Research Article

Received: 2 July 2010 Revised: 14 January 2011 Accepted: 21 January 2011 Published online in Wiley Online Library: 7 March 2011

(wileyonlinelibrary.com) DOI 10.1002/jsfa.4339

Some characteristics and functional properties

of rapeseed protein prepared by
ultrasonication, ultrafiltration and isoelectric
precipitation
Xu-Yan Dong,a,b Lu-Lu Guo,b Fang Wei,b Ju-Fang Li,b Mu-Lan Jiang,b
Guang-Ming Li,b Yuan-Di Zhaoa∗ and Hong Chenb∗

Abstract
BACKGROUND: The presence of complex protein constituents and difficulties in extracting protein from rapeseed meal limit
the application of rapeseed protein in food processing. However, double-low rapeseed (low erucic acid, low glucosinolate)
protein is a type of complete protein that is of potential use in the food industry. In this study the characteristics and functional
properties of rapeseed protein prepared by ultrasonic-assisted extraction, ultrafiltration and isoelectric precipitation were
analysed and compared with those of soybean protein.

RESULTS: The extraction efficiency with the ultrasonic-assisted method was significantly higher than that obtained with the
traditional method. Ultrafiltration and isoelectric precipitation yielded three different proteins: ultrafiltered protein RPs and
precipitated proteins RP5.8 and RP3.6. Chromatographic separation of RPs resulted in four fractions: RPsI, RPsII, RPsIII and
RPsIV. The distribution of the isoelectric point of rapeseed protein was investigated by two-dimensional electrophoresis. The
amino acid composition of RPs renders it suitable for human consumption. The hydrophobic/hydrophilic amino acid ratio
of rapeseed protein was higher than that of soybean protein. The functional properties (oil adsorption ability, emulsifying
capacity, foaming capacity and foam stability) of RPs, RP5.8 and RP3.6 were found to be better than those of soybean protein.

CONCLUSION: Ultrasonication and ultrafiltration were significantly better than the traditional method of rapeseed protein
extraction. The ultrafiltered rapeseed protein RPs had superior functional properties. The results of this study provide useful
indicators for rapeseed protein as a potential replacement for other proteins.

c 2011 Society of Chemical Industry

Keywords: rapeseed protein; ultrasonic-assisted extraction; ultrafiltration; isoelectric precipitation; functional properties

INTRODUCTION
Rapeseed (Brassica napus L.) is an important oilseed crop. In
recent years the annual output of rapeseed meal has exceeded
10 million tons in China, which is one of the largest producers of
this crop. The development of double-low rapeseed (low erucic
acid, low glucosinolate) in China has led to the availability of a
feed ingredient that has considerable potential to fully or partially
replace soybean meal in diets for monogastric animals. Double-
low rapeseed meal is a high-quality product that has a high protein
content and a well-balanced amino acid composition in line with
FAO guidelines. This rapeseed meal is rich in sulfur-containing
amino acids and lysine, both of which are generally limiting
constituents in legumes and cereals.1 Thus rapeseed protein is a
type of complete protein that can provide essential amino acids
for animals and is recognised as an important complementary
protein resource for soybean protein. In comparison with soy
protein, rapeseed protein is more complex, and there are great
differences in the isoelectric points and molecular weights of
its fractions. Rapeseed protein comprises two major classes of
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of the protein content, and 2S albumin (napin). Some minor types
of protein are also present,2,3 with isoelectric points and molecular
weights distributed over a wide range. Therefore protein extraction
is difficult, which limits the application of rapeseed protein in food
processing.
∗
Correspondence to: Yuan-Di Zhao, Wuhan National Laboratory for Optoelec-
tronics, College of Life Science and Technology, Huazhong University of Science
and Technology, 430074 Wuhan, Hubei, China. E-mail: zydi@mail.hust.edu.cn
Hong Chen, Institute of Oil Crops Research, Chinese Academy of Agricultural
Sciences, The Key Lab for Biological Sciences of Oil Crops, Ministry of Agriculture,
430062 Wuhan, Hubei, China. E-mail: chenhongref@yahoo.com
a Wuhan National Laboratory for Optoelectronics, College of Life Science and
Technology, Huazhong University of Science and Technology, 430074 Wuhan,
Hubei, China
b Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, The

Key Lab for Biological Sciences of Oil Crops, Ministry of Agriculture, 430062
protein: 12S globulin (cruciferin), which accounts for 25–65% Wuhan, Hubei, China

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Characteristics and functional properties of rapeseed protein
There are many reports on the extraction of rapeseed protein.
The traditional extraction method is based on an aqueous phase
technique. Rapeseed protein has been extracted with salt, alkali
or alcohol solution and has been mainly separated by isoelectric
precipitation and ultrafiltration. Yumiko et al.4 extracted rapeseed
protein under alkaline conditions and precipitated it at a pI of 5.8
to obtain an extract with a crude protein content of 708.4 g kg−1 .
The high albumin content of the mixture affected the isoelectric
precipitation yield, resulting in inadequate extraction. Berot et al.3
developed a rapid and suitable method for obtaining high yields
from large-scale purification processes of storage proteins. The
techniques used included nanofiltration, two cation exchange
chromatography steps, one size exclusion chromatography step to
refine cruciferin and one hydrophobic interaction chromatography
step to refine napin.
Membrane technologies are separation methods used in agroin-
dustries to concentrate and/or purify different feedstreams. Ultra-
filtration is one of the most fascinating separation technologies
used in the food industry.5,6 An ultrafiltration membrane has
differential selectivity and can retain proteins of high molecular
weight while allowing lower-molecular-weight proteins and small
molecules to pass through in the permeate.7 The extraction effi-
ciency can be significantly improved by using an ultrasonicator to
reduce the processing time and solvent consumption. Moreover,
ultrasonic-assisted extraction can be carried out at a lower tem-
perature, thereby preventing thermal damage to the extracts and
loss of volatile components during boiling.8,9 There have been a
few reports on the ultrasonic-assisted extraction of proteins. Small
pilot-scale ultrasound batch and continuous soy protein extrac-
tion trials were carried out by Moulton and Wang.10 They reported
that, in comparison with traditional extraction techniques, contin-
uous high-intensity ultrasonic-assisted technology resulted in the
extraction of 54 and 23% more protein by aqueous and alkaline
extraction respectively when comparable processing times and
volumes were used.
In this study the ultrasonic-assisted, ultrafiltration and isoelectric
precipitation techniques were applied in the extraction of proteins
from double-1ow defatted rapeseed. The functional properties of
rapeseed protein were investigated and compared with those
of soybean protein. The results of this study provide useful
information for the potential utilisation of rapeseed protein.
MATERIALS AND METHODS
Chemicals and instruments
Rapeseed was obtained from the Institute of Oil Crops Research,
Chinese Academy of Agricultural Sciences (Wuhan, China). The
protein molecular weight markers were from New England Biolabs
(Huashun, Wuhan, China). The KQ-500 DB ultrasonic cleaner was
from Ultrasound Instruments Ltd (Kunshan, China). The Labscale
TFF system used for ultrafiltration was from Millipore (Bedford,
MA, USA). The SPECORD 205 UV spectrophotometer was from
Jena Ltd (Jena, Germany). The KDN-08c Kjeldahl device was from
Jingkelong Science Instrument Company (Shanghai, China). All
other reagents used in the experiments were of analytical grade.
Preparation of rapeseed meal
Rapeseed meal was flaked with a roll mill and defatted by the
Soxhlet extraction method after decortication. The defatted meal
was dried in a vacuum oven at room temperature for up to 1 day.
The dry defatted meal was stored at 4 ◦ C in a hermetically sealed
plastic bag until further processing.
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Ultrasonic-assisted extraction of rapeseed protein
Rapeseed proteins were extracted from the dry defatted meal. The
extraction procedure is shown in Fig. 1. For protein production the
rapeseed meal was dispersed in phosphate buffer (pH 11.6) prior to
ultrasonic extraction. The extraction conditions (solid/liquid ratio,
ultrasonic power and ultrasonic treatment time) were optimised
via a single-factor experiment. Based on a three-level, three-
variable central composite rotatable design, response surface
methodology was used to evaluate the interactive effects of
solid/liquid ratio (1 : 20, 1 : 25 and 1 : 30), ultrasonic power (400,
450 and 500 W) and ultrasonic treatment time (70, 80 and 90 min)
on the protein content.11,12 The protein content was determined
by the method of Bradford.13 There were 15 experimental points
from the design of experimental factors and levels, comprising 12
factorial experiments and three zero points. The protein content in
the extraction solution was the response value, and the zero-point
experiment was done three times to estimate the error.
The slurry was centrifuged (Thermo Biofuge, Stratos, Waltham,
MA, USA) at 3000 × g for 15 min at 4 ◦ C. The supernatant was
concentrated by crossflow ultrafiltration with a membrane of
molecular weight cut-off 8 kDa. Ultrafiltered rapeseed protein RPs
was then obtained by vacuum freeze-drying of the collected
retentate. Traditional alkaline extraction and acidic precipitation
methods without the use of ultrasonication served as control.
The supernatant was acidified with 1 mol L−1 HCl to pH 5.8.
This pH was maintained for 15 min to allow protein aggregation,
then the suspension was centrifuged at 3000 × g for 20 min.
Precipitated rapeseed protein RP5.8 was obtained by vacuum
freeze-drying. Finally, the pH of the supernatant was adjusted to
3.6 and precipitated rapeseed protein RP3.6 was obtained after
vacuum freeze-drying.
Extraction of soybean protein
Soybean protein was extracted by ultrasonic-assisted alkaline
extraction and acidic precipitation. The extraction was performed
in alkaline (pH 8) buffer solution. The protein fraction was
precipitated at a slightly acidic pH of 4.5.10 After centrifugation
the protein precipitate was freeze-dried and then stored at 4 ◦ C.
Purification of RPs
The RPs fraction was dispersed in phosphate buffer (pH 10.5). After
centrifugation at 8000 × g for 10 min the supernatant was filtered
through a 0.22 µm filter membrane. The protein content was
adjusted to 10 mg mL−1 . The filtrate was subsequently applied to a
HiPrep26/60 Sephacryl S-200 high-resolution column (Amersham
Pharmacia Biotech Uppsala, Sweden) that had been equilibrated
with phosphate buffer (pH 10.5). The elution flow rate was
1 mL min−1 . Different fractions were collected with an automated
fraction collector, and their absorbance at 280 nm was recorded.
Protein content analysis
The crude protein contents of the defatted rapeseed and soybean
flakes and the processed protein samples were measured. Crude
protein (N × 6.25) was determined by the Kjeldahl method
(AOAC14 method 46-12).
Percentage of protein and extraction efficiency was calculated
using the following equations.
Percentage protein content (g)
=
of protein (%) total weight (g)
Extraction protein content in the extraction solution (g)
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=
efficiency (%) total protein content (g)
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Figure 1. Protein extraction process from defatted rapeseed meal.
Amino acid analysis
Amino acids were analysed by high-performance liquid chro-
matography using the method of Mason et al.15 The following
ratios were calculated:
E/T = essential amino acids/total amino acids
E/N = essential amino acids/non-essential amino acids
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE)
SDS-PAGE16 was carried out in the presence of the reducing agent
β-mercaptoethanol in a vertical electrophoresis unit. The sample
gel was composed of 50 g L−1 polyacrylamide and the separating
gel consisted of 120 g L−1 polyacrylamide. The gels were processed
at a constant voltage of 150 V for 2.5 h and stained with Coomassie
brilliant blue R-250 in 500 mL L−1 methanol and 100 mL L−1 acetic
acid. Destaining was carried out with 400 mL L−1 methanol and
100 mL L−1 acetic acid, and the gels were stored in 100 mL L−1
acetic acid and 50 mL L−1 glycerol.
Two-dimensional electrophoresis
The molecular weight distribution and isoelectric points
of RPs were analysed by two-dimensional electrophore-
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sis. Gel strips (ReadyStrip IPG strip pH 3–10, Bio-Rad
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Laboratories, Hercules, CA, USA) for isoelectric focusing
of the extracts in the first dimension were rehydrated
overnight at room temperature with 8 mol L−1 urea, 2 mol L−1
thiourea, 5 mL L−1 3-[(3-cholamidopropyl)dimethylammonium]-
1-propanesulfonate, 5.2 mL L−1 ampholyte, 0.2 g L−1 bromophe-
nol blue, 10 g L−1 DL-dithiothreitol (DTT) and 1 mg of protein
extract. Isoelectric focusing was performed in a Protean IEF cell
(Bio-Rad Laboratories) according to the manufacturer’s instruc-
tions. After isoelectric focusing, the gel strips were equilibrated
with 50 mmol L−1 Tris-HCl (pH 6.8), 6 mol L−1 urea, 20 g L−1 SDS,
0.2 g L−1 bromophenol blue, 20 g L−1 DTT and 300 mL L−1 glyc-
erol and then subjected to SDS-PAGE in the second dimension
according to the procedure of Laemmli.16 Electrophoresis in the
second dimension was performed using a vertical slab gel appa-
ratus (Model 220, Bio-Rad Laboratories) with 120 g L−1 acrylamide
gels containing 100 g L−1 SDS and 1.5 mol L−1 Tris-HCl (pH 8.8).
After focusing, the gels were stained overnight with 100 g L−1 am-
monium sulfate, 100 mL L−1 phosphoric acid, 1.2 g L−1 Coomassie
Brilliant Blue and 200 mL L−1 methanol. They were then destained
with 30 mL L−1 glacial acetic acid solution.
Analysis of functional properties
Solubility
The solubility of the proteins was analysed at different pH values
(1–14) according to the method described by Cepeda.17 Briefly,
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Characteristics and functional properties of rapeseed protein
10 mg of protein was added to 10 mL of buffer of different pH
values at room temperature. After centrifugation at 4000 × g
for 10 min the protein concentration in the supernatant was
determined by Coomassie brilliant blue staining. The protein
solubility was calculated by the standard concentration curve
method.11
Oil adsorption ability
The oil adsorption ability of the proteins was determined by
the method of Sze-Tao and Sathe.18 Briefly, 0.3 g of protein was
dispersed in 2 mL of soybean oil in a 10 mL centrifuge tube. The
contents were stirred for 30 s every 5 min. After 30 min the tube
was centrifuged at 3000 × g for 30 min. The volume of free oil was
recorded and the oil adsorption ability (mL g−1 ) was calculated.
Emulsifying capacity
To determine the emulsifying capacity, 50 mL of 10 g kg−1 protein
solution in distilled water was stirred at constant temperature
(20 ◦ C) in a 1 L laboratory reactor (IKA, Staufen, Germany). After
adjusting the pH, 50 mL of rapeseed oil was added. The mixture
was homogenised at 15 000 rpm for 5 min and then centrifuged
at 3000 × g for 5 min. The heights of the emulsified layer and total
liquid were recorded.19 The emulsifying capacity was calculated
as follows:
emulsifying capacity (%) = (volume of emulsified layer/
total liquid volume) × 100
Foaming capacity and foam stability
The foaming capacity and foam stability were analysed by the
method of Sze-Tao and Sathe.18 Briefly, 0.5 g of rapeseed protein
was added to 100 mL of buffer (pH 11.6) and stirred for 5 min.
The blend was then transferred to a 250 mL graduated cylinder.
The bubble volume in the graduated cylinder was recorded
immediately. The change in foam volume in the graduated cylinder
was recorded after 120 min of storage. The foaming capacity and
foam stability were calculated as follows (all volumes in mL):
foaming capacity (%) = [(volume after whipping
− volume before whipping)/volume
before whipping] × 100
foam stability (%) = [(volume after standing
− volume before whipping)/(volume after
whipping − volume before whipping)] × 100
The foaming capacity and foam stability of soybean protein
were also measured at pH 8.5.
Surface hydrophobicity measurement of RPs and its fractions
The surface hydrophobicity of soluble proteins was determined
using the fluorescent probe 8-anilino-1-naphthalenesulfonic acid
(ANS). RPs, RPsI or RPsII was added to 0.1 mol L−1 phosphate buffer
(pH 7.4), incubated for 30 min and centrifuged at 10 000 × g for
30 min. The protein content of the supernatant was determined
by staining with Coomassie brilliant blue, and a series of
different protein concentrations was obtained. A 1.2 mL aliquot
of protein solution was mixed with 60 µL of 8 mmol L−1 ANS
in 0.1 mol L−1 phosphate buffer (pH 7.4) and analysed by
fluorescence spectrometry. The operating conditions were an
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excitation wavelength of 380 nm, a fluorescence spectral range
of 400–700 nm and a scanning speed of 500 nm min−1 . The
excitation and emission slits were both set at a bandwidth of
3 nm. The fluorescence emission intensity at 470 nm (FI ) was
recorded together with that of the sample without ANS (FI0 ), and
the fluorescence emission intensity of the protein was calculated
as FI = FI − FI0 . By plotting the protein concentration as the
abscissa and FI as the ordinate, a linear graph was constructed.
The slope of the initial part of the curve was taken as the surface
hydrophobicity index of the protein molecules (S0 ).20
Statistical analysis
Results are expressed as mean ± standard deviation (SD) of three
separate determinations. Average data of triplicate observations
were subjected to one-way analysis of variance (ANOVA) followed
by Duncan’s multiple range test.
RESULTS AND DISCUSSION
Optimisation of conditions for ultrasonic-assisted extraction
The effects of ultrasonic temperature, pH, ultrasonic treatment
time, ultrasonic power and solid/liquid ratio on the extraction
efficiency of rapeseed protein are discussed.
The effect of ultrasonic temperature was evaluated with the pH
set at 11.5, the ultrasonic treatment time at 80 min, the ultrasonic
power at 450 W and the solid/liquid ratio at 1 : 20 (Fig. 2a). The
protein extraction efficiency increased significantly when the
ultrasonic temperature was raised from 25 to 35 ◦ C, remained
steady between 35 and 55 ◦ C and then decreased significantly
from 55 to 65 ◦ C. This final decline can be attributed to the
denaturation of rapeseed protein at high temperature. Thus it
seemed reasonable to set the ultrasonic temperature at 35 ◦ C.
The effect of pH was evaluated with the ultrasonic temperature
set at 35 ◦ C, the ultrasonic treatment time at 80 min, the ultrasonic
power at 450 W and the solid/liquid ratio at 1 : 20 (Fig. 2b). A
significant increase in protein extraction efficiency was observed
with increasing pH. Alkaline pH can loosen the texture of protein
and enhance its solubility. At pH > 12 the irreversible denaturation
of protein occurs and its nutritive value declines. Therefore the
extraction efficiency was highest at pH 11.5.
The effect of ultrasonic treatment time was evaluated with
the ultrasonic temperature set at 35 ◦ C, the pH at 11.5, the
ultrasonic power at 450 W and the solid/liquid ratio at 1 : 20
(Fig. 1c). The protein extraction efficiency increased steadily as the
ultrasonic treatment time was increased from 30 to 80 min, but no
further increase was observed at 90 min. Therefore the extraction
efficiency was highest when the ultrasonic treatment time was set
at 80 min.
The effect of ultrasonic power was evaluated with the ultrasonic
temperature set at 35 ◦ C, the pH at 11.5, the ultrasonic treatment
time at 80 min and the solid/liquid ratio at 1 : 20 (Fig. 2d). The
protein extraction efficiency increased steadily as the ultrasonic
power was increased from 250 to 450 W owing to the increase in
protein solubilisation. There was a slight decline in efficiency when
the ultrasonic power was raised to 500 W. This may be because
high ultrasonic power resulted in a thermal effect, with part of the
protein being denatured and unable to dissolve. Thus 450 W was
selected as the optimum ultrasonic power for protein extraction.
The effect of solid/liquid ratio was evaluated with the ultrasonic
temperature set at 35 ◦ C, the pH at 11.5, the ultrasonic treatment
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time at 80 min and the ultrasonic power at 450 W (Fig. 2e). The
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Figure 2. Effects of (a) ultrasonic temperature, (b) pH, (c) ultrasonic treatment time, (d) ultrasonic power and (e) solid/liquid ratio on extraction efficiency
of rapeseed protein.
protein extraction efficiency increased steadily with increasing
solid/liquid ratio. However, no significant increase was observed
when the solid/liquid ratio was set above 1 : 25.
From these experiments, various parameters were found to
play critical roles in the experimental conditions required for the
optimisation of the extraction efficiency of rapeseed protein, such
as ultrasonic power, ultrasonic treatment time and solid/liquid
ratio, which are generally considered to be the most important
factors. The levels of each factor investigated were selected
according to the results of the single-factor experiments. The
independent variables ultrasonic power (400, 450 and 500 W),
ultrasonic treatment time (70, 80 and 90 min) and solid/liquid ratio
(1 : 20, 1 : 25 and 1 : 30) at three levels of variation were tested using
response surface methodology (RSM). A regression equation was
generated by regression fitting of the response value and each
factor using SAS RSREG software (SAS Institute, Cary, NC, USA).
The following regression equation gave the empirical relationship
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between ultrasonic power (X1 ), ultrasonic treatment time (X2 ),
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solid/liquid ratio (X3 ) and protein extraction efficiency (Y):
Y = 71.3325 + 0.010687X1 + 2.468250X2
+ 0.539187X3 − 3.398062X1 X1 + 0.4295X1 X2
−3.147687X1 X3 − 0.179375X2 X2
+ 0.150250X2 X3 − 1.645062X3 X3
In the regression equation the significant effect of each variable
on the indicator was determined by the F test. If the probability
value was lower than 0.05, the effect of extraction efficiency was
significant. There was no significance in the lack of fit (P > 0.05) of
the model. These results indicated that the model could be used
to predict the response.
The effects of the three factors (ultrasonic power, ultrasonic
treatment time and solid/liquid ratio) on the extraction efficiency
were expressed by the coefficients of a second-order polynomial.
To aid visualisation, the response surface and contour plots of
extraction efficiency are shown in Fig. 3. The three response
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Figure 3. Response surface and contour plots of effects of (a) ultrasonic power and ultrasonic treatment time, (b) ultrasonic power and solid/liquid ratio
and (c) ultrasonic treatment time and solid/liquid ratio on extraction efficiency of rapeseed protein.

surfaces were all convex. The three factors had a parabolic relation
with the extraction efficiency of protein.
Analysis of the regression equation and RSM indicated that
the optimal conditions for ultrasonic-assisted extraction were
an ultrasonic power of 451 W, an ultrasonic treatment time of
protein was 71.33% under these optimised conditions. However,
owing to limitations of the ultrasonic synthesiser settings, the
optimal conditions chosen were an ultrasonic power of 450 W, an
ultrasonic treatment time of 84 min, a solid/liquid ratio of 1 : 24,
a pH of 11.5 and an ultrasonic temperature of 35 ◦ C. To verify
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84 min and a solid/liquid ratio of 1 : 24. The extraction efficiency of the results, an experiment was performed under these optimised

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Table 2. Analysis of amino acid composition (g kg−1 dry matter) of

rapeseed and soybean protein samples

Sample

FAO/WHO
Amino acid SP RPs RP5.8 RP3.6 recommendation

Aspartic acid 69.7 37.2 15.9 13.3

(Asp)
Glutamic acid 143.4 159.6 82.8 91.5
(Glu)
Serine (Ser) 39.4 33.5 32.5 25.7
Glycine (Gly) 25.5 33.3 34.7 23.5
Histidine (His) 37.5 48.0 33.9 48.0
Argnine (Arg) 51.8 37.1 43.5 37.1
Threonine (Thr) 22.2 20.2 25.6 20.2 40.0
Alanine (Ala) 27.6 30.4 27.2 30.4
Proline (Pro) 51.6 50.1 50.1 44.5
Tyrosine (Tyr) 29.4 19.5 27.0 20.0
Valine (Val) 25.6 28.6 26.5 23.6 49.6
Methionine (Met) 19.7 25.4 24.1 25.4
Isoleucine (Ile) 24.3 26.2 25.9 14.0 40.0
Leucine (Leu) 47.0 51.1 44.1 42.1 70.4
Phenylalanine 65.9 55.2 57.3 55.2
(Phe)
Lysine (Lys) 68.1 60.6 62.3 60.6 54.4
Sulfur amino 35.2
acids
Aromatic amino 98.9 74.7 98.3 89.2 60.8
acids
Essential amino 285.8 281.3 279.8 275.1
acids
Non essential 475.9 448.7 347.6 334.0
Figure 4. Gel exclusion chromatogram of ultrafiltrated rapeseed protein amino acids
(RPs).
E/T 375.2 385.3 445.9 457.7 360
E/N 600.5 626.9 804.9 847.8 600
Amino acid score 516.1 505.0 530.0 350.0
Table 1. Protein content of rapeseed and soybean protein samples
First limiting Val Thr Val Ile
Sample Protein content (g kg−1 dry matter) amino acid
Second limiting Thr Val Thr Val
Rapeseed meal 457.0 ± 4.2a amino acid
Rapeseed protein RPs 863.4 ± 5.1b
Rapeseed protein RP5.8 796.3 ± 1.8c
Rapeseed protein RP3.6 704.1 ± 3.2d concentrated and then separated and purified by gel exclusion
Soybean protein SP 896.3 ± 5.0e chromatography.
Values are mean ± SD of three determinations. Means with different A 10 mg mL−1 protein solution was obtained and purified on a
letters differ significantly at P < 0.01 by ANOVA. Sephacryl S-200 column (2.6 cm × 60 cm). The optimal operating
conditions were 10 mg protein mL−1 sample and 0.1 mol L−1
phosphate buffer (pH 10.5) as eluent. The eluate was collected,
conditions. Three repetitions of ultrasonication resulted in an dialysed and concentrated. As shown in Fig. 4, four different
extraction efficiency of 76.83 ± 2.53%, which was close to the fractions (RPsI, RPsII, RPsIII, and RPsIV) were collected and their
theoretical value. In comparison with the traditional water bath content ratio was found to be 8 : 3:1 : 1. From these results the
extraction method, the protein extraction efficiency increased proportions of RPsI and RPsII in the protein extract of rapeseed
by 35.43 ± 1.84% when ultrasonic-assisted extraction was used meal were estimated to be 61.54 and 23.08% respectively. Thus
(protein was precipitated at pH 5.8). Therefore ultrasonication was RPsI and RPsII are the major constituents of rapeseed protein.
significantly better than the traditional method of extraction.
Protein content analysis
Purification of rapeseed protein The rapeseed protein contents obtained with different extraction
The molecular weights of glucosinolate and antinutrients (such methods are shown in Table 1. After extraction the rapeseed
as polyphenols and phytic acid) in rapeseed are generally less protein content increased significantly. The ultrafiltered protein
than 1 kDa, so these compounds can easily co-precipitate at RPs had a higher protein content than the precipitated proteins
acidic pH during the extraction process. Rapeseed protein ob- RP5.8 and RP3.6 (P < 0.01), which had values 8.43% and 22.62%
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tained by ultrasonic-assisted extraction was directly ultrafiltered, less respectively. The protein content of RPs was 3.67% less than

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Characteristics and functional properties of rapeseed protein
Figure 5. Electrophoretic patterns of (1) soybean protein (SP),
(2) ultrafiltrated rapeseed protein (RPs), (3) rapeseed protein precipitated
at pH 5.8 (RP5.8) and (4) rapeseed protein precipitated at pH 3.6 (RP3.6).
that of soybean protein. The amount of protein extracted by
ultrasonication was 8.31% higher than that obtained by the
traditional method. This indicates that the ultrasonic-assisted
method was effective for rapeseed protein extraction. High-power
ultrasonication produces cavitation, which facilitates particle
disintegration and reactions that enhance liquefaction.9
Amino acid analysis
The amino acid composition is an important factor when assessing
protein quality and utility for humans. Using the energy and protein
requirement values of FAO/WHO as a reference,22 the amino acid
composition of rapeseed protein was compared with that of
soybean protein (Table 2). The limiting amino acids in both these
proteins were valine and threonine. However, in comparison with
soybean protein, rapeseed protein had more sulfur-containing
amino acids, indicating that the methionine content of rapeseed
protein is significantly higher than that of soybean protein and
that lysine is enriched in the former.
We used three indices (E/T, E/N and amino acid score) to
evaluate and compare the protein quality of samples.23 These
indices of both rapeseed and soybean proteins were higher than
the FAO/WHO reference values. Moreover, all three indices of
rapeseed protein were better than those of soybean protein,
indicating that the former is a quality protein with good nutritional
characteristics.
SDS-PAGE
β-Mercaptoethanol was used to reduce disulfide bonds in the
protein samples prior to SDS-PAGE. The electrophoretic patterns
of soybean protein (SP), ultrafiltrated rapeseed protein (RPs),
rapeseed protein precipitated at pH 5.8 (RP5.8) and rapeseed
protein precipitated at pH 3.6 (RP3.6) are shown in Fig. 5. The SDS-
PAGE pattern of SP included two main regions: 7S (14.4–22, 22–26,
26–34 and 34–44 kDa) and 11S (44–49, 49–55 and 55–67 kDa)
proteins. In comparison with RPs, the upper bands (>16.5 kDa)
of RP5.8 and RP3.6 became weak. Moreover, between 16.5 and
6.5 kDa the bands of RP5.8 and RP3.6 became light in colour,
especially the latter. It was thus inferred that RPs contained more
protein from rapeseed. Rapeseed protein is mainly composed of
the 12S globulin (cruciferin) and 2S albumin (napin) forms (Fig. 5).
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Figure 6. Two-dimensional electrophoretogram of RPs.
The 12S component of rapeseed protein has a structure similar
to that of glycinin, and the molecular weight (MW) of its native
conformation was approximately 300 kDa. The 2S component of
rapeseed protein is reported to be a strong basic protein, and its
MW ranged from 10 to 14 kDa, which is in agreement with the
values reported elsewhere.3 The polypeptides of each SP subunit
have MWs of 35 and 21 kDa respectively, and the MWs of each
β-conglycinin subunit are 48 and 50 kDa respectively.24
Two-dimensional electrophoresis
The MW distribution of RPs in the two-dimensional electrophore-
togram was identical to that from the standard SDS-PAGE analysis.
Moreover, the distribution of the subunit points was more spe-
cific and accurate. As shown in Fig. 6, the isoelectric points of β
polypeptides in the 12S component were distributed in the pH
range 5.2–8. There were a few proteins with a pI of 5.2, 5.4 or 5.6.
More subunits had a pI between 6 and 8, and their colour was
more intense in the electrophoretogram. However, α polypeptides
in the 12S component were slightly alkaline and their pI ranged
between pH 8 and 10, with mainly pH 8.2–8.6 and 9.4–9.8 at
high contents. Generally, the pI of the 12S component protein
was distributed in two regions: the pI range of weakly alkaline
components with MWs around 20 kDa was pH 8–10 and that of
slightly acidic components with MWs around 30 kDa was pH 6–7.
In the 12S component there were more proteins with pI > 6.8 than
with pI < 6.8. The 12S component was the main basic protein, and
its pI was around pH 8–9, but the pI was less dispersed at other
pH values. This may be the reason for the adverse effects of the
traditional alkaline extraction and acidic precipitation methods on
the extraction yield.
Rapeseed protein showed a wide range of isoelectric points,
which was related to the protein composition. Therefore we
could not precipitate rapeseed protein at a single isoelectric
point such as pH 4.5, 5 or 6 during extraction by the alkaline
extraction or acidic precipitation method. The results indicated
that a multistage precipitation method for extracting rapeseed
protein was necessary, which we verified on a laboratory scale. For
precipitation, solutions at optimum pH values around 6 and 8 could
precipitate most of the rapeseed protein from defatted rapeseed
1495
flakes. Further secondary precipitation at pH values around 5.2
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Functional properties
Table 3. Comparison of surface hydrophobicity and hydrophobic
amino acids of rapeseed and soybean protein samples Solubility
The solubility of rapeseed protein directly affects the amount that
Hydrophobic/
can be used as a food additive, because protein additives influence
Hydrophobic Hydrophilic hydrophilic
Surface amino acids amino acids amino acid the stability of food, and the application of insoluble proteins in
Sample hydrophobicity (%) (%) ratio the food industry is very limited. The solubility of rapeseed and
soybean proteins as a function of pH is shown in Fig. 7. Ultrafiltered
SP 1654.6 ± 5.6a 26.2 ± 2.5a 35.2 ± 3.2a 0.744 ± 0.008a RPs showed good solubility, higher than that of RP5.8 and RP3.6
RPs 1683.2 ± 7.6b 26.7 ± 3.5a 33.2 ± 2.8a 0.804 ± 0.009b at pH 2–8. Although RP3.6 was hardly solubilised at pH 3–9,
RPsI 1778.5 ± 6.3c ND ND ND approximately 90% could be solubilised at pH 12. Although the
RPsII 1765.2 ± 6.7c ND ND ND protein content was higher in the ultrafiltered protein than in the
Values are mean ± SD of three determinations. Means in a column with precipitated protein, the solubility of the ultrafiltered protein was
different letters differ significantly at P < 0.05 by ANOVA. greater than that of the precipitated protein at all pH values tested.
ND, not determined. SP showed minimum solubility at pH 5. In comparison with SP,
RPs showed higher solubility at all pH values tested. The results
indicated that RPs may be a useful replacement for SP at certain
pH values.4

Oil adsorption ability

The oil adsorption ability is a measure of the capacity of a protein
to bind to oil. This characteristic is influenced by factors such as
the method used to process the protein, amino acid composition,
particle size, temperature and the oil used.18 We added soybean
oil to both rapeseed and soybean proteins. The results showed
that the oil adsorption ability of RPs and RP5.8 was better than
that of SP (Fig. 8a). The amino acid composition and particle size
are important factors that influence the oil adsorption ability. The
particle size of RPs was small and thus it could easily diffuse to
the surface of the oil. The hydrophobic polypeptide part extended
into the lipid, resulting in better oil adsorption.

Figure 7. Solubility curves of rapeseed and soybean proteins: •, RPs;
,
RP5.8; , RP3.6; ◦, SP.
and 10 was attempted with the supernatants after centrifugation.
By this secondary precipitation we could extract a further small
amount of protein, thus improving the total protein extraction
yield.21
Surface hydrophobicity of RPs and its fractions
The surface hydrophobicity of proteins was determined using
fluorescence probes (Table 3). The surface hydrophobicity of RPs
and its fractions RPsI and RPsII was found to be relatively stronger
than that of SP, and the higher hydrophobicity of RPs resulted
in lower protein solubility.25 Thus RPs and its fractions had low
solubility.
The surface hydrophobicity of proteins is closely related to the
non-polar aliphatic compound content and aromatic amino acid
distribution on the protein surface. The main hydrophobic amino
acids include Ala, Val, Leu, Ile, Met, Pro and Phe, while the main
polar amino acids (hydrophilic) include Ser, Thr, Asn, Gln, Cys, His,
Tyr and Trp. As shown in Table 3, the hydrophobic and hydrophilic
amino acid contents and hydrophobic/hydrophilic amino acid
ratio were calculated for both RPs and SP. Comparison revealed
that the hydrophobic/hydrophilic amino acid ratio was higher in
RPs than in SP, which is in agreement with the data obtained with
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Emulsifying capacity
Emulsification is one of the most important and basic functions
that can significantly decrease the interfacial tension between oil,
water and the atmosphere in practical applications. This functional
property is affected by many factors, including protein content,
soluble nitrogen index and the presence of carbohydrates in the
protein. In this study we determined the emulsifying capacity of
rapeseed and soybean proteins (Fig. 8b). The emulsifying capacity
of RPs and RP5.8 was higher than that of SP. As the concentration
increased, the thickness and intensity of the interfacial film in
the emulsion were improved, leading to enhanced emulsification
and emulsion stability. Therefore, taking all functional properties
into consideration, RPs is considered to have better functional
properties for food applications.26
Foaming capacity and foam stability
The foaming capacity of a protein depends mainly on its soluble
fraction, but insoluble protein particles may also play a useful role
by improving surface viscosity, thereby leading to the formation
of a stable foam. Data on the foaming properties of rapeseed
and soybean proteins are presented in Fig. 8c. Our results showed
that RPs, RP5.8 and RP3.6 had higher foaming capacity and foam
stability than SP. The foaming capacity and foam stability of
RPs were about 50 and 60% respectively. The lower surface
viscosity of rapeseed protein is suitable for foam formation
and is the theoretical basis for the use of rapeseed as a food
additive.27 Functional properties are important characteristics of
protein products utilised in ground and canned meat formulations,
baked goods, doughnuts, pancakes, soups and whipped cakes and

fluorescence probes. desserts.28

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c 2011 Society of Chemical Industry J Sci Food Agric 2011; 91: 1488–1498
Characteristics and functional properties of rapeseed protein
Figure 8. Comparisons of (a) oil absorption ability, (b) emulsifying capacity and (c) foaming capacity and foam stability of rapeseed and soybean proteins.
CONCLUSION
Ultrasonic-assisted extraction and ultrafiltration methods were
significantly better than the traditional method for the extraction
of rapeseed protein. Qualitative analysis of the isoelectric point can
help in the preparation of rapeseed protein by alkaline extraction
and acidic precipitation. The hydrophobic/hydrophilic amino acid
ratio of rapeseed protein was higher than that of soybean
protein. Rapeseed protein isolates were found to have interesting
functional properties such as good solubility, emulsifying capacity
and emulsion stability. These results indicate the potential use
of rapeseed protein as a replacement for soybean protein in
food production. However, the protein value and digestibility of
rapeseed protein should be further studied.
ACKNOWLEDGEMENTS
This work was supported by the National High Technology
Research and Development Program of China (863 Program:
2007AA10Z328). We also gratefully appreciate receiving financial
support from the Director Foundation of Institute of Oil Crops
Research, Chinese Academy of Agricultural Sciences and the
Youth Dawn Plan of Science and Technology, Wuhan City, China
(201050231023). This work was also supported by the National
Natural Science Foundation of China (Grant No. 81071229),
Specialized Research Fund for the Doctoral Program of Higher
J Sci Food Agric 2011; 91: 1488–1498

c 2011 Society of Chemical Industry
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Education of China (No. 20100142110002), the Fundamental
Research Funds for the Central Universities (Hust, 2010ZD005).
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