Food Hydrocolloids 41 (2014) 178e187

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Functional protein from cumin seed (Cuminum cyminum):

Optimization and characterization studies
Hwee-Leng Siow, Chee-Yuen Gan*
Centre for Advanced Analytical Toxicology Services, Universiti Sains Malaysia, 11800 USM, Penang, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: Cumin seed protein isolate (CSPI) was found to be a potential source of bioactive protein. Box-Behnken
Received 19 December 2013 Design was used in order to optimize the protein extraction parameters: extraction time (X1: 0.5e1.5 h),
Accepted 6 April 2014 extraction temperature (X2: 20e40
C) and buffer-to-sample ratio (X3: 10e30 mL/g). Extraction condi-
Available online 18 April 2014
tions for maximum protein yield were corresponding to X1 ¼ 0.6 h, X2 ¼ 26.3
C and X3 ¼ 10 mL/g. A close
agreement between experimental (44.98 mg/g) and predicted (45.19 mg/g) values was found. The
Keywords:
physicochemical properties (i.e. amino acid composition, protein components, protein structure) and its
Bioactive protein
potential bioactivities (i.e. antioxidant and antidiabetic activity) were also evaluated, giving a better
Characterization
Cumin seed
understanding of general structure and properties of CSPI. 2S albumin, 7S globulin, 11S globulin and
Extraction lectin were found as the components of the extracted seed protein. Structure of CSPI was a mixture of
Optimization intramolecular b-sheet (1639 cm1), random coil (1642 cm1), a-helix (1655 cm1), b-turns (1660 cm1)
and antiparallel b-sheet aggregates (1690 cm1) as shown in the FTIR spectra. CSPI was predominant
with the Tyr, Glu, Asp, Arg, Leu and Phe, which could be considered as a high quality of natural protein. In
addition, CSPI showed appreciable DPPH free radical scavenging activity (47.7 %DPPHsc/mg) and reducing
power (12.4 mM/mg), which implied that CSPI could be used as a natural antioxidant agent. However,
CSPI showed a relatively low a-amylase inhibition activity (6.7%). These findings demonstrated that CSPI
could be a used as a potential nutraceutical or ingredient of functional and health-promoting foods.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction which is widely used as a condiment in the food preparation

The interest of natural plant-derived proteins is expected to
grow due to the growing of the consumers’ concerns over the food
safety as well as the increasing cost of food products derived from
animal. In recent years, the increasing demand of continuous
improvement regarding the nutritional and functional properties of
protein has also contributed to the acceleration of the research on
unconventional plant-derived protein (Henry & Kettlewell, 1996).
Seeds dietary proteins were reported as an important bioactive
protein with significant pharmacology and medicinal values
(Duranti, Consonni, Magni, Sessa, & Scarafoni, 2008).
Cumin (Cuminum cyminum), an annual plant of the family
Apiaceae, is one of the traditional medicinal herbs or spices in Asia,
Africa and Europe. The seeds have an elongated and oval shape
which is similar to fennel seeds. They have an aromatic odor and
give a spicy and bitter taste. This makes them as a flavoring agent,
* Corresponding author. Tel.: þ60 4 653 4261; fax: þ60 4 653 4688.
E-mail addresses: cygan@usm.my, mattgan81@gmail.com, mattgan81@yahoo.
com (C.-Y. Gan).
(Allahghadri et al., 2010). The therapeutic properties and physio-
logical effects of aqueous extract, oil or inorganic compounds iso-
lated from cumin seeds have been extensively studied, including
antimicrobial (Allahghadri et al., 2010), anti-carcinogenic
(Gagandepp, Dhanalakshmi, Mendiz, Rao, & Kale, 2003), anti-
diabetic (Dhandapani, Subramanian, Rajagopal, & Namasivayam,
2002) and antioxidant (Gagandepp et al., 2003). However, limited
information was found on the relative physicochemical and func-
tional attribute of cumin seed protein isolate (CSPI). The properties
of proteins are mainly contributed to their application and func-
tionalities (Amza, Amadou, Zhu, & Zhou, 2011; Yu, Ahmedna, &
Goktepe, 2007). In this aspect, the study of the physicochemical
and properties of the isolated protein (e.g. amino acid composition,
molecular weights (MW), protein structure and its potential bio-
activities) are required, to provide a better understanding of its
characteristics and potential applications in nutraceutical and food
industries. To date, there are no relevant study concerning on the
preparation and characterization of isolated protein from cumin
seed. The aims of the present study were to optimize the extraction
conditions using response surface methodology (RSM) in order to

http://dx.doi.org/10.1016/j.foodhyd.2014.04.017
0268-005X/Ó 2014 Elsevier Ltd. All rights reserved.
 H.-L. Siow, C.-Y. Gan / Food Hydrocolloids 41 (2014) 178e187 179

obtain a maximum yield of cumin seed protein isolate (CSPI), to
identify the protein components of CSPI, to determine the amino
acid compositions of CSPI, to investigate the protein structure of
CSPI using FTIR as well as to evaluate its potential as antioxidant
and antidiabetic agent.
2. Materials and methods
2.1. Materials
Cumin seeds were purchased from local market, Penang. All
other chemicals and reagent used in the experiment were of
analytical grade and purchased from SigmaeAldrich (Kuala Lum-
pur, Malaysia) company or otherwise mentioned.
2.2. Preparation of defatted cumin seed powder
The seeds were first ground into powder and sieved (30 mesh
size). The samples were then defatted using hexane with a sample-
to-solvent ratio of 1:10. After 3 h of incubation, the samples were
centrifuged at room temperature for 30 min with a constant speed
of 4500 rpm. Subsequently, the supernatant was removed and the
defatting procedure was repeated again with a sample-to-ratio of
1:5 and incubated with 1 h. The resulting flour was then air-dried
and stored at 4
C prior to extraction.
2.3. Extraction of cumin seed protein isolate
2.5. Experimental design
Three extraction parameters (i.e. extraction time (X1), incubator
temperature (X2) and buffer-to-sample ratio (X3)) were optimized
using response surface methodology (RSM). A Box-Behnken design
(BBD) was used in this regard. As shown in Table 1, the designated
parameters were selected on the basis of single factor experiment
results, using steepest ascent techniques.
A total of seventeen experimental designs (i.e. twelve factorial
points and five central points) were carried out (Table 1). Runs at
the central point of design were applied to estimate the possible
pure error. Also, the protein yield was used as the response variable
corresponding to the combination of the independent variables.
Three experimental replicates of each condition were performed
and the mean values were stated as experimental responses.
Experimental runs were randomized to minimize the effects of
unexpected variability in the observed responses.
The variables were coded according to the equation:
x ¼ ðXi  X0 Þ=DX (1)
where x is the coded value, Xi was the corresponding actual value,
X0 was the actual value in the centre of the domain, and DX is the
increment of Xi corresponding to a variation of 1 unit of x.
The mathematical model corresponding to the design was:
X
3 X
3 X
2 X
3
Y ¼ bo þ bi Xi þ bii Xi2 þ bij Xi Xj (2)
i¼1 i¼1 i ¼ 1 j ¼ 1þ1

The CSPI was extracted according to the method of Adebowale,
Adeyemi, Oshodi, and Niranjan (2007) with slight modifications.
The defatted cumin seed powder was suspended in phosphate
buffer solution (pH 8) and subsequently incubated at designated
time, temperature and buffer-to-sample ratio with constant agita-
tion at 200 rpm. The resulting slurry was then centrifuged at 5000 g
for 30 min after incubation. The supernatant was collected and the
extracted protein content of the sample was determined using
Bradford assay (Bradford, 1976). The extraction yield was expressed
as mg protein per gram of sample.
where Y was the dependent variables (i.e. extraction yield), bo was
the model constant, and bi, bii, and bij were the model coefficients.
They represented the linear, quadratic and interaction effects of the
variables, respectively. Analysis of the experimental design data
and calculation of predicted responses were carried out using
Design Expert software (version 6.0, USA). Additional confirmation
experiments were subsequently conducted to verify the validity of
the experimental model.
2.6. Characterization of CSPI

2.4. Single factor experiment
The effect of the extraction parameters (i.e. extraction time,
extraction temperature and buffer-to-sample ratio) was studied
under different combinations of extraction conditions as follows:
CSPI was prepared according to section 2.3 at the optimized
condition and the protein was precipitated by adjusting the pH to
4.5. Subsequently, the precipitate was collected after centrifugation
at 4000 g for 15 min at 4
C, lyophilized and ground into powder
prior to analyses.

2.6.1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis

2.4.1. Effect of extraction time
(SDS-PAGE)
The extraction of CSPI was carried out at the varying extraction
SDS-PAGE analysis of the CSPI was performed using 15%
time (0.5, 1, 1.5, 2, 2.5, 3, 3.5 h), while the extraction temperature
resolving gel and 4% stacking gel. Reducing and non-reducing
and buffer-to-sample ratio were fixed at 40
C and 30 mL/g,
electrophoresis experiments were conducted. For reducing SDS-
respectively.
PAGE analysis, samples (10 mg of protein) were added with 10 mL
of Laemmli sample buffer and 1 mL of 2-mercaptoethanol whereas
2.4.2. Effect of extraction temperature sample was only added with Laemmli sample buffer in non-
The extraction of CSPI was carried out at the varying extraction reducing condition. The samples were then heated at 95
C for
temperature (20
C, 30
C, 40
C, 50
C, 60
C) while the extraction
time and buffer-to-sample ratio were fixed at 2 h and 30 mL/g,
Table 1
respectively.
Experimental domain of Box-Behnken designs.

Xj Uncoded Coded Factor levels

2.4.3. Effect of buffer-to-sample ratio
1 0 1
The extraction of CSPI was carried out at the varying buffer-to-


sample ratio (10 mL/g, 20 mL/g, 30 mL/g, 40 mL/g, 50 mL/g) Temperature ( C) X1 x1 20 30 40
while the extraction time and extraction temperature were fixed at Time (h) X2 x2 0.5 1 1.5
Buffer to solid ratio (ml/g) X3 x3 10 20 30
2 h and 40
C, respectively.
180 H.-L. Siow, C.-Y. Gan / Food Hydrocolloids 41 (2014) 178e187
5 min using heating block (Grant, Fisher Scientific, UK). Samples
were loaded into the well and ran the electrophoresis at a constant
voltage of 80 V for 10 min, followed by 120 V for 120 min using Mini
Protean III Cell (Bio-Rad, USA). The gel was then stained for 2 h
using staining solution containing 0.1% Bio-Rad Coomassie Brilliant
Blue R-250, 40% (v/v) methanol and 10% acetic acid. Then the gel
was destained with solution containing 40% methanol and 10%
acetic acid. The image was subsequently captured using Fujifilm
luminescent image analyzer LAS-3000 (Fujifilm, Tokyo, Japan).
Multi Gauge Version 3.0 software (Fujifilm, Tokyo, Japan) was used
to analyze the electrophoresis pattern. The standard protein marker
(broad range MW of 6.0e202.4, Bio-Rad, USA) which contained
myosin (202.4 kDa), b-galactosidase (114.8 kDa), bovine serum al-
bumin (73.1 kDa), ovalbumin (47.9 kDa), carbonic anhydrase
(34.1 kDa), soybean trypsin inhibitor (27.0 kDa), lysozyme
(17.0 kDa) and aprotinin (6.0 kDa), was used to plot the standard
curve (molecular mass versus relative mobility).
2.6.2. Amino acid analysis
Sample (0.1 g) was heated at 110
C for 24 h after adding with
5 mL of 6 N HCl. The samples were then added with 400 mL of
50 mmol/mL of L-a-amino-n-butyric acid (AABA, internal standard)
and filtered through Whatman No. 1 filter paper following by
0.22 mm Millipore filters before the amino acid analysis, as
described by Zarkadas et al., 2007. Amino acid derivation and
analysis were conducted according to a standardized sequence: A
10 mL of samples were added with 20 mL of AccQ$FluorÔ reagent
(AQC: 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate). The
reaction was stood for 1 min at room temperature. Samples were
subsequently transferred to 150 mL glass low volume insert with
polyspring which was placed in the screw neck vial with cap. After
heating at 55
C for 10 min, samples (10 mL) were injected into the
column and elution was started with a flow rate at 1 mL/min.
Amino acid analysis was carried out using a Waters-HPLC-
System (USA) with Waters 1525 Binary Pump, Waters 717 plus
Autosampler, Waters 24675 Multi -l Fluorescence Detector and a
Waters AccQ$TagÔ Amino Acid Analysis Column
(3.9 mm  150 mm; packing material: silica based bonded with C18)
according to the AccQ$TagÔ method. Fluorescence was measured at
250 nm for excitation and 395 nm for emission. Breeze Workstation
version 3.20 was used to control the apparatus and solvent mixing as
well as plotting and evaluating. AccQ$TagÔ Eluent A and Acetoni-
trile/Water (60%/40%) were the two eluents used in this analysis. The
HPLC-system was calibrated using amino acids standard H (PIERCE,
USA) as reference. The amino acid composition was reported as per
1000 amino acid residues, which defined as molecules in 1000
molecules of individual amino acid. Each analysis was performed in
three replicates. Methionine and cysteine were determined sepa-
rately according to the performic acid procedure of Moore (1963).
2.6.3. Fourier transform infrared spectroscopy (FTIR)
CSPI lyophilized powder was desiccated prior to FTIR analysis.
FTIR spectra of the samples were recorded using Cary 600 Series
FTIR spectrometer with attenuated total reflectance (ATR) system
(Agilent Technologies, U.S.) from 650 to 4000 cm1. The measuring
resolution was 4 cm1 and iterations were performed for 32 times.
The scan speed was set at 5 kHz and sensitivity of 1. The resultant
spectra were analyzed using Resolution Pro version 5.2.0 software.
Deconvolution was performed with a half-bandwidth of 20 cm1
and an enhancement factor of 2.0.
2.6.4. Antioxidant activity of CSPI
2.6.4.1. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging
assay. The antioxidant activity of CSPI was determined using the
DPPH free radical scavenging assay, as described by Lee et al.
(2009). Prior to analysis, the samples were pre-diluted at a factor
of 2 and DPPH stock solution (0.1 mM) in ethanol was prepared. A
500 mL DPPH stock solution was added to 16.65 mL sample and
incubated at 30
C for 30 min in the dark. After incubation, the
sample was centrifuged at 14,000 g for 5 min. The absorbance of the
sample was then measured at 517 nm using a spectrophotometer
(Spectramax M5, Molecular Devices, USA). A blank sample
(16.65 mL of methanol and 500 mL DDPH solution) was prepared as
mentioned above. Gallic acid was used as a positive control in this
study. The antioxidant activity was expressed as percentage of
DPPH free radical scavenging activity (%DPPHsc/mg) and calculated
using formula:
h  i.
%DPPHsc =mg ¼ Ablank  Asample  100 ðAblank  mÞ (3)
where Asample is the absorbance of sample at t ¼ 30 min, Ablank is the
absorbance of blank sample and m is the weight of sample (mg).
2.6.4.2. Ferric reducing antioxidant power (FRAP) assay. The
reducing abilities of samples towards ferric ions were determined
according to the method of Benzie and Strain (1996). The working
FRAP reagent contained 10 mM TPTZ solution in 40 mM HCl, 20 mM
FeCl3$6H20 and 0.3 M acetate buffer at pH 3 at a ratio of 1:1:10. The
reagent was pre-warmed at 37
C and the samples were pre-diluted
at a factor of 2 prior to analysis. The pre-diluted sample (2.7 mL) was
then added to 200 mL of FRAP reagent and mixed thoroughly. The
sample was subsequently incubated at 37
C for 1 h and the
absorbance was measured at 593 nm using a spectrophotometer
(Spectramax M5, Molecular Devices, USA). Ferrous sulfate hepta-
hydrate (FeSO4$7H2O) at a concentration range of 0e2 mM was
prepared as standard. Gallic acid was used as a positive control in
this study. The ability of ferric reducing antioxidant potential was
expressed in mM FeSO4/mg.
2.7. a-Amylase inhibition assay
The a-amylase inhibition activity was determined according to a
modified version of an assay from the Worthington Enzyme
Manual (Worthington, Biochemical Corp., 1993). A total of 100 mL of
each sample extract and 100 mL of 0.02 M sodium phosphate buffer
(pH 6.9 with 0.006 M NaCl) containing a-amylase solution (0.5 mg/
mL) were incubated at 25
C for 10 min. A volume of 100 mL of 1%
starch solution in 0.02 M sodium phosphate buffer (pH 6.9 with
0.006 M NaCl) was then added to each tube. Subsequently, the
reaction mixtures were incubated at 25
C at 10 min. The reaction
was terminated by adding 200 mL of dinitrosalicylic acid color re-
agent, followed by incubation in a boiling water bath for 5 min and
cooled to room temperature. The reaction mixture was then added
with 3 mL of distilled water and the absorbance of the sample,
Control 1 or Control 2 was measured at 540 nm. The Control 1 was a
mixture of starch solution and sample without addition of enzyme
whereas Control 2 was a mixture of starch solution and enzyme
without addition sample. The a-amylase inhibitory activity was
calculated as follows:
 
AControl 2  AsampleAControl 1
% inhibition ¼  100 (4)
AControl 2
2.8. Statistical analysis
Statistical analysis was performed using SPSS for Windows,
Version 12.0 (SPSS Institute, Inc., Cary, NC). The means of results
were compared using one-way analysis of variance (one way
 H.-L. Siow, C.-Y. Gan / Food Hydrocolloids 41 (2014) 178e187 181

ANOVA) and significant level was of p  0.05. All the measurements Table 2
were performed in at least three replicates. BBD with the observed responses and predicted values for yield of protein (mg/g).

Std Run X1 X2 X3 Protein yield (mg/g)

3. Result and discussion Experimental Predicted

1 6 0.5 20 20 43.2  0.3 42.7

3.1. Single factor experiment 2 5 1.5 20 20 37.5  0.3 36.7
3 3 0.5 40 20 36.6  0.1 37.4
4 9 1.5 40 20 34.3  0.2 34.9
3.1.1. Effect of extraction time
5 8 0.5 30 10 45.5  0.6 44.6
As shown in Fig. 1a, the extracted protein content was signifi- 6 10 1.5 30 10 37.6  1.0 37.0
cantly (p < 0.05) increased for the first 1.5 h. Further extraction to 7 14 0.5 30 30 41.1  0.8 41.6
3 h did not show any significant (p > 0.05) changes in yield. When 8 13 1.5 30 30 39.9  0.1 40.7
the extraction was carried on for another 0.5 h, it could be observed 9 4 1 20 10 41.2  0.2 42.6
10 2 1 40 10 41.0  3.7 41.1
that there was a significant (P < 0.05) decrease. This result indi-
11 11 1 20 30 45.0  0.7 45.0
cated that a longer extraction time did not improve the protein 12 1 1 40 30 40.7  0.2 39.3
extraction efficiency. Hence, the extraction time within the range of 13 12 1 30 20 42.8  0.1 42.6
0.5e1.5 h was selected for further optimization study using steep- 14 7 1 30 20 42.3  0.5 42.6
15 17 1 30 20 41.6  0.1 42.6
est ascent techniques.
16 15 1 30 20 43.3  0.2 42.6
17 16 1 30 20 43.2  0.1 42.6
3.1.2. Effect of extraction temperature Data correspond to the mean  SD of three experiments.
Fig. 1b demonstrated that extraction temperature had signifi-
cant impact on the protein yield of CSPI. Protein yield was signifi-
cantly (p < 0.05) increased with the increasing of temperature up to reduced remarkably. This occurrence could be explained by the
30
C and the changes of protein yield were not significant increased intensity of thermal treatment (>50
C) which led to
(p < 0.05) thereafter. At 60
C, the protein yield was however protein denaturation and eventually decreased the protein solubi-
lity (Yu et al., 2007). In addition, phytic acid could also be eluted
from the seed at a higher temperature, thus encouraged the for-
mation of proteinephytic acid complexes in which decreased the
solubility of the protein (Elsheikh, Fadul, & El Tinay, 2000). This
finding is in agreement with previous studies which support the
fact that very low phytate content was released from the seed at
room temperature and shorter incubation time (Canan et al., 2011;
Deak & Johnson, 2007). Hence, the extraction temperature within
the range of 20e40
C was selected for further optimization study
using steepest ascent techniques.

3.1.3. Effect of buffer-to-sample ratio

Influence of the buffer-to-sample ratio is illustrated in Fig. 1c.
With the increase of buffer-to-sample ratio, the yield of protein was
proportionally increased. The ratio (10e30 mL/g) was selected
based on the steepest ascent technique because the main objective
of the study was to investigate the effect of each parameter
(including the buffer-to-sample ratio) in different responses. Other
parameters such as temperature and time could have a possible
interaction with the ratio. It was expected to observe that at lower
range of ratio, this effect could be observed. Also, from the
perspective of industry, lower ratio would bring benefit such as cost
saving. If 30e50 mL/g was chosen, there will not have any effect on
the response and it defeated the purpose of studying/optimizing
the effect this factor. Therefore, we used the steepest ascent range
(10e30 mL/g) in the optimization study.

3.2. Response surface methodology

The effects of three extraction parameters: time (X1), tempera-

Fig. 1. Single factor experiment: (a) effect of time on the yield of CSPI; (b) effect of
temperature on the yield of CSPI; (c) effect of buffer-to-sample ratio on the yield of CSPI.
ture (X2) and buffer-to-sample ratio (X3), on the protein yield of CS
were studied using a BBD design. The range of each factor is
selected using steepest ascent techniques on the basis of single
factor experiment. Protein yield (which was expressed as mg/g)
was selected as the response of interest. Table 2 shows the exper-
imental matrix design in a total of 17 runs, along with the experi-
mental and predicted results obtained. Results revealed that there
was a close agreement between experimental and predicted values.
The experimental protein yield was ranged from 34.5 to 45.5 mg/g
under different tested conditions. The protein yield seemed to be
182 H.-L. Siow, C.-Y. Gan / Food Hydrocolloids 41 (2014) 178e187

varied depending on the extraction conditions given. It could be 3.2.2. Interpretation of response surface model
observed that the maximum protein yield (45.5 mg/g) was obtained To visualize the effect of the independent variables on the
under the experimental conditions of X1 ¼ 1.5 h, X2 ¼ 30
C, and response of interest, three-dimensional (3-D) surface response and
X3 ¼ 10 mL/g. contour plots for protein yield as a function of extraction time and
temperature at varying buffer-to-sample ratio were illustrated in
3.2.1. Model fitting Fig. 2.
Analysis of variance (ANOVA) was performed to evaluate the It can be observed that the extraction temperature displayed a
significance of the coefficient of the models. As presented in Table 3, quadratic effect on the protein yield regardless of buffer-to-sample
ANOVA showed that the contribution of quadratic model was sig- ratio (Fig. 2). At buffer-to-sample ratio of 10 mL/g, the protein yield
nificant (p < 0.05). The fitted quadratic model for protein yield in was slightly increased with the increase of temperature from 20 to
coded variables is given in Eq. (5). The significance of each coeffi- 35
C, and then decreased thereafter (Fig. 2a). Fig. 2b and c dis-
cient was determined using the F-value and p-value. The corre- played a similar response surface pattern but the protein yield is
sponding variables would be more significant if the absolute F- started to decrease after the temperature reached 30
C. These
value becomes greater and the p-value becomes smaller (Atkinson noticeable effects reflect that the increase of temperature could
& Donev, 1992). Results revealed that the largest effect on the reduce the extraction efficiency. It was suggested that denaturation
protein yield was linear term of extraction time (X1) followed by of the protein might occur at higher temperature (>50
C). Apart
quadratic term of extraction time ðX1 2 Þ, linear term of extraction from that, higher content of phytic acid was eluted from the seed at
temperature (X2), quadratic term of extraction temperature ðX2 2 Þ this condition, thus interaction between the extracted protein and
and interaction term of X1X3. On the other hand, the linear and phytate occurred and resulted in precipitation (Elsheikh et al.,
quadratic terms of buffer-to-sample ratio (X3 and X3 2 ), as well as 2000). Phytate or phytic acids are natural constituents found in
the other interaction terms of X1X2 and X2X3 were not significant leguminous seeds, cereal grains and oil seeds which act as storage
(p > 0.05). It was suggested that the influence of the reaction time form of phosphorus (Alli & Baker, 1981). Results showed that higher
and temperature played the dominant roles in protein extraction protein yield was obtained in lower temperature of 25
C. This
while the buffer-to-sample ratio was insignificant within the finding is in agreement with previous studies which support the
experimental range. fact that very low phytate content was released from the seed when
protein extraction was performed at room temperature compared
Yield ¼ 42:63  2:13x1  1:79x2 þ 0:17x3  2:84x1 2  1:86x2 2 to higher extraction temperature (Deak & Johnson, 2007).
With the regard to the effect of time, its significant quadratic
 1:21x3 2 þ 0:85x1 x2 þ 1:68x1 x3 þ 1:04x2 x3
effect was shown along the incubation temperature (ranging from
(5) 20 to 40
C) as illustrated in Fig. 2. The protein yield was increased
The lack of fit test measures the failure of the model to represent for the first hour, however, the yield decreased when the incubation
the data in the experimental domain at points which were not time was extended until 1.5 h. Previous studies reported that the
included in the regression. The insignificant p-value of 0.074 prolonged extraction time was not conducive to increase protein
(p > 0.05) was shown in Table 3, indicating that the selected model yield (Eromosele, Arogundade, Eromosele, & Ademuyiwa, 2008). It
is fitted well and adequate to describe the observed data. The co- has been reported that the maximum extraction of phytic acid form
efficient of determination value (R2 ¼ 0.933) indicated that the rice bran was observed at extraction time of more than 1 h (Canan
fitted model can explain 93.3% of the variation in the data. Also, the et al., 2011). Therefore, the decrease in the protein yield with
value of adjusted determination coefficient ðR2 adj Þ was of 0.847, increasing time could be due to the formation of proteinephytic
indicating a high degree of correlation between the experimental acid complexes which make the protein less soluble (Elsheikh et al.,
and predicted values. Coefficient of variation (CV) represents the 2000). Also, it was possible to explain that denaturation of protein
ratio of standard deviation to the mean which shows the extent of would occur when protein was exposed to a high temperature for a
variability of the data. This model had a low CV of 2.87% suggesting long period of time. It can be concluded that the extraction of
a good precision and high reliability of the experiment performed. protein at room temperature with shorter extraction period (<1 h)
would produce higher yield of protein.
No significant (p > 0.05) changes in protein yield with the
Table 3 change of buffer-to-sample ratio. The range of protein yield was
ANOVA for quadratic model: estimated regression model of relationship between approximately between 35.04e42.18 mg/g, 34.85e42.18 mg/g and
response variable (yield) and independent variables (X1, X2, X3).
36.07e42.18 mg/g at buffer-to-sample ratio of 10 mL/g, 20 mL/g and
Source Sum of squares DF Mean square F value Prob > F 30 mL/g, respectively. Therefore, it is suggested that buffer-to-
Model 135.762 9 15.085 10.875 0.002 sample ratio of 10 mL/g is sufficient for CSPI extraction with the
X1 36.405 1 36.405 26.246 0.001 intention of waste minimization and cost reduction.
X2 25.615 1 25.615 18.467 0.004
X3 0.224 1 0.224 0.161 0.700
3.2.3. Verification or predictive models
X1 2 33.998 1 33.998 24.510 0.002
X2 2 14.619 1 14.619 10.539 0.014 Verification of the model was performed in order to validate its
X3 2 6.175 1 6.175 4.452 0.073 suitability in the experiment. A combination of extraction param-
X1X2 2.904 1 2.904 2.094 0.191 eters was suggested to optimize the protein yield taking into ac-
X1X3 11.329 1 11.329 8.167 0.024 count of the efficiency, the energy conservation and the feasibility
X2X3 4.361 1 4.361 3.144 0.120
Residual 9.710 7 1.387
of the experiment. With the desirability value of 0.972, the optimal
Lack of fit 7.715 3 2.572 5.157 0.074 conditions for the predicted maximum protein yield of w45.19 mL/
Pure error 1.995 4 0.499 g were corresponded to extraction temperature of 26.3
C, extrac-
Cor total 145.471 16 tion time of 0.6 h, and buffer-to-sample ratio of 10 mL/g. The
R-squared 0.933 experimental value of 44.98 mL/g was found closed to the predicted
Adj R-squared 0.847 value (45.19 mL/g) with only a small deviation. Hence, the model is
C.V. 2.870 valid to be used in optimizing the process of protein extraction from
Data correspond to the mean  SD of three experiments. cumin seed.
 H.-L. Siow, C.-Y. Gan / Food Hydrocolloids 41 (2014) 178e187 183

Fig. 2. Response surfaces: (i) three-dimensional plots and (ii) contour plots for extraction yield as a function of extraction temperature and time at buffer-to-sample ratio of (a)
10 mL/g, (b) 20 mL/g and (c) 30 mL/g.
184 H.-L. Siow, C.-Y. Gan / Food Hydrocolloids 41 (2014) 178e187

3.3. Characterization Table 4

Amino acid composition and the percentage of amino acid with different charac-
teristic in CSPI (per 1000 residues).
3.3.1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE) Amino acid Amount (per 1000 residues)
Separation of proteins based on their electrophoretic mobility Nonessential amino acid
was performed by means of SDS-PAGE. The electrophoresis pat- Asp 87.0  1.9
terns of CSPI under both reducing and non-reducing conditions Ser 48.6  1.3
Glu 134.2  3.7
were shown in Fig. 3. For non-reduced sample (lane 1), the MW of
Gly 44.2  1.2
protein bands is in the range of 10.4e184.1 kDa whereas the protein Arg 84.9  0.5
bands which were reduced were of 4.3e104.7 kDa after adding the Ala 36.7  0.4
reducing agent (lane 2). This indicates that the high MW of protein Pro 39.2  0.0
Cys 1.2  0.1
bands were dissociated into several polypeptides or monomers
Tyr 167.4  1.3
with respective MW. This could be supported by the observation of
several protein bands (184.1, 56.5, and 14.1 kDa) that were absent or Essential amino acid
Lys 34.6  0.9
not distinctly visible under reducing conditions.
Ile 51.7  0.2
According to Orrun  o and Morgan (2007), globulins are the major Leu 70.3  0.3
storage proteins which commonly found in seeds. Several promi- Phe 63.0  0.5
nent bands are likely corresponding to the acidic subunits (30e Met 19.7  0.6
33 kDa) and basic subunits (20e25 kDa) of 11S globulins under Thr 40.5  0.9
Val 48.9  0.2
reducing condition. Similar observations have been reported in His 27.9  1.2
sesame seeds and gingerbread plum seeds (Amza et al., 2011; Tai,
Percentage of amino acid with different characteristics
Wu, Chen, & Tzen, 1999). These occurrences indicated that
Acidica 221.0
dimeric structure is formed by interchain disulfide bonds within Basicb 147.4
the globulin protein structure. Hydrophobic c
329.5
Interestingly, the similar protein bands (w110.5 and Uncharged polard 301.9
w44.7 KDa) emerged for the CSPI treated with and without E/TN ratio 0.6
reducing agent. They were likely attributed to the presence of 7S Total essential amino acids 356.5
globulin, or vicilin. This could be explained by the absence of Total nonessential amino acids 643.5
disulphide bonds holding the polypeptides among 7S globulin in Data correspond to the mean  SD of three experiments.
a
CSPI. Previous studies reported that the subunits of 7S globulins Acidic: Aspartic acid, glutamic acid.
b
are hold by the non-covalent, weak secondary forces, such as Basic: Lysine, arginine, histidine.
c
Hydrophobic: Alanine, isoleucine, leucine, methionine, phenylalanine, proline,
hydrogen bonding, hydrophobic and electrostatic forces (Orrun o &
valine.
Morgan, 2007). d
Uncharged polar: Glycine, serine, threonine, tyrosine, cysteine.
Previous study showed that 2S albumin composed of poly-
peptides with sizes 8e16 kDa (Radovic, Maksimovic, Brkljacic,
Varkonji Gasic, & Savic, 1999). It is evident by the appearance of 3.3.2. Amino acid analysis
protein bands <17 kDa under both reducing and non-reducing Amino acid analysis was carried out in order to explore possible
condition. Addition of reducing agent has led to dissociation of correlations between amino acid composition and its biological
this protein to smaller MW (<10 kDa). Another major protein band activities. Based on the amino acid profile, Tyr and Glu were the
of 27.6 kDa was corresponding to lectins, also known as hemag- major constituent amino acids, at approximately 167.4 and 134.2
glutinins which possess sugar binding property and hemaggluti- per 1000 residues (Table 4). A relatively higher amount of Asp, Arg,
nating ability (Nasi, Picariello, & Ferranti, 2009). It was reported Leu and Phe is also presented in the result. On the contrary, the
that this component is considered as a toxin if injected into animals sulphur-containing amino acids (i.e. methionine and cysteine) were
in high doses. It could also result in negative consequences by found as the limiting amino acids.
triggering growth inhibition when incorporated in diet. However, it Glu and Asp were credited with strong antioxidant activity due
is possible to be reduced to a safe level by proper cooking (Liener, to the presence of excess electrons that can be donated during
1974). interaction with free radicals (Udenigwe & Aluko, 2012). The
presence of Tyr, Met, His and Lys have been reported to be
responsible for exhibiting strong antioxidant effects (Udenigwe &
Aluko, 2012). In addition, peptide sequences containing Glu and
Pro play a major role in exhibiting antidiabetic activity (Gault,
O’Harte, Harriott, & Flatt, 2002). In general, the antioxidant and
antidiabetic properties of CSPI could be attributed to the presence
of high functional amino acids content.
The amino acid characteristics profile (e.g., acidic, basic, hy-
drophobic and uncharged polar) of CSPI was also summarized in
Table 4. CSPI had highest amount of hydrophobic acid, followed by
uncharged polar and acidic amino acid while basic amino acid
shows the lowest amount. Result indicates that CSPI has a prom-
ising antioxidative potency due to hydrophobic properties of pep-
tides, which can improve their interaction with lipid target or
assessment of the peptides into target organs by means of hydro-
phobic association (Sarmadi & Ismail, 2010). Zuber (1988) reported
Fig. 3. SDS-PAGE profile of CSPI at non-reducing and reducing conditions. Lane M: that high content of hydrophobic acid led to a more compact
standard protein marker; lane 1: non-reduced CSPI; lane 2: reduced CSPI. interior core of protein, which accountable for a more stable
 H.-L. Siow, C.-Y. Gan / Food Hydrocolloids 41 (2014) 178e187
protein. The high level content of acidic amino acid is responsible
for the acidic characteristic of CSPI (Carbonaro, Cappelloni, Nicoli,
Lucarini, & Carnovale, 1997). In terms of essential amino acid, the
ratio of essential to total amino acid (E/TN) in CSPI was 0.6, which
had higher value than the minimum E/TN ratio (0.36) suggested by
FAO/WHO/UNU (WHO, 1985). In this respect, CSPI could be
considered as a high quality of natural protein.
3.3.3. Fourier transform infrared spectroscopy (FTIR)
Determination of the IR spectra of CSPI was performed to gain
insight into protein structure of CSPI as shown in Fig. 4a. The
spectrum of CSPI showed a broad peak in the region of 3100e
3500 cm1. It was likely corresponded to NeH stretching, which
was induced by flexural vibration frequencies of the intra- and
inter-molecular hydrogen bonds. Result showed that the distinct
peak (w3285 cm1) could be a useful indicative of a helical for-
mation that formed by the hydrogen bond between functional and
groups of C]O and NeH (Gupta et al., 2006).
In the region of 2850e2990 cm1, the bands absorptions rep-
resented the CH antisymmetric and symmetric stretching modes of
methyl (CH3) and methylene (CH2) that normally found in aliphatic
Fig. 4. (a) IR spectra of CSPI; (b) deconvoluted amide I region (1600e1700 cm1): (i) before deconvolution; (ii) after deconvolution.
185
side chain of proteins (Chen et al., 2013). It is therefore suggesting
that the peaks (2956, 2927 and 2855 cm1) found in the spectra
were corresponding to the stretching aforementioned.
Typical protein bands: amide I (1600e1700 cm1), amide II
(1500e1580 cm1) and amide III (1200e1400 cm1) were observed
in the IR spectrum of CSPI (Fig. 4a). They were corresponding to
specific stretching and bending vibrations of the protein backbone
(Byler & Suci, 1986). Amide I is predominantly arises from C]O
stretching while amide II is attributed to NeH bending vibrations
(60%) coupled to CeN stretching vibrations (40%). For amide III, it is
derived from a complex mix of NeH bending and CeN stretching
along with a minor contribution from CeO in plane bending and
the CeC stretching vibration.
The deconvolved amide I bands of CSPI were performed to
enhance the apparent resolution of overlapping peaks, which is
useful for more accurate determination of the number of peaks in a
region, the band positions as well as the areas under peaks (Fig. 4b).
Several peaks were assigned according to Byler and Suci (1986):
intramolecular b-sheet (1639 cm1) random coil (1642 cm1), a-
helix (1655 cm1), b-turns (1660 cm1) and antiparallel b-sheet
aggregates (1690 cm1). The band at 1605 cm1 was corresponded
186 H.-L. Siow, C.-Y. Gan / Food Hydrocolloids 41 (2014) 178e187
Table 5
Comparison of antioxidant and antidiabetic activity between CSPI, SPI and gallic
acid.
Sample Antioxidant activity Antidiabetic activity
DPPH (%DPPHsc/mg) FRAP (mM/mg) a-amylase inhibition (%)
CSPI 47.7  0.2 12.4  0.1 6.7  0.1
SPI a
ND ND ND
Gallic acid 246.8  0.1 12.0  0.1 Not determined
Data correspond to the mean  SD of three experiments.
a
ND means not detected.
to amino acid side-chain vibrations (Muga et al., 1990). In the amide
region II, a band located at 1570 cm1 was also observed. However,
they were predominantly resulting from complex vibration of
multiple functional groups, which is less useful for protein struc-
ture prediction compared with the amide I bands (Jackson &
Mantsch, 1995).
3.3.4. Antioxidant potential of CSPI
The antioxidant potential of CSPI was measured by using
DPPH and FRAP assay (Table 5). The former is based on the ef-
ficiency of antioxidant proteins in scavenging free radicals
generated by DPPH. The latter is determined based on the ability
of antioxidant protein to reduce ferric cyanide complex (Fe3þ) to
ferrous form (Fe2þ). The result was compared with the gallic acid
as a positive control (Table 5). It can be seen that CSPI gave a
lower scavenging ability (47.7 %DPPHsc/mg) than that of gallic
acid (246.8 %DPPHsc/mg). However, the ion metal reducing power
of CSPI (12.4 mM/mg) was found to be equally strong as gallic acid
(12 mM/mg). It was suggested that the strong antioxidant prop-
erties of protein isolate are likely attributed to the abundance of
functional amino acids found in CSPI. The present data was also
used to compare with the commercial soy protein isolate (SPI)
and the result showed that the antioxidant activity of SPI was not
detected. However, it was reported that antioxidative activity of
soy protein hydrolysate was found to have 50e60% radical
scavenging activity (Amadou et al., 2010), which was also lower
than that of CSPI. It was suggested that the extracted cumin
protein could have the potential to be used as a bioactive protein
in food application.
3.3.5. a-Amylase inhibition activity of CSPI
Regulating the glucose production from complex carbohydrates
is an effective indicator in controlling the diabetic. The a-amylase
is one of the main carbohydrate hydrolyzing enzymes in human,
which catalyzing the hydrolysis of 1,4-glucosidic linkage of com-
plex carbohydrates into simple sugars (Jumepaeng, Prachakool,
Luthria, & Chanthai, 2013). Thus, inhibition of this enzyme is
widely used in controlling the glucose level in diabetic patients. In
Table 5, CSPI showed a low inhibitory activity of a-amylase, at
approximately 6.7% inhibition. Conversely, the antidiabetic activity
of SPI was not detected. A recent study of Lu et al. (2012) however
reported a natural bioactive peptide derived from soybean had an
antidiabetic potential. This might be related to the inactive func-
tional amino acids which encrypted in the parent chain. However,
the protein was suggested to exhibit a higher activity after diges-
tion in the gastrointestinal system due to the release of the
bioactive peptide from the parental protein chain (Sarmadi &
Ismail, 2010). In addition, the inhibition activity of CSPI was re-
ported to be lower for bioactive peptide derived from barley pro-
tein isolate with anti-amylase activity of 57e77% (Alu’datt et al.,
2011). Future research will be conducted and examined the
possible roles of bioactive peptides derived from CSPI in inhibition
of a-amylase activity.
4. Conclusion
In summary, the study of single factors (i.e. time, temperature
and buffer-to-sample ratio) had a significant impact on the yield of
CSPI. Implementation of RSM was successfully optimized the yield
of CSPI. The model was also successfully verified and the most
efficient extraction conditions were corresponding to extraction
time of 0.6 h, extraction temperature of 26.3
C and buffer-to-
sample ratio of 10 mL/g. The physicochemical and properties of
CSPI showed that CSPI could be considered as a high quality of
natural protein. In addition, CSPI consists of a rich source of func-
tional amino acids, which indicating that it could be used as a
promising nutraceutical and food ingredient in terms of preventing
oxidative damage as well as controlling sugar level in diabetic
patients.
Acknowledgment
This project is supported by ERGS Grant (203/CAATS/6730103).
References
Adebowale, Y. A., Adeyemi, I. A., Oshodi, A. A., & Niranjan, K. (2007). Isolation,
fractionation and characterization of proteins from Mucuna bean. Food Chem-
istry, 104, 287e299.
Allahghadri, T., Rasooli, I., Owlia, P., Nadooshan, M. J., Ghazanfari, T., Taghizadeh, M.,
et al. (2010). Antimicrobial property, antioxidant capacity, and cytotoxicity of
essential oil from cumin produced in Iran. Journal of Food Science, 75, H54eH61.
Alli, I., & Baker, B. E. (1981). Constitution of leguminous seeds. A note on proteine
phytic acid interactions during isolation of acid-soluble protein from Phaseolus
beans. Journal of the Science of Food and Agriculture, 32, 588e592.
Alu’datt, M. H., Ereifej, K., Abu-Zaiton, A., Alrababah, M., Alamajwal, A., Rababah, T.,
et al. (2011). Antioxidant, antidiabetic, and anti-hypertensive effects of extrac-
ted phenolics and hydrolyzed peptides from barley protein fractions. Interna-
tional Journal of Food Properties, 15, 781e795.
Amadou, I., Olasunkanmi, S., Gbadamosi Shi, Y. H., Kamara, M. T., Jin, S., & Le, G. W.
(2010). Identification of antioxidative peptides from Lactobacillus plantarum Lp6
fermented soybean protein meal. Research Journal of Microbiology, 5, 372e380.
Amza, T., Amadou, I., Zhu, K. X., & Zhou, H. M. (2011). Effect of extraction and
isolation on physicochemical and functional properties of an underutilized seed
protein: gingerbread plum (Neocarya macrophylla). Food Research International,
44, 2843e2850.
Atkinson, A. C., & Donev, A. N. (1992). Optimum experimental designs. Oxford:
Clarendon Press.
Benzie, I. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a
measure of “antioxidant power”: the FRAP Assay. Analytical Biochemistry, 239,
70e76.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding.
Analytical Biochemistry, 72, 248e254.
Byler, D. M., & Suci, H. (1986). Examination of the secondary structure of proteins by
deconvolved FTIR spectra. Biopolymers, 25, 469e487.
Canan, C., Cruz, F. T. L., Delaroza, F., Casagrande, R., Sarmento, C. P. M.,
Shimokomaki, M., et al. (2011). Studies on the extraction and purification of
phytic acid from rice bran. Journal of Food Composition and Analysis, 24, 1057e
1063.
Carbonaro, M., Cappelloni, M., Nicoli, S., Lucarini, M., & Carnovale, E. (1997). Solu-
bilityedigestibility relationship of legume proteins. Journal of Agricultural and
Food Chemistry, 45, 3387e3394.
Chen, X., Ru, Y., Chen, F., Wang, X., Zhao, X., & Ao, Q. (2013). FTIR spectroscopic
characterization of soy proteins obtained through AOT reverse micelles. Food
Hydrocolloids, 31, 435e437.
Deak, N. A., & Johnson, L. A. (2007). Fate of phytic acid in producing soy protein
ingredients. Journal of the American Oil Chemists’ Society, 84, 369e376.
Dhandapani, S., Subramanian, V. R., Rajagopal, S., & Namasivayam, N. (2002).
Hypolipidemic effect of Cuminum cyminum L. on alloxan-induced diabetic rats.
Pharmacological Research, 46, 251e255.
Duranti, M., Consonni, A., Magni, C., Sessa, F., & Scarafoni, A. (2008). The major
proteins of lupin seed: characterisation and molecular properties for use as
functional and nutraceutical ingredients. Trends in Food Science & Technology, 19,
624e633.
Elsheikh, E. A. E., Fadul, I. A., & El Tinay, A. H. (2000). Effect of cooking on anti-
nutritional factors and in vitro protein digestibility (IVPD) of faba bean grown
with different nutritional regimes. Food Chemistry, 68, 211e212.
Eromosele, C. O., Arogundade, L. A., Eromosele, I. C., & Ademuyiwa, O. (2008).
Extractability of African yam bean (Sphenostylis stenocarpa) protein in acid, salt
and alkaline aqueous media. Food Hydrocolloids, 22, 1622e1628.
 H.-L. Siow, C.-Y. Gan / Food Hydrocolloids 41 (2014) 178e187
Gagandeep, Dhanalakshmi, S., Mendiz, E., Rao, A. R., & Kale, R. K. (2003). Chemo-
preventive effects of Cuminum cyminum in chemically induced forestomach and
uterine cervix tumors in murine model systems. Nutrition and Cancer, 47, 171e
180.
Gault, V. A., O’Harte, F. P. M., Harriott, P., & Flatt, P. R. (2002). Characterization of the
cellular and. metabolic effects of a novel enzyme-resistant antagonist of
glucose-dependent insulinotropic polypeptide. Biochemical and Biophysical
Research Communications, 290, 1420e1426.
Gupta, A., Mehrotra, R., Klimov, E., Siesler, H. W., Joshi, R. M., & Chauhan, V. S.
(2006). Thermal stability of dehydrophenylalanine-containing model peptides
as probed by infrared spectroscopy: a case study of a-helical and a 310-helical
peptide. Chemistry and Biodiversity, 3, 284e295.
Henry, R. J., & Kettlewell, P. (1996). Cereal grain quality. London: Chapman and Hall.
Jackson, M., & Mantsch, H. H. (1995). The use and misuse of FTIR spectroscopy in the
determination of protein structure. Critical Review in Biochemistry and Molecular
Biology, 30, 95e120.
Jumepaeng, T., Prachakool, S., Luthria, D. L., & Chanthai, S. (2013). Determination of
antioxidant capacity and a-amylase inhibitory activity of the essential oils
from citronella grass and lemongrass. International Food Research Journal, 20,
481e485.
Lee, O. H., Lee, B. Y., Lee, J., Lee, H. B., Son, J. Y., Park, C. S., et al. (2009). Assessment of
phenolics-enriched extract and fractions of olive leaves and their antioxidant
activities. Bioresource Technology, 100, 6107e6113.
Liener, I. E. (1974). Phytohemagglutinins: their nutritional significance. Journal of
Agricultural and Food Chemistry, 22, 17e22.
Lu, J., Zeng, Y., Hou, W., Zhang, S., Li, L., Luo, X., et al. (2012). The soybean peptide
aglycin regulated glucose homeostasis in type 2 diabetic mice via IR/IRS1
pathway. Journal of Nutritional Biochemistry, 23, 1448e1457.
Moore, S. (1963). On the determination of cystine as cysteic acid. The Journal of
Biological Chemistry, 238, 235e237.
Muga, A., Surewicz, W. K., Wong, P. T. T., Mantsch, H. H., Singh, V. K., & Shinohara, T.
(1990). Structural studies with the uveopathogenic peptide M derived from
retinal S-antigen. Biochemistry, 29, 2925e2930.
187
Nasi, A., Picariello, G., & Ferranti, P. (2009). Proteomic approaches to study structure,
functions and toxicity of legume seeds lectins. Perspectives for the assessment
of food quality and safety. Journal of Proteomics, 72, 527e538.
Orrun  o, E., & Morgan, M. R. A. (2007). Purification and characterization of the 7S
globulin storage protein from sesame (Sesamum indicum L.). Food Chemistry,
100, 926e934.
Radovic, R. S., Maksimovic, R. V., Brkljacic, M. J., Varkonji Gasic, I. E., & Savic, P. A.
(1999). 2S albumin from buckwheat (Fagopyrum esculentum moench) seeds.
Journal of Agricultural and Food Chemistry, 47, 1467e1470.
Sarmadi, B. H., & Ismail, A. (2010). Antioxidative peptides from food proteins: a
review. Peptides, 31, 1949e1956.
Tai, S. S., Wu, L. S., Chen, E. C., & Tzen, J. T. (1999). Molecular cloning of 11S globulin
and 2S albumin, the two major seed storage proteins in sesame. Journal of
Agricultural and Food Chemistry, 47, 4932e4938.
Udenigwe, C. C., & Aluko, R. E. (2012). Food protein-derived bioactive peptides:
production, processing, and potential health benefits. Journal of Food Science, 77,
R11eR24.
WHO. (1985). Energy and protein requirements. Report of a Joint FAO/WHO/UNU
Expert Consultation. Geneva: World Health Organization (WHO Technical
Report Series, No. 724).
Worthington Biochemical Corporation. (1993). Alpha amylase. In V. Worthington
(Ed.), Worthington enzyme manual (pp. 36e41). Freehold.
Yu, J., Ahmedna, M., & Goktepe, I. (2007). Peanut protein concentrate: production and
functional properties as affected by processing. Food Chemistry, 103, 121e129.
Zarkadas, C. G., Gagnon, C., Gleddie, S., Khanizadeh, S., Cober, E. R., &
Guillemette, R. J. D. (2007). Assessment of the protein quality of fourteen soy-
bean [Glycine max (L.) Merr.] cultivars using amino acid analysis and two-
dimensional electrophoresis. Food Research International, 40, 129e146.
Zuber, H. (1988). Temperature adaptation of lactate dehydrogenase Structural,
functional and genetic aspects. Biophysical Chemistry, 29, 171e179.