JOURNAL OF FUNCTIONAL FOODS

5 (2 0 1 3) 2 1 922 7

Available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/jff

Antioxidant activities of enzymatic rapeseed protein

hydrolysates and the membrane ultrafiltration fractions
Rong Hea,b, Abraham T. Girgihb,c, Sunday A. Malomob,c, Xingrong Jud, Rotimi E. Alukob,c,*
a

College of Food Science, Jiangnan University, Wuxi 214122, China

The Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
c
Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2
d
College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing 210003, China
b

A R T I C L E I N F O

A B S T R A C T

Article history:

In this study, rapeseed protein isolate was hydrolyzed with various proteases to obtain

Received 16 July 2012

hydrolysates that were separated by membrane ultrafiltration into four molecular size frac-

Received in revised form

tions (<1, 13, 35, and 510 kDa). Alcalase hydrolysis significantly (p < 0.05) produced the

27 August 2012

highest yield of protein hydrolysate while Flavourzyme produced the least. The <1 kDa

Accepted 12 October 2012

fraction was the most abundant after the membrane ultrafiltration of the protein hydroly-

Available online 11 November 2012

sates, which indicates that the proteases were efficient at reducing the native rapeseed proteins into low molecular weight peptides. Antioxidant properties of the resulting

Keywords:

hydrolysates and membrane fractions were characterized and results showed the Pep-

Rapeseed protein isolate

sin + Pancreatin (P + P) protein hydrolysate had significantly highest (p < 0.05) scavenging

Antioxidant properties

activity against DPPH radical among the unfractionated enzymatic hydrolysates. But the

Enzymatic hydrolysis
Membrane ultrafiltration
Amino acid composition
Peptide yield

P + P hydrolysate was not as effective as other hydrolysates during long-term inhibition

of linoleic acid oxidation. For most of the samples, fractionation into the <1 kDa peptides
significantly (p < 0.05) improved DPPH and superoxide scavenging properties when compared to the unfractionated protein hydrolysates. Only the <1 kDa fraction showed ferric
reducing antioxidant power and the effect was dose-dependent. Overall, Alcalase and
Proteinase K seem to be more efficient proteases to release antioxidant peptides from rapeseed proteins when compared to P + P, Flavourzyme and Thermolysin.
 2012 Elsevier Ltd. All rights reserved.

1.

Introduction

Oxidative stress occurs as a result of an imbalance between

the productions of reactive oxygen species (ROS) and availability of antioxidant endogenous compounds. Depletion of
endogenous antioxidant compounds and/or excessive production of ROS can damage membranes, proteins, enzymes,
and DNA resulting in the development of chronic disease
conditions (Ray, Huang, & Tsuji, 2012; Yongvanit, Pinlaor, &
Bartsch, 2012). In an effort to design preventive and curative

strategies, reduction in the degree of oxidative stress has been

identified as a key factor in the therapeutic management of
brain disorders, diabetes, cardiac hypertrophy and cardiovascular disease (Bains & Hall, 2012; Lepping et al., 2011; Maulik &
Kumar, 2012). Hence there is the need for development of
antioxidants from natural sources that can prevent the
deleterious effects of ROS. Food-derived antioxidant peptides
that commonly contain 220 amino acid residues are considered natural antioxidant resources in comparison to synthetic
compounds such as butylated hydroxyanisole and butylated

* Corresponding author at: Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2.
Tel.: +1 204 474 9555; fax: +1 204 474 7593.
E-mail address: rotimi.aluko@ad.umanitoba.ca (R.E. Aluko).
1756-4646/$ - see front matter  2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jff.2012.10.008

220

JOURNAL OF FUNCTIONAL FOODS

hydroxytoluene (Haenen et al., 1996; Sarmadi & Ismail, 2010).

Peptides derived from natural sources have been the focus of
growing interest because of their potential health benefits
associated with low molecular weight, low cost, high activity,
easy absorption, and little or no negative side effects (Sarmadi
& Ismail, 2010). Enzymatic hydrolysis of proteins is one effective approach that can be used to release antioxidant peptides
without affecting nutritive value. Various studies have been
conducted to investigate antioxidant properties of food protein-derived peptides and hydrolysates, especially from
animal protein sources like milk, egg, fish, and blood plasma
(Lin, Guo, You, Yin, & Liu, 2012; Najafian & Babji, 2012; Qian
et al., 2011; Sun, Luo, Shen, Li, & Yao, 2012), as well as some
plant proteins like algae, soy, corn, and sunflower (Li et al.,
2010; Park, Lee, Baek, & Lee, 2010; Ren, Zheng, Liu, & Liu,
2010; Sheih, Fang, Wu, & Lin, 2010). The antioxidant properties of these peptides largely depend on enzyme specificity,
degree of hydrolysis, and the nature of the peptides released
including molecular weight, amino acid composition, and
hydrophobicity (Sarmadi & Ismail, 2010).
Rapeseed protein isolate (RPI), which is obtained from rapeseed meal, is considered a suitable source of dietary protein due to its excellent balance of essential amino acid
composition and high bioavailability (Barbin, Natsch, &
Muller, 2011; Dong et al., 2011; Yoshie-Stark, Wada, Schott,
& Wasche, 2006). The inclusion of RPI as edible films to
maintain quality of Seolhyang strawberries (Shin, Jang, Song,
Song, & Bin Song, 2011), and as substitute for milk protein to
reduce vascular and oxidative disturbances have been recently reported (Magne et al., 2009). Moreover, several studies have reported that enzymatic hydrolysis of RPI yielded
peptides and hydrolysates that possess antioxidant (Makinen, Johannson, Gerd, Pihlava, & Pihlanto, 2012; Pan, Jiang,
& Pan, 2011) and antitumor properties in Hela cells (Xue,
Liu, Wu, Zhuang, & Yu, 2010) as well as in vitro inhibition
of angiotensin converting enzyme, a causative agent of
hypertension (Makinen et al., 2012; Yamada et al., 2010). In
regards to antioxidant activity, previous works have limited
information on relationships between antioxidant activities
and molecular size of peptides obtained from different rapeseed protein hydrolysates.
The close interrelationships of protein hydrolysate antioxidant activities with amino acid composition and sequence as
well as peptide molecular weight has generated increased
interest in evaluating efficiency of proteases in releasing antioxidant peptides from RPI. Therefore, this study was aimed at
determining the ability of several proteases to convert RPI
into antioxidant peptides followed by evaluating the relationships of measured antioxidant activities with peptide size and
amino acid composition of the protein hydrolysates.

2.

Materials and methods

2.1.

Materials

The defatted rapeseed meal (DRM) was supplied by COFCO

Eastocean Oil & Grains Industries Co., Ltd., (Zhang Jiagang,
China). The meal was grounded to pass through a 15 mm
screen sieve. Alcalase, Proteinase K, Pepsin, Pancreatin,

5 ( 2 0 1 3 ) 2 1 9 2 2 7

Thermolysin, Flavourzyme, 2,2-diphenyl-1-picrylhydrazyl

(DPPH), Triton X-100, pyrogallol, ethylenediaminetetraacetic
acid (EDTA), hydrogen peroxide, 1,10-phenanthroline, ferrous
sulfate, linoleic acid, ammonia thiocyanate, ferrous chloride,
and reduced L-glutathione (GSH) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and the other analytical
grade reagents were obtained from Fisher Scientific (Oakville,
ON, Canada).

2.2.

Preparation of rapeseed protein isolates (RPI)

RPI was produced from DRM according to the method described by Yoshie-Stark, Wada, and Wasche (2008) with slight
modifications. Briefly, DRM was dispersed in deionized water
(1:15 w/v), adjusted to pH 10.0 with 1 M NaOH, and then
mixed at 45 C for 2 h. The slurry was centrifuged at 10,000g
for 30 min, the supernatant recovered, adjusted to pH 4.5 with
1 M HCl and centrifuged again. The precipitated proteins were
recovered and re-dispersed in deionized water, adjusted to pH
7.0 with 1 M NaOH and freezedried to produce RPI powder.
Protein content of RPI was determined by the modified Lowry
method (Markwell, Haas, Bieber, & Tolbert, 1978).

2.3.
Preparation of rapeseed protein hydrolysates and
membrane fractions
Hydrolysis of RPI was conducted with Alcalase, Proteinase K,
Pepsin + Pancreatin (P + P), Thermolysin and Flavourzyme under different conditions using a pH-stat method (Chabanon,
Chevalot, Framboisier, Chenu, & Marc, 2007). RPI (5% w/v, protein basis) was suspended in deionized water in a reaction
vessel equipped with a stirrer, heated to the appropriate
temperature and adjusted to the appropriate pH prior to the
addition of the proteolytic enzyme; the reaction conditions
are shown in Table 1. Each enzyme was added to the slurry
at an enzyme/substrate ratio (E/S) of 1:25 (based on the protein content of the protein isolate). Digestion was performed
at the above conditions for 4 h; pH of the reaction mixture
was kept constant by the pH-stat with 2 M NaOH except for
the Pepsin reaction. After digestion, the enzyme was inactivated by adjusting the reaction mixture to pH 4.0 with 2 M
HCl followed by immersing the reaction vessel in boiling
water bath for 10 min and undigested proteins were precipitated by centrifugation at 8000g for 60 min. A portion of the
supernatant containing target peptides was freezedried as
the rapeseed protein hydrolysate (RPH) while the remaining
portion was passed through ultrafiltration membranes with
molecular weight cut-off (MWCO) of 1, 3, 5, and 10 kDa using
an Amicon stirred ultrafiltration cell. Ultrafiltration was performed sequentially: first through the 1 kDa and retentate
passed through 3 kDa; retentate from 3 kDa was passed
through the 5 kDa whose retentate was passed through the
10 kDa membrane. The permeate from each MWCO membrane was collected as <1, 13, 35, and 510 kDa peptide fractions, respectively. All the permeates were freezedried and
stored at 20 C until needed for further analysis. The protein
contents of the freezedried RPH and peptide fractions were
determined using the modified Lowry method (Markwell
et al., 1978). The above digestion and fractionation protocols
were performed in triplicate. The percent yield of RPH was

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JOURNAL OF FUNCTIONAL FOODS

Table 1 Enzyme hydrolysis conditions and yield of unfractionated rapeseed protein hydrolysate and fractions obtained
from membrane ultrafiltration.*
Protease

pH

T (C)

Yield (%)
1

Alcalase
Proteinase K
Pepsin +
Pancreatin
Thermolysin
Flavourzyme

8.0
7.5
2.0
7.5
8.0
6.5

50
37
37
37
50
50

RPH

<1 kDa

13 kDa2

35 kDa2

510 kDa2

76.67 0.63a
72.44 1.14b
68.01 1.27c

26.35 1.38a
22.08 0.93b
17.54 0.29c

21.81 0.20a
14.40 1.08b
13.39 0.42b

14.42 0.05a
11.76 1.17b
11.68 0.10b

10.46 0.21a
7.51 0.47b
8.20 0.31b

64.97 0.96d
36.18 0.15e

16.60 1.04c
9.46 1.16d

13.33 0.25b
6.47 0.42c

13.63 0.73a
4.64 0.41c

9.60 1.01a
3.49 0.52c

* Results are presented as mean standard deviation (n = 3). For each column, mean values that contain different alphabets are significantly
different at p < 0.05.
1
Percentage ratio of protein hydrolysate weight/rapeseed protein isolate weight.
2
Percentage ratio of membrane fraction weight/protein hydrolysate weight.

determined as the ratio of peptide weight of freezedried RPH

to the protein weight of substrate RPI. Similarly, percent
yields of the ultrafiltration membrane fractions were calculated as the ratio of peptide weight of each fraction to peptide
weight of the RPH before fractionation.

2.4.

Amino acid composition analysis

The amino acid profiles of the samples were determined using

an HPLC system, after samples were hydrolyzed with 6 M HCl
as described by Bidlingmeyer, Cohen, and Tarvin (1984). The
cysteine and methionine contents were determined after performing acid oxidation (Gehrke, Wall, & Absheer, 1985) while
the tryptophan content was determined after alkaline hydrolysis (Landry & Delhaye, 1992).

2.5.

The scavenging activity of RPH and its peptide fractions

against the DPPH radical was determined using a previously
described method (Aluko & Monu, 2003) with slight modifications for a 96-well clear flat-bottom plate. Samples were dissolved in 0.1 M sodium phosphate buffer, pH 7.0 containing
1% (w/v) Triton X-100. DPPH was dissolved in methanol to a
final concentration of 100 lM. Peptide samples (100 ll) were
mixed with 100 ll of the DPPH solution in the 96-well plate
to a final assay concentration of 0.2, 0.4, 0.6, 0.8, and 1.0 mg/
ml and incubated at room temperature in the dark for
30 min. The absorbance values of the control (Ac) and samples (As) were measured at 517 nm. The control consisted of
sodium phosphate buffer in place of the peptide sample while
GSH was used as the positive control. The percent DPPH radical scavenging activity of the samples was determined using
the following equation:
Ac  As
 100
Ac

The concentration of sample that reduced DPPH radical

scavenging activity by 50% (IC50) was calculated from a nonlinear regression plot of percentage scavenging activity versus
sample concentration.

Superoxide radical scavenging assay

The superoxide radical scavenging activity of sample was

determined by the method developed by Gao, Yuan, Zhao,
and Gao (1998) with slight modifications. An aliquot of 80 ll
(final assay concentration of 1, 2, 3, 4, and 5 mg/ml) of sample
or GSH dissolved in 0.1 M NaOH were mixed with 80 ll of
50 mM TrisHCl buffer (pH 8.3) containing 1 mM EDTA directly
into a clear bottom 96-well plate in the dark. A 40 ll aliquot of
1.5 mM pyrogallol dissolved in 10 mM HCl was then added to
each well. Absorbance of sample and GSH was measured
immediately at 420 nm within 4 min at room temperature.
TrisHCl buffer was used as control and superoxide scavenging activity of peptides was calculated as follows:
Superoxide scavenging activity %

DPPH radical scavenging assay

DPPH radical scavenging activity %

2.6.

DA=mincontrol  DA=min sample

 100
DA=mincontrol

The concentration of sample that reduced superoxide radical scavenging activity by 50% (IC50) was calculated from a
non-linear regression plot of percentage activity versus sample concentration.

2.7.

Chelation of metal ions

The metal chelating activity was measured using a modified

method of Xie, Huang, Xu, and Jin (2008). Peptide sample solution or GSH (final assay concentration of 1, 2, 3, 4, and 5 mg/
ml) was combined with 0.05 ml of 2 mM FeCl2 and 1.85 ml
double distilled water in a reaction tube. Ferrozine solution
(0.1 ml of 5 mM) was added and mixed thoroughly. The mixture was then allowed to stand at room temperature for
10 min from which an aliquot of 200 ll was removed and
added to a clear bottom 96-well plate. A control was also conducted by replacing the sample with 1 ml of double distilled
water. The absorbance values of control (Ac) and sample
(As) at 562 nm were measured using a spectrophotometer.
Percentage chelating effect (%) was calculated using the following equation:
Metal chelating effect%

Ac  As
 100
Ac

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JOURNAL OF FUNCTIONAL FOODS

The effective concentration of sample that reduced absorbance by 50% (EC50) was calculated from a non-linear regression plot of percentage activity versus sample concentration.

2.8.

Ferric reducing power

The reducing power of peptide samples was measured

according to a previously reported method (Zhang, Wang, &
Xu, 2008), which was modified as follows. Peptide or GSH
samples (250 ll) dissolved in 0.2 M sodium phosphate buffer
at pH 6.6 were mixed with 250 ll of buffer and 250 ll of 1%
potassium ferricyanide solution. Control reaction consisted
of double distilled water instead of sample. The final peptide
concentration in the assay mixture was 2, 4, and 6 mg/ml. The
resulting mixture was heated at 50 C and incubated for
20 min. After incubation, 250 ll of 10% of aqueous TCA was
added. Thereafter, 250 ll of peptide/TCA mixture was combined with 50 ll of 0.1% ferric chloride and 200 ll of water
and allowed to stand at room temperature for 10 min. The
solution was centrifuged at 1000g and 200 ll of the supernatant transferred to a clear bottom 96-well plate. The absorbance of the supernatant was read at 700 nm.

2.9.

Inhibition of linoleic acid oxidation

Linoleic acid oxidation was measured using the method

described by Li, Jiang, Zhang, Mu, and Liu (2008b). Peptide
samples (final concentration of 1 mg/ml) were dissolved in
1.5 ml of 0.1 M sodium phosphate buffer (pH 7.0) and the mixture added to 1 ml of 50 mM linoleic acid dissolved in 99.5%
ethanol. For the control assay, 1.5 ml of buffer were added
to the ethanolic linoleic acid solution and mixed. The mixtures were kept at 60 C in the dark for 7 days. At 24-h intervals, 100 ll of the assay solution was mixed with 4.7 ml of
75% aqueous ethanol, 0.1 ml of ammonium thiocyanate
(30% w/v) and 0.1 ml of 0.02 M ferrous chloride dissolved in
1 M HCl. An aliquot (200 ll) of this solution mixture for each
sample was added to clear bottom 96-well microplates and
the degree of color development was measured at 500 nm
after 3 min incubation at room temperature. An increased
absorbance implied an increase in the level of linoleic acid
oxidation.

2.10.

Statistical analysis

Antioxidant assays were conducted in triplicate and analyzed

by one-way analysis of variance (ANOVA). The means were
compared using Duncans multiple range test and significant
differences accepted at p < 0.05.

3.

Results and discussion

3.1.

Yield of RPH and membrane permeates

Five different endopeptidases were employed to hydrolyze

RPI, and resultant RPH was ultrafiltered to separate the
hydrolysates according to their peptide sizes. The hydrolysis
reaction, conditions and peptide yields are shown in Table

5 ( 2 0 1 3 ) 2 1 9 2 2 7

1. Irrespective of the protease employed, there were significant decreases (p < 0.05) in peptide yield as size of peptide increased, indicating hydrolysis of RPI effectively produced low
molecular weight peptides. With exception of Thermolysin
and P + P, yields of peptides with sizes <3 kDa were twice
those of peptides with sizes >3 kDa. The results are similar
to those reported by Girgih, Udenigwe, and Aluko (2011) that
showed yields of hempseed hydrolysate peptides that passed
through the 3 kDa membrane were 3 that of retained
peptides. On the other hand, the enzymes yielded different
results for their various protein hydrolysates. Overall, the
yields of RPH and peptide fractions generated from Alcalase
hydrolysis were always significantly higher (p < 0.05) than values obtained for the other enzymes, which suggests Alcalase
as a more effective protease to release peptides from rapeseed
proteins. Least RPH and peptide fraction yields were obtained
from Flavourzyme hydrolysis, which indicates rapeseed
proteins were resistant to the endoprotease activity of the
enzyme or the exoprotease activity (generates mostly free
amino acids) was predominant. Previous reports have
reported higher yields of Flavourzyme hydrolysates from
bombay duck and barley proteins (Bamdad, Wu, & Chen,
2011; Jin, Wu, & Wang, 2012), which indicates that type of protein substrate probably dictates rate of the enzymes activity.
Based on the results that showed >60% yields of RPH, hydrolysis of rapeseed proteins with Alcalase, Proteinase K, P + P,
and Thermolysin may be more desirable than Flavourzyme.
However, yield alone is not a determining factor for choice
of enzyme during production of bioactive peptides because
potency is also critical.

3.2.

Amino acid composition

The function of any peptide is mostly dependent on its amino

acid composition. The presence of Tyr, Met, His, and Lys has
been shown to contribute greatly to the potency of antioxidant
peptides (Samaranayaka & Li-Chan, 2011; Udenigwe & Aluko,
2011). His in particular is credited with strong radical scavenging activity due to the decomposition of its imidazole ring
(Samaranayaka & Li-Chan, 2011). In addition, the hydrophobic
properties of peptides can enhance their interaction with lipid
targets or entry of the peptides into target organs through
hydrophobic associations, which is favorable to achieving
antioxidant effects (Sarmadi & Ismail, 2010). Hydrolysates obtained from Proteinase K, Thermolysin and Flavourzyme contained higher concentration of hydrophobic amino acid (HAA)
when compared to Alcalase and P + P hydrolysates (Table 2).
The amino acid compositions of various enzymatic hydrolysates revealed that they were rich in Glu, Asp, Arg, and Leu
(Table 2). A previous report has indicated that acidic amino
acids such as Glu and Asp have strong antioxidant effects
due to the presence of excess electrons that can be donated
during interaction with free radicals (Udenigwe & Aluko,
2011). Proteinase K, Thermolysin and Flavourzyme hydrolysates also contained higher concentrations of essential amino
acid (EAA), which indicates better nutritional values when
compared to Alcalase and P + P hydrolysates. The proportion
of amino acids present in the Alcalase RPH is very similar to
values previously reported by Pan et al. (2011), but total amino
acid content is higher in the present report.

JOURNAL OF FUNCTIONAL FOODS

223

5 ( 20 1 3) 2 1 922 7

Table 2 Amino acid composition of rapeseed protein hydrolysates (g/100 g sample).

AA

Alcalase

Proteinase K

Pepsin + Pancreatin

Thermolysin

Flavourzyme

Aspartic/asparagine
Threonine
Serine
Glutamic/glutamine
Proline
Glycine
Alanine
Cysteine
Valine
Methionine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Histidine
Lysine
Arginine
Tryptophan

6.40
3.50
3.54
12.63
4.34
3.82
3.26
1.13
3.51
1.41
2.51
5.11
2.70
3.06
2.58
4.23
5.23
0.94

7.32
3.96
4.26
13.58
4.85
4.30
3.69
1.16
3.87
1.60
2.75
5.69
3.12
3.43
2.96
4.55
5.68
1.00

6.57
3.44
3.49
11.68
4.10
3.92
3.17
0.95
3.66
1.23
2.63
5.06
2.80
3.23
2.49
4.22
5.29
0.81

7.44
4.00
4.00
14.08
4.87
4.35
3.67
1.08
3.97
1.25
3.02
5.84
3.28
3.65
2.98
4.84
6.20
0.93

7.23
3.99
3.86
13.93
4.84
4.21
3.52
1.00
4.40
1.24
3.26
5.41
3.08
3.36
2.73
4.87
6.22
0.89

HAA
PCAA
NCAA
AAA
EAA

27.95
12.04
19.03
6.70
24.27

31.13
13.19
20.89
7.55
26.85

27.64
12.00
18.25
6.84
24.28

31.56
14.02
21.50
7.85
27.50

31.00
13.82
21.16
7.34
27.42

Combined total of hydrophobic amino acids-alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan, proline, methionine, and
cysteine (HAA). Positively charged amino acids- arginine, histidine, lysine (PCAA). Negatively charged amino acids-ASX and GLX (NCAA).
Aromatic amino acids- phenylalanine, tryptophan, and tyrosine (AAA).

3.3.

Radical scavenging activities (RSA)

DPPH is a stable nitrogen centered free radical compound,

and is widely used in the evaluation of peptide, phenolic
and food antioxidant capacity (Karadag, Ozcelik, & Saner,
2009). The ability of RPH and membrane fractions to scavenge
DPPH radical is shown in Fig. 1A. IC50 is a parameter widely
used to measure the antiradical efficiency, and a low IC50 is
indicative of high scavenging activity. For the RPH, the P + P
digest was the most active (lowest IC50 value) against DPPH
followed by Proteinase K, Thermolysin and Alcalase, while
Flavourzyme RPH was the least active. Fractionation of the
RPHs from P + P, Thermolysin and Flavourzyme led to improved DPPH RSA for the <1 kDa peptides (lower IC50 values),
suggesting that the short-chain peptides had higher activity
than the long-chain peptides. The DPPH RSA of the membrane fractions was found to be dependent on their molecular
size for all the protease treatments. Overall, the <1 kDa peptide fractions exhibited the highest DPPH RSA with the lower
IC50 values of 0.450.6 mg/ml, while 310 kDa fractions had
lowest activities with the higher IC50 values in the range of
0.70.9 mg/ml. The high RSA obtained for the <1 kDa peptide
fractions in comparison to the >3 kDa peptide fractions is
similar to similar findings observed for hemp seed and wheat
gluten hydrolysate peptide fractions (Girgih et al., 2011; Kong,
Zhou, & Hua, 2008). The results are also similar to those reported by Li et al. (2008b) who showed that peptides in the
lowest molecular size fraction IV from chickpea protein
hydrolysate had the highest DPPH RSA when compared to
higher molecular peptides present in fractions IIII. In addition, it was observed that the <1 kDa peptide fraction from

P + P digest had significantly highest (p < 0.05) potency against

DPPH radical with the lowest IC50 value of 0.45 mg/ml, when
compared to the protein hydrolysates and peptide fractions
from the other four enzymes (Fig. 1A). Previous work by Pan
et al. (2011) reported a DPPH RSA IC50 value of 0.71 mg/ml
for rapeseed hydrolysate produced from Alcalase-mediated
hydrolysis, which is similar to the 0.62 mg/ml value obtained
for Alcalase RPH in this work. For all the RPH samples and
peptide fractions, the values of IC50 were found to be in the
range of 0.450.91 mg/ml, which are significantly higher
(p < 0.05) than the DPPH RSA of GSH (IC50 0.023 mg/ml), but
lower than values reported for alfalfa leaf (Xie et al., 2008)
and wheat gluten protein hydrolysates (Kong et al., 2008).
Superoxide radical is one of the important free radicals
in vivo that could promote oxidative reactions due to its ability
to reduce transition metals and react with the hydroxyl
radical, and cause damages to vital cell components
(Elias, Kellerby, & Decker, 2008). The scavenging effect of RPHs
and their peptide fractions for superoxide radicals was investigated, and the results are shown in Fig. 1B. Among the RPHs,
the Proteinase K digest was significantly (p < 0.05) the most
potent against superoxide radical with an IC50 value
1.2 mg/ml whereas the P + P RPH was the least potent
(IC50 = 3.2 mg/ml). With exception of the 510 kDa fraction
from Thermolysin and the <1 kDa from Flavourzyme, fractionation generally led to similar or significantly (p < 0.05)
lower (higher potency) IC50 values, which suggests an influence of peptide size on superoxide scavenging ability. In general, the peptides with size <3 kDa had significantly higher
(p < 0.05) superoxide scavenging activities than the peptides
with >3 kDa sizes. Li et al. (2008b) also reported that fraction

1.0

0.8

DE DE

B B

CD
DE

DE

EF

F
G

GHI
HI

GH

GHI
J I

0.6

GHI

GHI
J

K
L

0.4

0.2
M

e
hi
on
at

rz

G
lu
t

ou
Fl
av

er

Pe

ps

Th

in
+P
a

ot
Pr

ym
e

in
m
ol
ys

re
nc

ei
na

A
lc
a

se

at

in

0.0

3-5 kDa

1-3 kDa

<1 kDa

5-10 kDa

A
B

BC

BC

C
DE

ED

F
F

FG

H
I
J

IJ

IJ

J IJ

compounds such as DNA and proteins; these reactions have

been implicated in the pathogenesis of at least some neurodegenerative diseases, in vivo (Mandel, Amit, Reznichenko,
Weinreb, & Youdim, 2006). Therefore, the chelation of transition metal ions by antioxidative peptides could prevent or
reduce the negative effects of the Fe2+-catalyzed generation
of ROS. Fig. 2 illustrates the ability of GSH, RPH and peptide
fractions to chelate Fe2+. Clearly, the unfractionated hydrolysate (RPH) and high molecular weight (510 kDa) fractions
exhibited the strongest chelating capacity (least EC50 values),
while low MW fractions (<3 kDa) possessed weaker (higher
EC50 values) Fe2+ chelating activity (p < 0.05). Similar results
were observed by Tang, Wang, and Yang (2009) on hemp protein hydrolysate with a report that the increased peptide
chain length could lead to higher iron chelating effects. The
strong metal chelating properties of long-chain peptides
may be due to synergistic effects of higher number of amino
acid residues when compared to the shorter peptides. Our results, which showed Flavourzyme RPH with the lowest EC50
value when compared only to the other RPHs are similar to
those reported by Dong et al. (2008) who observed that silver
carp protein hydrolysates from Alcalase and Flavourzyme had
the highest Fe2+ metal chelating activities.

3.5.

Ferric reducing antioxidant power (FRAP)

1
K
L

ne
th
ta

lu
G
5-10 kDa

Fe2+ chelating capacity

Transition metal ions, for instance Fe2+, can catalyze generation of ROS that promote oxidative damage to critical cellular

14

BC

CD

12

DE

10

F
FG

8 GHI

GH

HI

HI

IJ

IJ

HIJ

K
KL

io
ta
th
lu

ou
av
Fl

ne

e
rz
ym

in
m
er
Th

+P
an
Pe
p

si
n

ol

cr
e

as
in
te
Pr
o

ys

at

se

in

0
la

IV (lowest molecular size) from chickpea protein hydrolysate

had the highest superoxide radical scavenging activity when
compared to fractions IIII of higher molecular size. The
Proteinase K 13 kDa fraction was the most potent against
superoxide radical with the lowest IC50 value of 0.63 mg/ml,
which is about 50% more than the value obtained for GSH
(0.32 mg/ml), suggesting that this peptide fraction could be
an effective inhibitor of superoxide-induced damage to cells.
However, in vivo tests are required to confirm utility of these
peptide fractions, especially as health promoting agents in
human nutrition.

16

ca

Fig. 1 Free radical scavenging activities of rapeseed protein

hydrolysates and their membrane ultrafiltration peptide
fractions against DPPH radicals (A) and Superoxide radicals
(B). Scavenging activity is expressed as inhibitory
concentration (mg/ml) of peptide that reduced absorbance at
517 nm (DPPH) or 420 nm (superoxide) by 50% (IC50) when
compared to uninhibited (control) reaction. Bars
(mean standard deviation, n = 3) with different alphabets
have mean values that are significantly different at p < 0.05.

Al

3-5 kDa

chelating activity (EC50,mg/mL)

Fl

av
ou

rz

io

ym

in
m
Th
er

1-3 kDa

2+

< 1 kDa

The FRAP is often used to evaluate the ability of an antioxidant to donate an electron or hydrogen, and some research
have indicated that there is a direct correlation between
antioxidant activities and reducing power of peptide (Li
et al., 2010; Tang et al., 2012). In the present study, only
the 1 kDa peptide fractions showed FRAP activity while

Fe

RPH

Pe

ps

in

+P

Pr
ot

an

ei

cr

ol

ea

ys

tin

K
se
na

al
a
lc
A

0
se

Superoxide scavenging activity (IC50, mg/mL)

RPH

3.4.

5 ( 2 0 1 3 ) 2 1 9 2 2 7

JOURNAL OF FUNCTIONAL FOODS

la
se

DPPH radical scavenging activity (IC50, mg/mL)

224

RPH

<1 kDa

3-5 kDa

1-3 kDa

5-10 kDa

Fig. 2 Ferrous ion chelating capacities of rapeseed protein

hydrolysates and their membrane ultrafiltration peptide
fractions. Chelating activity is expressed as effective
concentration (mg/ml) of peptide that reduced absorbance at
562 nm by 50% (IC50) when compared to uninhibited
(control) reaction. Bars (mean standard deviation, n = 3)
with different alphabets have mean values that are
significantly different at p < 0.05.

JOURNAL OF FUNCTIONAL FOODS

2.8

225

5 ( 20 1 3) 2 1 922 7

3.6.

Inhibition of linoleic acid oxidation

1.6

1.2

0.8

E
F

0.4

G
H

ne
th
ta
lu

av
Fl

ou

rz

ol
m
er
Th

io

ys

ym

in

ti n
ea
cr

Pe

ps

in

Pr

+P

ot

an

ei

Al

na

ca

la

se

se

0.0

2 mg/mL

4 mg/mL

6 mg/mL

Fig. 3 Ferric reducing antioxidant power (FRAP) of the

<1 kDa peptide fraction obtained from membrane
ultrafiltration of rapeseed protein hydrolysates. Glutathione
was tested at 1 mg/ml. Bars (mean standard deviation,
n = 3) with different alphabets have mean values that are
significantly different at p < 0.05.

the RPHs and other membrane fractions had no detectable

activity. Similar to our current results, Cumby, Zhong, Naczk, and Shahidi (2008) also observed the highest reducing
power from canola protein hydrolysate hydrolyzed by Flavourzyme. Thus, the FRAP of the RPHs were concentrated
in the peptides with <1 kDa size and presence of other peptides (>1 kDa) diluted the activity and could have been
responsible for the lack of activity by the RPHs. On the
other hand, <1 kDa fraction exhibited significant (p < 0.05)
dose-dependent FRAP activity as shown in Fig. 3, though
GSH exhibited significantly higher (p < 0.05) activity at a
lower concentration of 1 mg/ml. Our results are similar to
previous reports that showed the 5001500 Da peptide fraction of corn protein hydrolysate (Li, Han, & Chen, 2008a)
and the <1 kDa fraction of African yam bean protein hydrolysate (Ajibola, Fashakin, Fagbemi, & Aluko, 2011) had the
strongest FRAP. A hydrolysate derived from alfalfa leaf proteins with a molecular weight <3 kDa also possessed moderate reducing power with an absorbance of 0.69 at 2 mg/
ml (Xie et al., 2008), which is higher than the absorbance
values obtained in this work for similar peptide concentration. Overall, the <1 kDa fraction from P + P and Flavourzyme at 46 mg/ml concentrations showed significantly
higher (p < 0.05) FRAP than similar fraction from the other
proteases. Thus, the differences in enzyme specificity during proteolysis of rapeseed proteins may have produced
peptides with varied levels of hydrogen or electron donating
amino acid residues. For example, it was reported that Pepsin digestion of buckwheat proteins had a negative effect
on FRAP activity of the protein hydrolysate but subsequent
addition of Pancreatin to the Pepsin digest led to recovery
of activity (Ma & Xiong, 2009).

In biological systems, lipid peroxidation proceeds via a radical-mediated abstraction of hydrogen atoms from methylene
carbons in polyunsaturated fatty acids, which initiates a sequence of reactions that generates aldehydes, ketones and
other potentially toxic substances (Niki, 2010; Winczura,
Zdzalik, & Tudek, 2012). Therefore, inhibition of lipid peroxidation is also an important indicator for measuring antioxidant activity of peptides. The lipid peroxidation inhibition
activities of RPH and GSH were evaluated at 1 mg/ml using a
linoleic acid system and the results obtained after 7 days of
incubation are shown in Fig. 4. From day 2 of the incubation,
there was a rapid increase in absorbance values for the control (uninhibited) reaction, which indicates rapid autoxidation
of linoleic acid oxidation. Addition of peptide inhibitors was
effective in attenuating linoleic acid oxidation up till day 5
of the incubation. Comparing days 6 and 7, the P + P RPH
was least effective in reducing linoleic acid oxidation, though
level of absorbance was still significantly less (p < 0.05) than
that of the control. Next to lose antioxidant power (increased
absorbance) at days 6 and 7 is the Flavourzyme RPH but still
had significantly higher (p < 0.05) inhibitory activity than the
P + P RPH and control. The loss in the ability to inhibit linoleic
acid oxidation may be due to depletion of free electrons,
which again suggests differences in the peptide composition
of the RPHs. This is because the inhibitory activities of Alcalase, Proteinase K, and Thermolysin RPHs did not decrease
significantly throughout the 7-day incubation period when
compared to those of P + P and Flavourzyme RPHs. On the last
day of incubation, GSH was significantly more effective than
the RPHs. A absorbance of control and GSH reached the highest point on the day 4, and gradually decreased in the subsequent 3 days following. These results are in agreement with
the observations of Pownall, Udenigwe, and Aluko (2010)

0.6

0.4

Absorbance at 500 nm

Absorbance at 700 nm

2.1

0.2

C
D
E
0.1
0

Incubation time (days)

Alcalase

Proteinase K

Pepsin+Pancreatin

Thermolysin

Flavourzyme

Control

Glutathione

Fig. 4 Inhibition of linoleic acid oxidation by enzymatic

rapeseed protein hydrolysates at a concentration of 1 mg/
ml. The control contains only linoleic acid and no
antioxidant compound. Results are presented as
mean standard deviation (n = 3). Lines (mean standard
deviation, n = 3) with different alphabets have mean values
that are significantly different at p < 0.05 on days 6 and 7.

226

JOURNAL OF FUNCTIONAL FOODS

and Zhu, Su, Guo, Peng, and Zhou (2011) that have showed
ability of food protein-derive peptides to attenuate linoleic
acid oxidation. However, our results showed that the RPHs
had superior inhibition of linoleic acid oxidation when compared to Alcalase-digests of wheat gluten that lost inhibitory
activity after 3 days and at a concentration of 4 mg/ml (Zhu
et al., 2011).

4.

Conclusions

Our results confirm that the specificity of protease activity

and peptide size have substantial influence on the antioxidant activities of RPHs. The Alcalase and Proteinase K peptide
fractions had less ability to reduce ferric ion but higher capacity to scavenge superoxide radicals and inhibit linoleic acid
oxidation. In contrast, the P + P and Flavourzyme peptide
fractions had high ferric ion reducing capacity but low capacity to scavenge superoxide radicals and inhibit linoleic acid
oxidation. The <1 kDa peptide fractions were generally the
most effective scavengers of free radicals but had weaker iron
chelating ability when compared to peptide fractions with
sizes >3 kDa. The reduced capacity to inhibit linoleic acid oxidation as incubation progressed beyond 5 days was more
prominent P + P and Flavourzyme RPHs, which suggests
depletion of excess electrons at a much faster pace than the
other RPHs. Based on these results, the RPHs obtained from
Alcalase or Proteinase K hydrolysis may be considered potential alternative peptide ingredients to synthetic antioxidants
and can find applications in the food and nutrition industry.
However, a direct relationship still needs to be established between their in vitro and in vivo activities in order to find practical use for the peptides.

Acknowledgements
Funding for this work was provided through Ministry of Science and Technology of Agriculture Lee Technical Achievements
Transformation
Fund
project
(Project
No.
2011GB2C100012) and Natural Science Fund for Colleges and
Universities in Jiangsu Province (Project No. BK2010573). The
research program of Dr. R.E. Aluko is funded by the Natural
Sciences and Engineering Research Council of Canada
(NSERC) through a Discovery Grant.

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