975.0cB 920.0eC 853.0cD
Alkaline B 1,514.0dA 1,393.0dB 1,304.3fC 1,238.0fD 992.3eA 927.3dB 876.7fC 853.3cD
Alkaline C 1,537.3dA 1,399.3dB 1,310.7fC 1,266.7eD 1,020.0dA 951.0cdB 896.7efC 875.3cD
Alkaline D 1,735.3bA 1,650.0bB 1,513.7dC 1,442.0cD 1,119.7cA 1,078.0bB 1,008.3dC 975.3bD
Alkaline None ND ND 1,825.5b ND ND ND 1,452.2b ND
Flour control 1,873.2aA 1,873.2aA 1,873.2aA 1,873.2aA 1,520.3aA 1,520.3aA 1,520.3aA 1,520.3aA
(continued on next page)
y Means of pasting properties (peak, trough, breakdown, final, and setback) of batter flour having the same chemical treatment and fraction replacement of
disrupted, nondisrupted lentil flour, and wheat flour with different enzymatic treatments (column) with different lowercase letters are significantly (P < 0.05)
different according to least squares differences (LSD). Means of pasting properties of similar disruption treatment and various fraction replacement (same row)
with different capital letters are significantly (P < 0.05) different according to LSD. cP = centipoise.
z A = protease enzymes from Rhizopus spp. (EC 3.4.23.21); B = papaya (EC 3.4.22.2); C = Aspergillus mellus (EC 232.642.4); and D = Aspergillus saitoi (EC
3.4.23.18).

Tables IV and V present the pasting properties of batter flour
treatments prepared with fractions (i.e., 5, 10, 15, and 20%) of
sequential acid + enzymatic, acid + alkaline + enzymatic, and al-
kaline + enzymatic treated flours as well as fractions of nontreated
chickpea and lentil flours. Results demonstrated a decrease in values
of pasting properties with the increase in percentages of disrupted
chickpea or lentil flours. For example, samples substituted with 5,
10, 15, and 20% chickpea flour disrupted with acid and enzyme C
had peak viscosities of 1,547.0, 1,429.3, 1,381.3, and 1,338.3 cP,
respectively. Lentil samples having similar treatment and fraction
replacement had peak viscosity values of 1,728.7, 1,610.3, 1,425.0,
and 1,358.0 cP, respectively. Similar trends were reported for acid +
alkaline + enzyme and alkaline + enzyme treatment across treat-
ments and trough, breakdown, and final viscosity. Results suggest
a limited role of disrupted legume flours in providing protection to
the starch granule configuration, resulting in decreased pasting
viscosities, except for gel retrogradation (i.e., setback) of legume-
substituted samples. Moreover, fractional substitution of legumes,
whether treated or nontreated, appears to influence protein–starch
interaction during pasting. This result might be because during
paste formation, molecular proteins and lipids were reported to
promote the formation of an insoluble polymeric matrix conferring
rigidity to the starch granules and also to provide protection to the
starch granule integrity (Tester and Morrison 1990). Hamaker and
Griffin (1990) and Marshall et al. (1990) also indicated that the
structure of proteins plays a key role in affecting cereal functional
properties. The decrease in pasting properties was attributed to the
lack of ability for protein to form the appropriate bonding necessary
to protect swollen starch granule integrity from rupture and to the
decreased contribution of total starch available for swelling (Saleh
and Meullenet 2007). Our results are in agreement those of Wurz-
burg (1987), who reported that faster swelling of starch granules at
lower temperature could result in low pasting properties. The in-
crease in retrogradation of the enzymatically treated samples was
attributed to modifications of legume proteins providing support to
the rearranged starch granules during the cooling step of the pasting
measurements.
Pasting properties results also showed that for the same chemical
treatment and fraction replaced, various enzymes significantly (P <
0.05) varied in their effect on the changing batter flour pasting
properties. Additionally, for similar chickpea sample replacements,
enzymes A and C had significantly (P < 0.05) lower pasting vis-
cosities than the control nondisrupted chickpea flour, whereas
enzymes B and D had significantly (P < 0.05) higher pasting vis-
cosities. For instance, a 15% replacement of nontreated chickpea
had a peak viscosity of 1,491.0 cP compared with 1,495.0 and
1,381.3 cP for acid + enzymes A and C and with 1,557.7 and
1,694.0 cP for acid + enzymes B and D, respectively. For lentil
treated samples, enzymatic-treated samples had significantly (P <
0.05) lower pasting viscosities compared with nondisrupted lentil
flour samples. These results clearly demonstrated the variability of
enzymatic treatment in modifying the pasting profile of treated
legumes, a result that was demonstrated by the varying effects of
disruption treatments on chickpea and lentil proteins (Fig. 1).
CONCLUSIONS
This study investigated the effects of substituting wheat flour with
fractions of disrupted chickpea and lentil flours on the rheological
properties of batter systems. Sequential acidic, alkaline, and
enzymatic treatments were performed on chickpea and lentil flours.
Flow curves of batters containing flour blends of substituted legumes
at different mixing ratios and disruption treatments showed
shear-thinning behavior. The viscosity values of the batters de-
creased with increasing disrupted legume content. The addition
of disrupted legumes significantly decreased batter flour vis-
cosity and pasting properties and increased WHC of wheat-based
batter. These changes in batter viscoelastic properties were attributed

168 CEREAL CHEMISTRY

 TABLE V
(continued from previous page)
Breakdown Final Viscosity Setback
5% 10% 15% 20% 5% 10% 15% 20% 5% 10% 15% 20%
608.7aA 562.3bB 548.3bC 540.0aD 2,444.0bA 2,355.0cB 2,286.7cC 2,093.7cD 1,212.0dA 1,172.0dB 1,089.3dC 1,041.0dD
600.0bA 584.0aB 556.7bC 541.0aD 2,296.0dA 2,279.0dB 2,163.0dC 2,034.3dD 1,310.3cB 1,368.0cA 1,312.0cB 1,244.7cC
593.3bA 564.0bB 511.7cC 480.3bD 2,384.0cD 2,513.3bA 2,493.7bB 2,401.0bC 1,338.7bD 1,497.0bB 1,516.7bA 1,470.3bC
500.0cA 452.3dB 401.7dC 377.0dD 2,118.7eA 2,013.0eB 1,901.0fC 1,823.7fD 1,113.3dA 1,059.3dB 1,010.0eC 972.0eD
605.7aA 540.3cB 510.0cC 447.7cD 2,615.3aB 2,703.0aA 2,585.7aC 2,472.3aC 1,548.0aC 1,630.3aA 1,569.0aB 1,495.3aD
ND ND 1,251.5a ND ND ND 1,854.6g ND ND ND 603.1f ND
352.9dA 352.9eA 352.9eA 352.9eA 1,950.5eA 1,950.5fA 1,950.5fA 1,950.5eA 430.2eA 430.2eA 430.2eA 430.2gA
608.7cA 562.3bB 548.3bC 540.0bC 2,444.0aA 2,355.0cB 2,286.7dC 2,093.7dD 1,212.0dA 1,172.0dB 1,089.3dC 1,041.0eD
646.3bA 614.3aB 546.7bcC 655.7aA 2,391.7bA 2,408.7bB 2,355.0cC 2,245.0cD 1,358.7aA 1,405.3aB 1,388.0bC 1,381.0bC
744.0aA 579.7bB 555.3bC 520.0bD 2,425.3abA 2,465.7aB 2,418.0bC 2,314.3bD 1,364.3aA 1,405.7aB 1,386.7bC 1,320.7cD
665.0bA 601.7aB 480.3dC 431.3dD 2,348.0cA 2,260.0cB 2,113.0eC 2,057.0dD 1,284.3bA 1,251.3cB 1,168.3cC 1,130.3dD
579.3dA 570.3bB 539.0cC 498.7cD 2,330.0cA 2,463.0aB 2,489.3aC 2,468.0aD 1,257.7cA 1,377.3bC 1,432.7aB 1,440.7aB
ND ND 1,202.3a ND ND ND 1,802.6g ND ND ND 600.3e ND
352.9eA 352.9eA 352.9cA 352.9eA 1,950.5dA 1,950.5dA 1,950.5dA 1,950.5fA 430.2eA 430.2eA 430.2eA 430.2fA
608.7aA 562.3aB 548.3bC 540.0aC 2,444.0bA 2,355.0cB 2,286.7cC 2,093.7cD 1,212.0cA 1,172.0cB 1,089.3cC 1,041.0cD
609.3aA 580.3aB 552.3bC 517.3aD 2,410.3cA 2,452.0bB 2,384.0bC 2,251.0bD 1,384.7bA 1,477.0bB 1,464.0bB 1,398.0bD
521.7bA 465.7bB 427.7dC 384.7cD 2,118.7eA 2,015.7dB 1,929.7deC 1,798.0fD 1,126.3dA 1,088.3dB 1,053.0dC 944.7eD
517.3bA 448.3bB 414.0dC 391.3cD 2,169.3dA 2,033.0dB 1,943.0deC 1,893.0eD 1,149.3dA 1,082.0dB 1,046.3dC 1,017.7dD
615.7aA 572.0aB 505.3cC 466.7bD 2,820.0aA 2,758.3aB 2,591.0aC 2,471.7aD 1,700.3aA 1,680.3aB 1,582.7aC 1,496.3aD
ND ND 1,452.2a ND ND ND 1,925.5e ND ND ND 473.3e ND
352.9cA 352.9cA 352.9cA 352.9eA 1,950.5eA 1,950.5fA 1,950.5eA 1,950.5dA 430.2eA 430.2eA 430.2eA 430.2fA

to the increase in the percentage of soluble proteins and to the in-
crease in WHC.
This study can provide important information for the potential
use of legumes to enhance the rheological properties of coating
applications including adhesion and WHC of legume-substituted
batter.
ACKNOWLEDGMENTS
The authors thank the Deanship of Academic Research at the University
of Jordan, Amman, Jordan, for supporting this research (project number
1496). The authors also thank Miss Dania Ehsan Al-Alaf for the kind help in
data collection.
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