Original article

Ultrasonic assisted alkali extraction of protein from defatted rice

bran and properties of the protein concentrates
Thutiyaporn Chittapalo & Athapol Noomhorm*
Food Engineering and Bioprocess Technology Programme, School of Environment, Resources and Development, Asian Institute of Technology,
P.O. Box 4, Klong Luang, Pathumthani 12120, Thailand
(Received 28 October 2008; Accepted in revised form 22 May 2009)
Summary Alkaline extraction for the preparation of protein concentrate from rice bran was compared with a range of
ultrasonic treatments. Results revealed that the extraction time decreased, and the reaction rate constant
increased, with increasing ultrasonic power. The reaction rate constants were 0.0065, 0.0130, 0.0237 and
0.0924 at 40, 60, 80 and 100 W respectively. The defatted rice bran protein concentrate (DRBPC) using
ultrasonication (100 W for 5 min) and conventional methods showed no signicant dierence in bulk
densities (P > 0.05) but it had higher yield (%) and was lighter brown using ultrasonication (P 0.05). The
SEM showed that the residual rice bran after extracting protein using ultrasonication exhibited more damage
than the conventional method. The functional properties of both samples were not signicantly dierent
(P > 0.05) in terms of foam and emulsifying stability. However, the water and oil absorption, foam capacity
and emulsion activity were signicantly dierent (P 0.05). The nitrogen solubility index of both DRBPC
samples gave similar proles with the lowest solubility at pH 46.
Keywords Alkaline extraction, protein concentrate, rice bran protein, ultrasonic assisted extraction, ultrasonication power.
Introduction
The application of ultrasonication is widely used in food
processing, especially for extraction. Many researchers
have been investigating the advantages of ultrasonic
assisted extraction (UAE) compared with the conven-
tional method. UAE can produce higher yields at higher
rates in batch extraction of isoavones from the root of
Radix Puerariae (Lee & Lin, 2007), gingerols (Bala-
chandran et al., 2006), anthraquinones from Morinda
citrifolia roots (Hemwimol et al., 2006), rice starch
isolate (Wang & Wang, 2004), oil and coixenolide from
adlay seed (Hu et al., 2007) and antioxidant substance
from chilli (Li-E et al., 2008). In addition, it can be used
to extract volatile compounds which have lower molec-
ular weights (Jerkov c et al., 2007). Moultuon & Wang
(1982) have studied both continuous and batch ultra-
sonic extraction of defatted soybean akes protein and
found that both processes gave higher yields than the
conventional method.
Thailand is one of the worlds major rice traders,
exporting an average of 8 million tons of rice annually.
The by products obtained from the milling of rice
includes approximately 60% hulls, 35% bran and 5%
polish (Shih & Daigle, 1999). Defatted rice bran is a by
product of rice bran oil extraction. The chemical
composition of defatted rice bran includes nutrients,
especially protein which has high lysine content and is
considered hypoallergenic. Rice bran is richest in
phenolics when extracted using methanolic extracts
(Bhanger et al., 2008). Several studies have been con-
ducted on the preparation of protein concentrates and
isolates such as extracted protein from full-fatted rice
bran and defatted rice bran using an alkaline method
(Bera & Mukherjee, 1989; Prakash & Ramanatham,
1994; Gnanasambandam & Hettiarachchy, 1995; Chan-
di & Sogi, 2007) which is the most common method used
because the reagents required for the process are easily
available. Adebiyi et al. (2008) found that crude rice
bran protein had higher antioxidative activity than the
phytate-free rice protein. Moreover, the preparation of
protein concentrates by enzymatic extraction (Wang
et al., 1998; Parrado et al., 2006), and the extraction of
rice protein concentrates from rice our have been
investigated (Ju et al., 2001; Agboola & Mills, 2005).
However, all of these methods have low extraction rates
and result in low yields.
The aim of this study was to determine the extraction
time of the conventional alkaline hydrolysis and ultra-
sonic assisted extraction methods at various powers. The
various powers (40100 W) and the resulting extraction
*Correspondent: Fax: +66 25246200;
e-mail: athapol@ait.ac.th
International Journal of Food Science and Technology 2009, 44, 18431849 1843
doi:10.1111/j.1365-2621.2009.02009.x
2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology
times (540 min) were used to determine the reaction
constants for each power. Finally, the physical and
functional properties of defatted rice bran protein
concentrate (DRBPC) samples extracted using both
methods were investigated.
Materials and methods
Materials
Defatted rice bran was obtained from Thai Edible Oil
Co., Ltd., Ayuthaya, Thailand, and all chemical
reagents used in this experiment were of analytical
grade.
Methods
Each extraction time was determined in three replicates.
Effect of ultrasonic power on protein extraction
Conventional treatment: Defatted rice bran was dispersed
in distilled water with a sample to water ratio of 1:5. The
pH was then adjusted and maintained at 11 with 2N
NaOH for 10, 20, 30, 40, 60 and 120 min with stirring.
Next, the product was centrifuged (Centrikon T-324;
Kontron Instrument, Milano, Italy) at 3,850 g for
15 min at 4 C and ltered with Whatman lter paper
(No.4) to remove the insoluble material. Finally the
supernatant was analysed for the amount of protein
using Biuret protein assay.
Ultrasonic treatment: About 20 g of defatted rice bran
was dispersed in 100 mL of distilled water (ratio 1:5) in a
conical stainless steel container. The pH was then
adjusted and maintained at 11. The ultrasonic
experiments were carried out at 20 kHz using an
ultrasonic generator (Model VCX750 Vibracell; Sonic
& Materials, Inc., Newtown, CT, USA) with an
ultrasonic horn diameter of 38 mm. Ultrasonication
powers of 40, 60, 80 and 100 W were generated at a time
interval of 540 min. The horn tip was immersed around
2 cm into the solution to be processed. The ultrasonic
generator ran on a cycle of 59 s on and 10 s o (pulse
59 10 s) with the temperature controlled by an ice bath
at 26.62 2.74 C. Next, the product was centrifuged
at 3,850 g for 15 min at 4 C and ltered with
Whatman lter paper (No.4). Finally the supernatant
was analysed for the amount of protein using Biuret
protein assay.
Biuret protein assay
Standard bovine serum albumin (BSA) solution was
pipetted into each of ve tubes at volumes of 0.0, 0.2,
0.4, 0.6 and 0.8 mL, and then distilled water was added
to each tube for a total volume of 1 mL. After that,
4 mL of Biuret reagent was added to each tube, shaken
and left aside for 30 min. Absorption of the solutions
was read using a spectrophotometer (Model UV2 UV
vis spectrometer; UNICAM, Cambridge, UK) at
570 nm and plotted against BSA concentration. The
protein content in the samples was determined by
pipetting 1 mL of protein solution into each of the
tubes. After that, 4 mL of Biuret reagent was added to
each tube, shaken and left aside for 30 min. The
absorption was read using the spectrophotometer at
570 nm. Protein quantication was determined using a
standard curve.
Preparation of defatted rice bran protein concentrates
Defatted rice bran protein concentrate samples were
prepared using two dierent methods, the conventional
and UAE methods. For the conventional method, the
alkaline extraction and acid precipitation methods
described respectively by Chen & Houston (1970) and
Youssef et al. (1974) were used. Briey, defatted rice
bran was dispersed in distilled water with a ratio of 1:5,
then the pH of the slurry was adjusted and maintained
at 11 with 2N NaOH and stirred with a mixer
(Servodyne Model 50000-25; Cole-Parmer Instrument
Co., Vernon Hills, IL, USA) at 300 rpm for 1 h. Next,
the slurry was centrifuged at 10,000 g for 15 min and
ltered through Whatman lter paper (No.4). The
ltrate was precipitated for protein using 2N HCl at
pH 4.5, then centrifuged at 10,000 g for 15 min. The
solid was recovered and washed with a small amount of
distilled water and again centrifuged at 10,000 g. The
protein concentrate paste was freeze dried (PL9000;
Heto PowerDry, Waltham, MA, USA) and kept in a
polyethylene bag at 4 C until used.
For UAE, defatted rice bran and distilled water was
placed in a conical stainless steel container with a ratio
of 1:5 (based on a batch volume of 100 mL) and put in
an ice bath to control temperature, and pH was adjusted
to 11. The ultrasonic machine (Model VCX750 Vibra-
cell; Sonic & Materials, Inc.) was set at 100 W for 5 min
(pulse 59 10 s) and then pH was readjusted after
2.57 min. Similar to the conventional method, the steps
of centrifuging to remove insoluble material, ltering,
acid precipitation, centrifuging again, washing sediment,
freeze drying and storing were used.
Proximate composition
The moisture content (oven-drying procedure), ash
content, crude protein (N 5.95) and fat content of
the defatted rice bran and DRBPC samples were
determined using the Association of Ocial Analytical
Chemists (AOAC) methods (AOAC, 2000).
Colour
Freeze dried DRBPC samples were measured for colour
using a colorimeter CR-10 (Konica Minolta Sensing
UAE of protein from defatted rice bran T. Chittapalo and A. Noomhorm 1844
International Journal of Food Science and Technology 2009 2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology
Inc., Osaka, Japan). Colour parameters were L* (light-
ness), a* (redness greenness) and b* (yellowness blue-
ness). All measurements were taken under the
conditions of standard illuminant D65 and 10 observer.
Bulk density
Defatted rice bran protein concentrate samples with
known weights were added into graduated measuring
cylinders. The cylinders were gently tapped and the
volumes occupied by the samples determined. The bulk
densities were calculated as weight per unit volume
(g mL
)1
).
Scanning electron microscopy
Defatted rice bran protein concentrate samples were
sprinkled onto double-backed, cellophane tape attached
to a bronze stub before coating with platinum and
palladium (Sputter coated E-102 Hitachi, Tokyo,
Japan). The samples were then photographed under a
scanning electron microscope (Hitachi S-2500, Tokyo,
Japan) at an accelerating voltage of 15 KV.
Water and oil absorption
Three grams of water were added into 0.5 g DRBPC
samples and mixed well. The slurry was then centrifuged
at 750 g for 15 min and the pellets were drained for
30 min. The water absorptions were determined using
gain in weight per unit weight of protein concentrate
(g g
)1
). For oil absorption, the same procedure was
followed using oil instead of water.
Nitrogen solubility index
The nitrogen solubility index (NSI) was determined
using the method of Gnanasambandam & Hettiarach-
chy (1995). DRBPC samples (1%) were weighed and
dispersed in distilled water in 50 mL centrifuge tubes,
and the pH was adjusted to 2, 4, 6, 8, 10 and 12 using 0.1
and 1 N HCl or NaOH. The suspensions were shaken
using an orbital shaker (Model SI 50; Stuart Scientic,
Redhill, UK) at 250 rpm for 30 min. The protein
solutions were then centrifuged at 5,000 g for 15 min
(Centrikon T-324; Kontron Instrument, Milano, Italy)
and ltered through Whatman lter paper (No.4). The
supernatant was analysed for nitrogen content using the
Kjedhal method (AOAC, 2000) and nitrogen solubility
was calculated using the following equation:
Nitrogen solubility (%)
Nitrogen in supernatant
Nitrogen in 100 g of sample
100
Emulsifying activity and emulsion stability
Emulsifying activity (EA) and emulsifying stability (ES)
of DRBPC samples were evaluated using the turbidi-
metric methods of Pearce & Kinsella (1978). Summa-
rising, the DRBPC samples were initially prepared for
6 mL protein solutions (0.1%w v) and stirred for 1 min;
then 2 mL of commercial soybean oil were added and
homogenised (Model X-520; Polyscience, Niles, IL,
USA) for 1 min at maximum speed. Fifty microlitres
were then pipetted from the bottom of the tubes into
0.1% (w v) sodium dodecyl sulphate at 0 and 10 min
after homogenising. The absorbance of the diluted
emulsions was measured using a spectrophotometer
(Model UV2 UV VIS spectrometer; UNICAM) at
500 nm. The EA was expressed directly at the initial
time after being homogenised and emulsion stability was
calculated using the following equation:
ES
A
0
DA
Dt
where A
0
is the absorbance of the diluted emulsion
immediately after homogenisation, and DA is the
reduction in absorbance at the interval time (Dt).
Foam capacity and foam stability
Foam capacity and foam stability of DRBPC were
assessed using the same methods used by Kato et al.
(1983). Air (90 cm
3
min
)1
) was introduced to 5 mL of
0.2% protein in a 0.1 M phosphate buer (pH 7.4) in a
glass tube for 15 s. The foam capacity was determined
by measuring the volume immediately after the air was
added. Foam stability was then calculated using the
following equation:
FS
V
0
DV
Dt
where V
0
is the volume of foam at time 0 (after air was
introduced), and DV is the reduction in volume of foam
at the interval time (Dt).
Statistical analysis
The analysis of variance (anova) and dierences
between means assessed by least signicant dierence
were computed (Statistix software version 7.0; Analy-
tical Software, Tallahassee, FL, USA). The evaluations
were based on a signicance level of P 0.05.
Results and discussion
Effect of varying ultrasonic powers
Figure 1 shows that, using the conventional method,
most of the protein was extracted within 60 min with no
signicant dierence in the yield after 60 min
(P > 0.05). Using ultrasonic power, the protein yield
increased with increasing power and elapsed time. The
yield plateaued at 0.378 mg mL
)1
in <10 min for all
powers except 40 W which showed a continuous
increase until the experiment was terminated at
40 min. This result agrees with the study of Moultuon
& Wang (1982) which found that the amount of protein
UAE of protein from defatted rice bran T. Chittapalo and A. Noomhorm 1845
2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology International Journal of Food Science and Technology 2009
extracted from defatted akes using batch and contin-
uous sonication was higher than when using the
conventional method. Figure 1 also shows that, the
highest rate of protein extraction was at 100 W,
followed by 80, 60 and 40 W at 5, 10, 20 and 40 min
respectively. The rate of extraction using the conven-
tional and ultrasonication methods decreased exponen-
tially. Therefore, the rate was dependent on the
concentration of soluble protein in the rice bran. As
shown in Fig. 2, the change in extracted protein was
reasonably described by rst order kinetic model which
was:
ln C=C
0
kt
where, C is the amount of protein using the Biuret
method measured at any time during the transient stage
of the process, C
0
is the initial protein, k is the reaction
rate constant and t is extraction time.
The slope of the plot of ln (C C
0
) vs. time (Fig. 2)
revealed the reaction rate constant (k) and its correla-
tion equations were listed in Table 1. The reaction rate
constant increased, with increasing ultrasonic power.
The reaction rate constants were 0.0065, 0.0130, 0.0237
and 0.0924 at 40, 60, 80 and 100 W respectively. In all
cases, a linear regression with coecient of determina-
tion value (R
2
) ranged between 0.9127 and 0.9931.
Proximate composition
The chemical composition of defatted rice bran and
DRBPC samples are shown in Table 2. The moisture
and bre contents of both DRBPC samples using
ultrasonication and conventional methods were signif-
icantly dierent (P 0.05) while there were no dier-
ences in ash, protein and lipid content (P > 0.05). The
protein content of both DRBPC samples (76.09 and
77.30%) were less than that reported by Lew et al.
(1975) who used the same extraction and precipitation
pH.
Physical properties
The colours of both DRBPC samples (Table 3) were
signicantly dierent (P 0.05). The protein concen-
trate extracted using the conventional method was
darker and was more reddish yellow than that extracted
using the ultrasonication method. However, the bulk
densities of both samples were not signicantly dierent
(P > 0.05). The small dierence in values might have
resulted from the smaller particle size of the protein
0.15
0.2
0.25
0.15
0.3
0.35
0.4
0 20 40 60 80 100 120 140
Time (min)
P
r
o
t
e
i
n

(
m
g

m
L

1
)
0 W (Non UAE) 40 W 60 W 80 W 100 W
Figure 1 The effect of ultrasonic power on the time of protein
extraction.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 20 40 60 80
Time (min)
l
n

(
C
/
C
o
)
0 W (Non UAE) 40 W 60 W 80 W 100 W
Figure 2 The plot of ln (C C
0
) over time.
Table 1 Correlation equations describing the relationship between
reaction rate constants and ultrasonic power
Ultrasonic power (W) Equation R
2
0 ln (C C
0
) = 0.0058t + 0.3215 0.9591
40 ln (C C
0
) = 0.0065t + 0.6214 0.9127
60 ln (C C
0
) = 0.0130t + 0.6438 0.9895
80 ln (C C
0
) = 0.0237t + 0.6014 0.9931
100 ln (C C
0
) = 0.0924t + 0.3936 0.9348
Table 2 Proximate composition of the defatted rice bran and defatted
rice bran protein concentrates
Composition*
Defatted rice
bran
Protein concentrate
Ultrasonication
method
Conventional
method
Ash 9.34 0.05
a
2.73 0.13
b
2.89 0.19
b
Fibre 10.22 0.14
a
1.07 0.03
b
0.89 0.05
c
Protein 9.26 0.10
b
76.09 1.44
a
77.30 2.77
a
Lipid 1.04 1.12
a
0.82 0.04
a
0.95 0.04
a
Values are expressed as mean SD (n = 3).
Means with different letters within the same row indicate signicant
differences (P 0.05).
*Compositions were calculated on a moisture free basis.
UAE of protein from defatted rice bran T. Chittapalo and A. Noomhorm 1846
International Journal of Food Science and Technology 2009 2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology
concentrate extracted using the conventional method
(data not shown). The production of DRBPC using the
ultrasonication method resulted in a higher yield which
was about 1.65 times than that of the conventional
method.
Rice bran is composed of the aleurone layer, and
some of the embryo and endosperm. Figure 3 shows
three types of rice bran: defatted rice bran (a), defatted
rice bran after using UAE (b) and defatted rice bran
after using the conventional method (c). There were a lot
of starch granules that were polygonal in shape on the
surface of the rice bran in all three types. The cell walls
were composed mainly of the polysaccharide cellulose
which was swollen and dissolved by sodium hydroxide
in Fig. 3b, c only. The residual of rice bran after
extracting the protein using the ultrasonication method
(b) exhibited more damage than when using the
conventional method (c) because the production and
denseness of cavitation bubbles during the application
of ultrasound provided a greater penetration of alkaline
into the cellular materials and then the cell walls were
disrupted; therefore, intracellular products were released
(Romdhane & Gourdon, 2002). These gures agree with
the study of Chemat et al. (2004) in which the SEM
showed the mechanical eects of ultrasound, mainly
appearing on cell wall.
Functional properties
The water absorption (Table 4) of both DRBPC sam-
ples revealed no signicant dierence (P > 0.05).
DRBPC samples using ultrasonication and conventional
methods had water absorption at 2.51 and 2.14 g g
)1
respectively. The water absorption of the DRBPC
samples in this study were less than the DRBPC samples
from Basmati 370, Basmati 386 and HBC19 reported by
Chandi & Sogi (2007). Oil absorption of DRBPC
extracted using the ultrasonication method was higher
than the conventional method (P 0.05), which was
1.52 and 1.31 g g
)1
respectively. These results show that
the DRBPC sample using ultrasonication method
retained more water than oil, and was also more
Table 3 Physical properties of defatted rice bran protein concentrate
Properties
Protein concentrate
Ultrasonication
method
Conventional
method
Colour
L 46.17 0.42
a
42.25 0.12
b
a* 5.37 0.16
b
5.75 0.14
a
b* 10.7 0.41
a
7.42 0.17
b
Bulk density (g mL
)1
) 0.47 0.01
a
0.46 0.01
a
% Yield 4.45 0.27
a
2.70 0.32
b
Values are expressed as mean SD (n = 3).
Means with different letters within the same row indicate signicant
differences (P 0.05).
% yield was calculated based on the weight of defatted rice bran.
Cell wall
(a) (b) (c) Figure 3 SEM of defatted rice bran, which
were raw material (a), after using ultrasonic
assisted extraction (b) and after using con-
ventional method (c).
Table 4 Functional properties of defatted rice bran protein concen-
trate
Properties
Protein concentrate
Ultrasonication
method
Conventional
method
Water adsorption (g g
)1
) 2.52 0.04
a
2.14 0.04
b
Oil adsorption (g g
)1
) 1.52 0.01
a
1.31 0.01
b
Emulsifying activity 0.32 0.20
b
0.51 0.04
a
Emulsifying stability (min) 14.36 0.28
a
14.35 0.3
a
Foam capacity (mL) 5.72 0.18
b
11.56 0.89
a
Foam stability (min) 0
a
0
a
Values are expressed as mean SD (n = 3).
Means with different letters within the same row indicate signicant
differences (P 0.05).
UAE of protein from defatted rice bran T. Chittapalo and A. Noomhorm 1847
2009 The Authors. Journal compilation 2009 Institute of Food Science and Technology International Journal of Food Science and Technology 2009
hydrophilic and lipophilic than that using the conven-
tional method.
The EA of the DRBPC sample extracted using the
conventional method was higher than that using the
ultrasonication method (P 0.05). It might have been
inuenced by sonic waves during extraction. All EA
values were higher than the values reported by Tang
et al. (2003). This dierence is probably because of the
dierent kinds of rice bran used. ES of the DRBPC
sample using the conventional and ultrasonication
methods showed no signicant dierence (P > 0.05)
at 14.36 and14.35 min respectively.
Foam capacity of DRBPC using the conventional
method was higher than that using the ultrasonication
method, around two-fold, at 11.56 and 5.72 mL respec-
tively. This might have been caused by the nitrogen
solubility of the DRBPC sample using the conventional
was higher than that using the ultrasonication method at
the present pH. However, foam capacity of the ultraso-
nicated sample was similar to rice bran fromBasmati 370
reported by Chandi & Sogi (2007). Both samples were
almost negligible of foam stability. The result is in
accordance with previously published literature (Tang
et al., 2003) which indicated that to have foam capacity;
proteins should solubilise in the aqueous phase and
rapidly unfold to forma cohesive layer of protein around
gas air droplets. This requires exible protein molecules
with fewsecondary and tertiary structures. To have foam
stability, protein molecules should form continuous
intermolecular polymers enveloping the air bubbles, as
intermolecular cohesiveness and elasticity are important
to produce stable foams. Chandi & Sogi (2007) reported
that the half-life of foam stability of rice protein concen-
trate was pH 7 which was higher than in this experiment.
As a function of pH (Fig. 4), both DRBPC samples
showed similar nitrogen solubility index (NSI) proles
with the lowest at pH 46 (24%), but below and above
this pH the NSI increased. At pH 2, the NSI of the
DRBPC sample using the ultrasonication method was
about 30% while that of the conventional method was
about 40% which agrees with the report of Gnanasam-
bandam & Hettiarachchy (1995) and Tang et al. (2003).
At pH 8 and above, the NSI of both methods gradually
increased; however, the NSI of the conventional was
higher than the ultrasonication method. The maximum
nitrogen solubilities of DRBPC samples were observed
at pH 12 with 95% for the conventional and 85% for the
ultrasonication method.
Conclusion
By the kinetics of protein extraction as a function of
ultrasonic power, it was found that the reaction rate
constant increased when increasing the ultrasonic
power. The rate of extraction using the conventional
and ultrasonication methods decreased exponentially.
Therefore, the change in extracted protein quantity was
reasonably described by rst order kinetic model. The
optimum condition for preparing DRBPC using the
ultrasonication method was 100 W for 5 min while
using conventional method was 0 W for 60 min. The
DRBPC samples were then compared physical and
functional properties. It was found that they were
similar in bulk density, foam and ES and NSI prole.
However, they were dierent in colour, yield, water and
oil absorption, foam capacity and EA.
Acknowledgments
We are grateful to Payap University and the Royal Thai
Government for nancial support.
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0
0 2
Sonication
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4 6
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N
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