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JOURNAL OF FUNCTIONAL FOODS 5 ( 2 0 1 3 ) 1 8 1 0 –1 8 2 1

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Purification and identification of antioxidant


peptides from corn gluten meal

Hong Zhuanga,1, Ning Tanga,1, Yuan Yuanb,*


a
Department of Food Science and Engineering, Jilin University, Changchun, China
b
Department of Food Quality and Safety, Jilin University, Changchun, China

A R T I C L E I N F O A B S T R A C T

Article history: Corn gluten meal was hydrolyzed by alkaline protease and Flavourzyme to obtain the anti-
Received 27 May 2013 oxidant peptides. The antioxidant activities of the hydrolysates or peptides were evaluated
Received in revised form by free radical scavenging capacity (1,1-diphenyl-2-picrylhydrazyl/2,2-azino-bis(3-ethyl-
23 August 2013 benzothiazoline-6-sulphonic acid) diammonium salt/hydroxyl radical/superoxide radical
Accepted 26 August 2013 anion), metal ion (Fe2+/Cu2+) chelating activity and lipid peroxidation inhibitory capacity.
Available online 18 September 2013 The hydrolysates were separated by ultrafiltration, and those with molecular weight
<10 kDa exhibited highest antioxidant activity in all relevant assays. The hydrolysates were
Keywords: subsequently purified by gel filtration chromatography, and fraction F3 showed the highest
Corn gluten meal antioxidant activity. Three peptides were identified from fraction F3 using LC–ESI–Q–TOF
Antioxidant peptides MS/MS as Leu-Pro-Phe (375.46 Da), Leu-Leu-Pro-Phe (488.64 Da) and Phe-Leu-Pro-Phe
Purification
(522.64 Da). These peptides exhibited good free radical scavenging activity and lipid perox-
Identification
idation inhibitory effect. Thus, corn gluten meal may be used as a potential source of anti-
oxidant peptides for food and nutraceutical applications.
 2013 Elsevier Ltd. All rights reserved.

1. Introduction initiate several diseases, including diabetes, atherosclerosis,


arthritis, coronary heart diseases and cancer (Chandrasekara
Oxidation is an essential process for life to perform biological & Shahidi, 2011). In addition, free radical-induced lipid oxida-
functions such as catabolism of proteins, fats and carbohy- tion is a major concern in the food industry, because the oxi-
drates (Gulcin, 2009). During the oxidation, free radicals such dation of fats and oils during processing and storage of food
as superoxide anion radicals (O2) and hydroxyl radicals (OH) products has a negative effect on the quality and nutritive va-
are generated in the living body (Evans, Goldfine, Maddux, & lue of lipids (Rajapakse, Mendis, Jung, Je, & Kim, 2005). There-
Grodsky, 2003). These free radicals, which are physiologically fore, it is important to inhibit the formation of excessive free
produced, play important roles in biological systems that ex- radicals occurring in the living body and food stuffs (Zhong,
ert diverse functions like signaling roles and providing de- Ma, Lin, & Luo, 2011). Antioxidants can act at different levels
fense against infections (Johansen, Harris, Rychly, & Ergul, in an oxidative sequence, protecting the human body from
2005). Nevertheless, when these radicals are produced in ex- free radicals and retarding the progress of many chronic dis-
cess, they can accumulate in cells and cause harm over time. eases which are influenced by oxidative reactions (Pihlanto,
This can result in protein damage, DNA mutation, oxidation 2006). However, synthetic antioxidants such as butylated
of membrane phospholipids (Lee, Koo, & Min, 2004) and hydroxytoluene (BHT) and butylated hydroxyanisole (BHA),
modification in low density lipoproteins, which in turn can which are widely used in the food industry have potential

* Corresponding author: Tel.: +86 13756062695; fax: +86 043187836364.


E-mail address: downing@163.com (Y. Yuan).
1
These authors contributed equally to this work and are regarded as joint first authors.
1756-4646/$ - see front matter  2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jff.2013.08.013
JOURNAL OF FUNCTIONAL FOODS 5 ( 2 01 3 ) 18 1 0–18 2 1 1811

risks on human health (Pihlanto, 2006; Shahidi & Zhong, meal for another 66 min. At the end of the hydrolysis period,
2008). Thus, more attention has been focused on the develop- the mixture was heated in boiling water for 10 min to inacti-
ment safe and effective functional foods and antioxidative vate the proteases. The hydrolysates were then centrifuged at
agents from natural sources. 10,000g for 10 min at 4 C. The obtained supernatant was ul-
In recent years, hydrolyzed proteins from many animal tra-filtered through a membrane with a cut off molecular
and plant sources such as peanut kernels (Hwang, Shyu, weight of 30 kDa. This process yielded two fractions: retentate
Wang, & Hsu, 2010), rice bran (Revilla et al., 2009), sun flower (fraction 1, represented hydrolysates >30 kDa) and permeate
protein (Megı́as et al., 2008), frog skin (Qian, Jung, & Kim, (MW < 30 kDa). The permeate was further ultra-filtered
2008), milk casein (Blanca, Ana, Lourdes, & Isidra, 2007), egg through a membrane with a cut off molecular weight of
yolk protein (Sakanaka & Tachibana, 2006) and canola (Cum- 10 kDa to obtain the second retentate (fraction 2, represented
by, Zhong, Naczk, & Shahidi, 2008) have been found to possess hydrolysates between 30 and 10 kDa) and permeate (fraction
antioxidant activity. Corn gluten meal is a major by-product 3, represented hydrolysates <10 kDa). The obtained hydroly-
of corn wet milling, containing 60% (w/w) protein. However, sates with different molecular weight distribution were
its low water solubility and severely imbalanced amino acid lyophilized and stored at 20 C until use (no longer than
composition makes it difficult to be used as a food additive. 90 d).
In China, Over 840,000 tonnes of corn gluten meal are pro-
duced every year (Lin et al., 2011). At present, corn gluten 2.3. Determination of antioxidant activities
meal is mainly used as feedstuff or discarded. Producing
hydrolysates with antioxidant properties from corn gluten 2.3.1. DPPH radical scavenging activity assay
could effectively increase its value in the marketplace (Li, The DPPH radical scavenging activity of hydrolysates or pep-
Han, & Chen, 2008a). In the present study, corn gluten meal tides was measured using a modified method of Zhang, Li,
was hydrolyzed by alkaline protease and Flavourzyme to pro- Miao, and Jiang (2011). DPPH radical (2 ml 0.1 mM) dissolved
duce hydrolysates with antioxidant activities. The antioxi- in 95% ethanol was added to 2 ml of sample solution. The
dant activities of hydrolysates with different molecular mixture was shaken and left for 30 min at room temperature.
weight distribution were compared. Moreover, the hydroly- Absorbance of the resulting solution was measured at
sates that exhibited strong antioxidant activities were frac- 517 nm. For the blank, 2 ml of distilled water were used
tionated by gel filtration chromatography. Antioxidant instead of the sample. The radical scavenging activity was
activities of the fractions were determined and peptides that calculated as follows:
showed highest antioxidant activity were identified by LC– DPPH scavenging activityð%Þ
MS/MS. Furthermore, the peptides were synthesized accord- ¼ ½ðBlank absorbance  Sample absorbanceÞ=Blank absorbance
ing to the obtained amino acid sequences to confirm their  100:
antioxidant activities.

2.3.2. ABTS radical scavenging activity assay


2. Materials and methods The ABTS radical scavenging activity of hydrolysates or pep-
tides was determined according to the method of Tironi and
2.1. Materials
Añón (2010) with some modifications. The ABTS radical cat-
ion was generated by mixing ABTS stock solution (7 mM) with
Corn gluten meal was obtained from Dacheng Ltd. (Changc-
potassium persulphate (2.45 mM) and allowing the resultant
hun, Jilin, China). Alkaline protease and Flavourzyme were
mixture in the dark at room temperature for 16 h before
purchased from Pangbo Biological Engineering Co. Ltd. (Nan-
use. The ABTS radical solution was diluted in 0.2 M phosphate
ning, Guangxi, China). 1,1-Diphenyl-2-picrylhydrazyl (DPPH),
buffered saline (pH 7.4), to an absorbance of 0.70 ± 0.02 at
2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diam-
734 nm. The diluted ABTS radical solution (2 ml) was mixed
monium salt (ABTS), ferrozine, linoleic acid and Sephadex G-
with 200 ll of sample. After 10 min, the absorbance was read
25 were purchased from Sigma to Aldrich (Shanghai, China).
at 734 nm. For the blank, 200 ll of distilled water were used
All other chemicals used were of analytical grade.
instead of the sample. The ABTS scavenging activity of
samples was calculated as follows:
2.2. Preparation of hydrolysates with different molecular
ABTS scavenging activityð%Þ ¼ ½ðBlank absorbance
weight distribution
 Sample absorbanceÞ=Blank absorbance  100

The method of preparing the corn gluten meal hydrolysates


was previously developed in our laboratory (Zhuang, Tang, 2.3.3. Hydroxyl radical scavenging activity
Dong, Sun, & Liu, 2013). Corn gluten meal was mixed with The hydroxyl radical scavenging activity of hydrolysates or
deionized water (1:25, w/v). The obtained mixture was ad- peptides was determined according to the method of Wang,
justed to pH 9.5 by adding 0.1 M NaOH and heated in a water Wang, Dang, Zheng, and Zhang (2013) with some modifica-
bath at 55 C. Then the alkaline protease (8%, w/v) was added tions. Briefly, 1 ml of 6 mM FeSO4 solution was mixed with
to hydrolyze the corn gluten meal over a 75 min period. After 1 ml of sample solution and 1 ml of 6 mM H2O2 solution.
being heated in a boiling water bath for 10 min, the pH and The mixture was shaken and left for 10 min at room
temperature were adjusted to 7, 50 C, respectively. Then Fla- temperature. Then 1 ml of 6 mM salicylic acid was added to
vourzyme (4.2%, w/v) was added to hydrolyze the corn gluten the mixture, after 30 min, the absorbance was determined
1812 JOURNAL OF FUNCTIONAL FOODS 5 ( 2 0 1 3 ) 1 8 1 0 –1 8 2 1

at 510 nm. For the blank, 1 ml of distilled water was used in- 2.3.7. Lipid peroxidation inhibition assay
stead of the sample. The hydroxyl radical scavenging activity The lipid peroxidation inhibition activity of corn peptide was
of samples was calculated as follows: measured in a linoleic acid emulsion system according to the
method of Chandrasekara and Shahidi (2012). Samples (1 ml)
Hydroxyl radical scavenging activityð%Þ
were dissolved in 10 ml of 50 mM phosphate buffer (pH 7.0),
¼ ½1  ðAs  A0 Þ=Ab Þ  100%
was added to a solution of 0.13 ml of linoleic acid and 10 ml
where Ab is the absorbance of the blank, As is the absorbance of 99.5% ethanol. The total volume was then adjusted to
of the sample and A0 is the absorbance of the sample without 25 ml with distilled water. The mixture was incubated in at
salicylic acid. 40 ± 1 C for 7 days in a dark room. The degree of oxidation
of linoleic acid was measured using the ferric thiocyanate
2.3.4. Superoxide radical anion scavenging activity method of Chandrasekara and Shahidi (2012). The reaction
Superoxide radical anions were generated by the pyrogallol mixture (0.1 ml) was mixed with 4.7 ml of 75% ethanol,
autoxidation method with some modifications (Li, Jiang, 0.1 ml of 30% ammonium thiocyanate, and 0.1 ml of 20 mM
Zhang, Mu, & Liu, 2008b). Briefly, 1 ml of hydrolysates or pep- ferrous chloride solution in 3.5% HCl. After 3 min incubation,
tides was mixed with 3 ml of 50 mM Tris–HCl buffer (pH 8.2) the color development, which represents the linoleic acid oxi-
containing 1 mM ethylenediaminetetraacetic acid (EDTA) dation, was measured at 500 nm.
and pyrogallic acid (0.5 ml, 4.5 mM). Then the mixture was
shaken rapidly at room temperature. The absorbance of the 2.4. Purification of hydrolysates
mixture was measured at 320 nm every 30 s for 4 min, and a
slope was calculated as the absorbance/min. For the blank, The freeze-dried hydrolysates (50 mg) obtained by treatment
1 ml of distilled water was used instead of the sample. The with alkaline protease and Flavourzyme was dissolved in
superoxide radical anion scavenging activity was calculated 1 ml deionized water and fractionated by gel filtration chro-
as follows: matography on a Sephadex G-25 column (2.6 · 100 cm), and
then eluted with deionized water at a flow rate of 1 ml/
Superoxide anion radical scavenging activityð%Þ
min. The absorbance was measured at 280 nm to determine
¼ ð1  As =Ab Þ  100%
the elution profile of the sample. The eluent with peak at
where Ab is the change speed of absorbance of the blank 280 nm was collected, vacuum concentrated and freeze-
group in the superoxide radical anion generation system dried.
and As is the change speed of absorbance of the sample.
2.5. Identification of peptides by LC/MS/MS
2+
2.3.5. Fe chelating activity
The ability of the hydrolysates or peptides to chelate Fe2+ ions The LC/MS/MS system consisted of a Waters UPLC system
was evaluated by the method of Sabeena Farvin, Baron, Niel- and a Synapt G2 HDMS system with a hybrid quadrupole/
sen, and Jacobsen (2010) with some modifications. Three mil- travelingwave ion mobility/orthogonal acceleration time-
lilitres of sample solution were mixed with 0.1 ml of 2 mM of-flight configuration (Waters, Manchester, UK). The chroma-
ferrous chloride solution, after 3 min, the reaction was initi- tography was performed on a reverse phase C18 column
ated by the addition of 5 mM ferrozine (0.2 ml). The mixture (Waters 4.6 · 150 mm No. 8012515). A binary mobile phase
was shaken vigorously and left at room temperature for was used that consisted of acetonitrile and deionized water
10 min. Absorbance of the resulting solution was measured with 0.1% formic acid. The mass spectrogram instrument
at 562 nm. For the blank, 3 ml of distilled water were used in- was equipped with an electrospray ionization (ESI) source
stead of the sample. The chelating capacity was calculated as operated in the positive ion mode with a capillary voltage of
follows: 3 kV and cone voltage of 25 V. The TOF analyzer was operated
in resolution mode with a scan speed of 5 scans/s and cali-
Fe2þ chelating activityð%Þ ¼ ½ðBlank absorbance brated in the m/z 100–1000 range with sodium formate solu-
 Sample absorbanceÞ=Blank absorbance  100: tion infused at 20 ml/min. The sodium formate solution was
prepared by mixing 0.05 mM sodium hydroxide solution with
0.05% formic acid in propanol/water (90/10; v/v). The collision
2.3.6. Cu2+ ion chelating activity
energy in the trap and transfer ion guide was set at 15 eV and
The ability of hydrolysates or peptides to chelate Cu2+ was
0 eV, respectively. Generic IMS conditions were applied to all
determined according to Zhu, Chen, Tang, and Xiong (2008)
the different sets of compounds analyzed. The mobility
with some modifications. In the chelation assay, 1 ml of
T-wave cell was operated at a pressure of 2.8 mbar of nitro-
2 mM CuSO4 was mixed with 1 ml of 10% pyridine and 20 ll
gen. The wave velocity was ramped from 300 to 600 m/s,
of 0.1% pyrocatechol violet. After the addition of 1 ml of sam-
and the wave height from 15 to 40 V. Data acquisition and pro-
ple, the disappearance of the blue colour was monitored by
cessing were carried out using Masslynx 4.1.
measuring the absorbance at 632 nm after 5 min of reaction.
An equivalent volume of distilled water instead of the sample
2.6. Synthetic peptides
was used for the blank.

Cu2þ chelating activityð%Þ ¼ ½ðBlank absorbance The peptide derived from corn gluten meal was synthesized
 Sample absorbanceÞ=Blank absorbance  100: by the solid phase procedure peptide using FMOC protected
JOURNAL OF FUNCTIONAL FOODS 5 ( 2 01 3 ) 18 1 0–18 2 1 1813

amino acids synthesis methods. The peptides were synthes- differences in substrate, protease type as well as the en-
ised on Fmoc-Met-Wang resin and elongated under standard zyme-to-substrate ratio, all of which will directly affect the
FMOC solid phase peptide conditions in the peptide synthes- extent of hydrolysis.
iser as described previously (Aggarwal, Janssen, Wadkins, The ABTS decolorization assay can be used to determine
Harden, & Denmeade, 2005). The synthesized peptide was antioxidant activity of both lipophilic and hydrophilic mole-
purified by HPLC. The molecular masses of the isolated pep- cules, and is based on the reaction of hydrogen donating anti-
tides were determined by mass spectrometry. oxidants with the ABTS radical, which is intensely coloured
and is determined by measuring absorbance at 734 nm (Pihl-
3. Results and discussion anto Leppala, 2001). As can be seen from Fig. 1B, the ABTS
radical scavenging activity of all hydrolysates was increased
3.1. Antioxidant activities of hydrolysates with different significantly (p < 0.05) with increasing concentration (1–
molecular weight distribution 5 mg/ml). The hydrolysates with Mw < 10 kDa exhibited the
highest ABTS scavenging activity. The scavenging activity
DPPH is a stable free radical even at room temperature. DPPH was 78 ± 2.53% at the concentration of 5 mg/ml. However,
radical with a single electron shows strong absorbance at compared with crude hydrolysates and hydrolysates with
517 nm in ethanol, and its absorbance is reduced gradually Mw > 30 kDa, there was no significant difference (p > 0.05) in
while the free radicals are scavenged by accepting an electron the scavenging activity. The scavenging activities of crude
or hydrogen atom and the colour of the solution changes hydrolysates and hydrolysates of >30 kDa were
from modena to yellow in the presence of a proton-donating 73.52 ± 1.55%, 75.09 ± 1.49%, respectively, at 5 mg/ml. Among
substance (Xie, Huang, Xu, & Jin, 2008). The relatively stable the hydrolysates, hydrolysates of 10–30 kDa showed the low-
DPPH radical has been widely used to test the ability of com- est scavenging activity (52.29 ± 1.9%). However, the ABTS
pounds to act as free radical scavengers or hydrogen donors scavenging activity of all hydrolysates was still lower than
and thus to evaluate the antioxidant activity (Li et al., ascorbic acid (87.26 ± 1.22%). Ruiz-Ruiz, et al. (2008) reported
2008b). As shown in Fig. 1A, all hydrolysates exhibited a con- that hard-to-cook bean hydrolysates with low molecular
centration-dependent DPPH radical scavenging activity. The weight (<1 kDa) showed the highest ABTS radical scavenging
scavenging activity increased significantly (p < 0.05) with the activity (1985.5 mM TEAC/mg protein) among the fractions.
increase in the concentration (100–500 lg/ml). Hydrolysates The amaranth fraction with lower molecular weight pre-
with a molecular weight (Mw) of <10 kDa showed the highest sented a higher ABTS radical scavenging capacity, which
DPPH radical scavenging activity, the scavenging activity was was 83% at 310 lg/ml (Tironi & Añón, 2010). Similar results
34.16 ± 0.79% at 500 lg/ml, which was significantly higher were observed by Cheung, Cheung, Tan, and Li-Chan (2012)
(p < 0.05) than hydrolysates without ultrafiltration or hydroly- that low molecular size peptides below 1400 Da is the domi-
sates with Mw > 30 kDa. However, there was no significant dif- nant factor contributing to the potent ABTS radical scaveng-
ference in scavenging activity between the hydrolysates with ing activity in pacific hake protein hydrolysates.
Mw < 10 and 10–30 kDa (33.39 ± 1.0%). However, the scaveng- Hydroxyl radical is the most reactive free radical, and it
ing activity was significantly lower (p < 0.01) than ascorbic can be formed from a superoxide anion and hydrogen perox-
acid which exhibited 88.29 ± 0.47% inhibition of DPPH radical ide in the presence of metal ions, such as those of copper or
at 500 lg/ml (data was not shown). The result from this study iron. The hydroxyl radical can easily react with biomolecules,
indicated that the hydrolysates with Mw < 10 and 10–30 kDa such as amino acids, lipids, DNA and proteins (You, Zhao,
contained more amino acid groups that could readily donate Regenstein, & Ren, 2010). Therefore, the removal of hydroxyl
electrons to DPPH radical when compared to the larger pep- radicals is probably one of the most effective defenses of a liv-
tides. Since DPPH radical scavenging reaction is a single elec- ing body against various diseases. Determination of hydroxyl
tron transfer (SET) reaction. A number of studies have already radical scavenging activity provides useful information on
shown that the antioxidant activity of peptides is dependent antioxidant activities. The hydroxyl radical scavenging activ-
on their molecular weight distribution (Pena-Ramos, Xiong, ities of different hydrolysates are shown in Fig. 1C. All hydrol-
& Arteaga, 2004). Similarly, many studies have suggested that ysates showed similar hydroxyl radical scavenging activity;
high degree of hydrolysis and low molecular weights were the activities of crude hydrolysates, hydrolysates with
found to correlate well with DPPH radical scavenging activity Mw > 30, 10–30 and <10 kDa were 50.76 ± 1.31%,
(Bougatef et al., 2009; Liu, Kong, Xiong, & Xia, 2010; Raghavan 54.46 ± 1.31%, 55.97 ± 1.5% and 59.44 ± 2.70%, respectively, at
& Kristinsson, 2008; Udenigwe & Aluko, 2011). On the con- 5 mg/ml. The hydroxyl radical scavenging activity was signif-
trary, it was found that barley glutelin hydrolysates of icantly lower (p < 0.01) than that of ascorbic acid
>10 kDa exhibited a DPPH radical scavenging ability of 61.9% (92.32 ± 0.94%). Previous studies have reported that soy pro-
at 1.0 mg/ml. This indicated that large-size peptides pos- tein hydrolysates with molecular weight between 30 and
sessed much greater (p < 0.05) DPPH scavenging activity than 50 kDa showed the highest hydroxyl radical scavenging
the small-size peptides (Xia, Bamdad, Gänzle, & Chen, 2012). capacity (69.75%) (Moure, Dominguez, & Parajo, 2006). How-
In addition, Sabeena Farvin et al. (2010) found that yoghurt ever, peptide fraction from Alcalase chickpea protein hydroly-
hydrolysates with higher molecular weight >30 and sate with the lowest molecular weight showed the highest
10–30 kDa showed significantly (p < 0.05) higher DPPH radical hydroxyl radical scavenging activity (81% at 1.5 mg/ml) (Li
scavenging activity (99.3% and 97.7% at 200 lg/ml) when com- et al., 2008b). Egg yolk protein treated by Bacillus sp. protease
pared to the lower molecular weight fractions. The discrep- scavenged 74% of hydroxyl radical at 0.5 mg/ml (Sakanaka &
ancy of these findings could be explained by the existing Tachibana, 2006). In addition, Peng, Xiong, and Kong (2009) re-
1814 JOURNAL OF FUNCTIONAL FOODS 5 ( 2 0 1 3 ) 1 8 1 0 –1 8 2 1

Hydroxyl radical scavenging activity (%)


ABTS radical scavenging activity (%)
DPPH radical scavenging activity (%)
40 100 80
A B C
80
30 60
60
20 40
40
10 20
20

0 0 0
0 100 200 300 400 500 0 1 2 3 4 5 0 1 2 3 4 5
Concentration (ug/ml) Concentration (mg/ml) Concentration (mg/ml)

20 100 50
D E F
Hydrogen peroxide scavenging

Cu chelating activity (%)


Fe chelating activity (%)
80 40
15
60 30
activity (%)

10
40 20
5
20 10

2+
2+

0 0 0
0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5
Concentration (mg/ml) Concentration (mg/ml) Concentration (mg/ml)

5
Control G
Absorbance at 500nm

4 hydrolysates
>30 kDa
10-30 kDa
3
<10 kDa
BHT hydrolysates >30 kDa 10-30 kDa <10 kDa
2

0
0 1 2 3 4 5 6 7
Incubation time (day)

Fig. 1 – Antioxidant activities of hydrolysates with different molecular weight distribution. (A, B, C, D)-Free radical (DPPH/
ABTS/O2/OH) scavenging activities of hydrolysates with different molecular weight at different concentrations. (E, F)-The
metal ion (Fe2+/Cu2+) chelating activities of hydrolysates with different molecular weight at different concentrations. G-Lipid
peroxidation inhibition activity of hydrolysates with different molecular weight at 1 mg/ml.

ported that the hydroxyl radical scavenging activity observed different molecular weights was between 24.7% and 85.6%
in their study for whey protein hydrolysate fractions could be and was increased with decreasing molecular weight (Moure
attributed to the hydrolysates, which they reported to possess et al., 2006). The scavenging activity against superoxide rad-
poor Fe2+ chelating activities. ical for wheat germ protein hydrolysates (0–600 lg/ml) ran-
Numerous biological reactions generate superoxide radi- ged from 0% to 75.40% (Cheng, Wang, & Xu, 2006).
cal anion which is a highly toxic species. This radical is po- Udenigwe, Lu, Han, Hou, and Aluko (2009) found that the
tential precursors of highly reactive species, such as higher molecular weight peptide fractions from flaxseed pro-
hydroxyl radical, and thus study of the scavenging of this tein showed stronger superoxide radical anion scavenging
radical is important (Kanatt, Chander, & Sharma, 2007). In activity with lower IC50. However, no relationship between
this study, superoxide radical anion was generated from superoxide radical anion scavenging ability and peptide
autoxidation of pyrogallol. The scavenging activities of dif- molecular weight could be found in barley glutelin hydroly-
ferent hydrolysates were presented in Fig. 1D. Differed from sates (Xia et al., 2012). Our results indicated that the corn
other free radical scavenging activity assay, all hydrolysates gluten meal hydrolysate is not a good superoxide radical an-
did not show concentration-dependent superoxide radical ion scavenger. This may be due to the fact that molecular
anion scavenging activity. The scavenging activity was very weight might not be the most important factor leading to
low, but hydrolysates with Mw < 10 kDa still exhibited the scavenging activity of peptides in the superoxide radical an-
highest activity (13.96 ± 0.4% at 5 mg/ml) among the ion scavenging activity assay.
hydrolysates. In addition, the scavenging activities of all Transition metal ions, such as Fe2+ and Cu2+, can catalyze
hydrolysates were significantly (p < 0.01) lower than ascorbic the generation of reactive oxygen species, resulting in lipid
acid (88.94 ± 0.77%). The superoxide radical anion scavenging peroxidation and DNA damage (Stohs & Bagachi, 1995). Espe-
activity of soy protein hydrolysates from the fractions with cially, Fe2+ generates hydroxyl radical by the Fenton reaction
JOURNAL OF FUNCTIONAL FOODS 5 ( 2 01 3 ) 18 1 0–18 2 1 1815

that could accelerate the lipid peroxidation chain reaction. shown in Fig. 1G, hydrolysates (crude, 10–30 and <10 kDa)
Moreover, Fe2+ catalyzes the breakdown of lipid peroxides, exhibited significant (P < 0.05) lipid peroxidation inhibitory
which leads to the formation of off-flavour. Therefore, the activity in the linoleic acid system. However, the hydrolysates
chelation of transition metal ions by peptides would retard with Mw > 30 kDa accelerated the oxidation of linoleic acid in
the oxidation reaction. In the present study, the Fe2+ and the first 4 days. At the end of the 7-day lipid peroxidation, the
Cu2+ chelating activities of the different fractions were com- inhibition activities were 44.51%, 9.01%, 65.35% and 67.04% for
pared with that of EDTA. As shown in Fig. 1E, the lower molec- crude hydrolysates, hydrolysates with MW > 30, 10–30 and
ular weight hydrolysates (10–30 and <10 kDa) showed <10 kDa, respectively. Among the hydrolysates, hydrolysates
significantly higher Fe2+ chelating activity than the higher with MW < 10 kDa exhibited the highest inhibitory activity
molecular weight fractions or crude hydrolysates. The chelat- (p < 0.05). However, its inhibitory activity was significantly
ing activities were 84.82 ± 0.26% and 89.65 ± 3.61%, respec- lower (p < 0.01) than that of the positive control, BHT, a well
tively, at 5 mg/ml. There was no significant (p > 0.05) known antioxidant, during 7 days of oxidative reaction. The
difference between hydrolysates with Mw < 10 kDa; EDTA inhibitory activity was 94.99% at the end of 7 days. Some
showed a chelation capacity of 90.9%. This result clearly researchers reported peptides <3000 Da are more effective in
shows that the lower molecular weight fractions contained inhibiting the lipid peroxidation (Mendis, Rajapakse, & Kim,
small peptides that could chelate metal ions like Fe2+. As ob- 2005). However, Cheung et al. (2012) found that pacific hake
served in the present study (Fig. 1F), the Cu2+ chelating activ- hydrolysates with a range of molecular weights were all effec-
ities of all hydrolysates were significantly lower (p < 0.01) than tive in inhibiting lipid peroxidation in the linoleic acid model
Fe2+ chelating activities. At a concentration of 1 mg/ml, only system. In addition, it has been reported that peptide se-
hydrolysates with Mw < 10 kDa showed chelating activity quence was important for the antioxidant activity of low
(6.31 ± 0.51%). The activities of crude hydrolysates, hydroly- molecular weight peptides in the lipid peroxidation model
sates with Mw > 30, 10–30 and <10 kDa were 7.85 ± 1.82%, system. Our results indicated that hydrolysates with lower
11.35 ± 1.17%, 36.64 ± 1.0% and 37.47 ± 1.62%, respectively, at molecular weight were more effective in inhibiting the perox-
5 mg/ml, which were significantly lower than EDTA. The idation of linoleic acid. These results agreed with the free rad-
cause for the poor copper binding was not clear, but it may ical scavenging activities and metal ion chelating activities
be related to the greater number of coordination sites that analyzed above.
are required for copper chelation as compared to those for Numerous studies have investigated the effect of molecu-
iron chelation (Kong & Xiong, 2006). Chang, Wu, and Chiang lar size on bioactivity through fractionation by ultrafiltration
(2007) reported a Fe2+ chelating ability ranging from 8% to (Je, Park, & Kim, 2005; Kim, Je, & Kim, 2007; Liu et al., 2010;
63% for hydrolysates derived from porcine haemoglobin at Mendis et al., 2005). Although low molecular size peptides
5.0 mg/ml. Zein and chickpea protein hydrolysates showed have often been associated with high antioxidant activity,
poor Fe2+ chelating ability, even at 30–40 mg/ml concentration there is a lack of evidence to suggest which molecular weight
(Kong & Xiong, 2006; Li et al., 2008b). Recently, it was reported range encompasses the most potent peptides. Some research-
that high His contents (20–30%) and small peptides (105– ers have reported that peptides below 1500 Da as possessing
1205 Da) provided the highest copper chelating activity (Tor- potent antioxidative activity (Je et al., 2005), while some sug-
res-Fuentes, Alaiz, & Vioque, 2011). In addition, some studies gested hydrolysates with high molecular weight (>30 or 10–
have reported that higher DH, and thus lower molecular 30 kDa) are more effective to scavenge free radicals (Sabeena
weights, result in increased metal ion chelating ability (Ale- Farvin et al., 2010) and others reported <3000 Da to be the po-
mán, Giménez, Pérez-Santin, Gómez-Guillén, & Montero, tent fraction (Mendis et al., 2005). In addition, Wu, Chen, and
2011; Nalinanon, Benjakul, Kishimura, & Shahidi, 2011), while Shiau (2003) found that mackerel hydrolysates of molecular
others proposed high molecular weights for potent metal ion weights around 1400 Da exhibited much stronger inhibition
chelators, which could form a caged structure for metal ion against lipid oxidation than lower molecular weight fractions
entrapment (Bamdad, Wu, & Chen, 2011). It has been reported at 900 and 200 Da. This discrepancy indicated that molecular
that a ‘‘cage structure’’ in metallothionein would facilitate weight may not be the most important factor that influenced
binding of copper by thiol groups. the antioxidant activity of hydrolysates or peptides. The spe-
Assays measuring DPPH radical scavenging, ABTS radical cific peptide composition and properties might play a more
scavenging and metal ion chelating have been widely used crucial role.
to assess the antioxidant potential of hydrolysates or pep-
tides. However, each of these assays measures an antioxidant 3.2. Antioxidant activities of different fractions that
property representing a different mechanism, which may not fractionated by gel filtration chromatography
reflect the efficiency of the samples in retarding or inhibiting
lipid oxidation in a food system. Lipid oxidation is the main To purify corn antioxidant peptides, hydrolysates with molec-
cause of deterioration of food quality, leading to rancidity ular weight <10 and 10–30 kDa, which exhibited higher anti-
and shortening of shelf life (Pihlanto, 2006). Lipid oxidation oxidant activities, were separated by gel filtration
products react with proteins causing their oxidation. Carbo- chromatography. The hydrolysates were dissolved in distilled
hydrates are also susceptible to oxidation, but they are less water and loaded onto a Sephadex G-25 gel filtration column
sensitive than lipids and proteins. Therefore, in this study, (2.6 cm · 100 cm), which had been previously equilibrated
the ability of the hydrolysates to suppress lipid peroxidation with distilled water. Five fractions, named F1–F5, were col-
in a linoleic acid model system was investigated. BHT (butyl- lected separately (Fig. 2). Each fraction was pooled, concen-
ated hydroxytoluene) was used as a positive control. As trated and freeze dried, and their antioxidant activities were
1816 JOURNAL OF FUNCTIONAL FOODS 5 ( 2 0 1 3 ) 1 8 1 0 –1 8 2 1

determined. As shown in Fig. 3, all fractions exhibited con- model system. Lower absorbance at 500 nm indicated higher
centration-dependent free radical scavenging activities. The lipid peroxidation inhibition. F3 still showed the highest anti-
scavenging activities were increased significantly with the in- oxidant activity, which exhibited 82.03% inhibition of linoleic
crease in the concentrations. Fraction F3 with lower molecu- acid peroxidation. The activities of F1, F2, F4 and F5 were
lar weight exhibited the highest free radical scavenging 72.8%, 44.73%, 78.16% and 58.34%, respectively. However, the
activity. The DPPH radical scavenging activity, ABTS radical activity of all fractions was significantly lower (p < 0.01) than
scavenging activity and hydroxyl radical scavenging activity BHT.
of fraction F3 was 33.71 ± 1.15%, 86.95 ± 1.27% and
43.85 ± 2.84%, respectively, at 1 mg/ml. Meanwhile the frac- 3.3. Identification of peptides in F3
tion F5 also exhibited the similar but lower free radical scav-
enging activity. However, in comparison with ascorbic acid, The fraction F3 that exhibited the highest antioxidant activi-
the free radical scavenging activities of all fractions were sig- ties was indentified by LC–MS/MS. The peptides in F3 fraction
nificantly lower (p < 0.01). These agreed with the ultrafiltra- were separated by LC. The peptides with the strong signal
tion results that hydrolysates or peptides (obtained from were analyzed by MS/MS. As shown in Fig. 4, three peptides
corn gluten meal) with lower molecular weight showed high- were identified from fraction F3: Leu-Pro-Phe (375.46 Da),
er free radical scavenging activities. In addition, in the metal Leu-Leu-Pro-Phe (488.64 Da) and Phe-Leu-Pro-Phe
ion chelating activity assay (Fig. 3D), all fractions exhibited a (522.64 Da). Bioactive peptides usually contain 2–20 amino
concentration-dependent chelating activity. The Fe2+ chelat- acid residues that could cross the intestinal barrier and exert
ing activities of all fractions increased significantly (p < 0.05) biological effects (Pihlanto Leppala, 2001; Shahidi & Zhong,
with increasing fraction concentrations (200–1000 lg/ml). 2008). It is believed that the antioxidant peptides possess
Similar to free radical scavenging activities, fraction F3 still some metal-chelation or hydrogen/electron donating activity,
showed the highest activity. The Fe2+ chelating activities were which could make them interact with free radicals and termi-
84.73 ± 1.89%, 39.64 ± 6.06%, 89.62 ± 0.32%, 78.3 ± 1.3% and nate the radical chain reaction. In the present study, the iden-
78.29 ± 0.98% for F1–F5, respectively, at 1 mg/ml. There was tified peptides exhibited a high content of hydrophobic amino
no significant (p > 0.05) difference between fraction F3 and acid residues such as Leu, Pro and Phe, which comprise 100%
EDTA which showed a chelation capacity of 89.9% at 1 mg/ of the total residues. It has been reported that hydrophobic
ml. The lipid peroxidation inhibition activity of the fractions amino acids have a significant effect on radical scavenging
was also analyzed. As can be seen from Fig. 3E, all fractions (Rajapakse et al., 2005). For peptides, high content of hydro-
showed lipid peroxidation inhibitory activity in a linoleic acid phobic amino acids may increase their antioxidant activity.

0.8 B F3 0.8 C
F5
Absorbance at 280nm
Absorbance at 280nm

0.6 0.6

0.4 F1 0.4

F4
F2
0.2 0.2

0.0 0.0
0 50 100 150 200 250 300 0 50 100 150 200 250 300
Elution Volume (ml) Elution Volume (ml)

5 A
4
Absorbance

2
<10 kDa
10-30 kDa
1

200 300 400 500 600 700 800


Wavelength (nm)

Fig. 2 – Elution profile of hydrolysates with MW < 10 kDa (B) and 10–30 kDa (C) separated by gel filtration chromatography on
Sephadex G-25 column. The column (2.6 · 100 cm) was equilibrated and eluted with distilled water at a flow rate of 1 ml/min.
JOURNAL OF FUNCTIONAL FOODS 5 ( 2 01 3 ) 18 1 0–18 2 1 1817

40 100 50
10ug/ml A 10ug/ml B 10ug/ml C
100ug/ml 100ug/ml 100ug/ml

Hydroxyl radical scavenging activity (%)


DPPH radical scavenging activity (%)

ABTS radical scavenging activity (%)


1000ug/ml 80 1000ug/ml 40 1000ug/ml
30

60 30

20

40 20

10
20 10

0 0 0
F1 F2 F3 F4 F5 F1 F2 F3 F4 F5 F1 F2 F3 F4 F5
Fraction Fraction Fraction

100 4
F1 D Control E
F2 F1
80 F3 F2
F4 3
Fe chelating activity (%)

F3
F5
Absorbance at 500nm

F4
60 F5
BHT
2

40
2+

1
20

0 0
0 200 400 600 800 1000 0 1 2 3 4 5 6 7
Concentration (µg/ml) Incubation time (day)

Fig. 3 – Antioxidant activities of fractions isolated by gel filtration chromatography. (A, B, C)-Free radical (DPPH/ABTS/OH)
scavenging activities of fractions isolated by gel filtration chromatography at different concentrations. (D)-Fe2+ chelating
activities of fractions isolated by gel filtration chromatography at different concentrations. (E)-Lipid peroxidation inhibition
activity of fractions isolated by gel filtration chromatography at 1 mg/ml.

Similar results were also reported by You et al. (2010). There- 2010). This could explain the very strong Fe2+ chelating capac-
fore, these hydrophobic amino acids present in the sequences ity of corn peptides. The presence of aromatic amino acid
may have significant antioxidant properties. As can be seen phenylalanine allows direct electron transfer to reactive oxy-
from the obtained sequences, all peptides contained the se- gen species (ROS). The results are in accordance with Chen,
quence of Leu-Pro-Phe. So this sequence may play an impor- Chi, Zhao, and Lv (2012) who also found that Phe played
tant role in the antioxidant activity of corn peptide. In important roles in the antioxidant activity of the peptides.
addition, the presence of a terminal Leu was also shown to
be associated with the antioxidant activity of corn peptides 3.4. Antioxidant activities of synthesized peptides
(Ma, Xiong, Zhai, Zhu, & Dziubla, 2010). Moreover, peptides
that contained a terminal Leu obtained from corn gluten meal Peptides Leu-Pro-Phe, Leu-Leu-Pro-Phe and Phe-Leu-Pro-Phe
exhibited high antioxidant activity were also reported by Tang were identified by HPLC and mass spectrometry. The HPLC re-
et al. (2010). Meanwhile, the peptides that contained a termi- sult showed that the purities of the peptide LPF, LLPF and FLPF
nal Leu obtained from other protein sources also exhibited were 98.18% 98.04% and 98.06%, respectively. Mass spectra of
the antioxidant activity (Alemán et al., 2011). Many explana- those peptides showed the correct molecular weight of syn-
tions have been put forth to explain the antioxidant proper- thetic peptides. As shown in Table 1, all three peptides exhib-
ties of peptides. For example, the antioxidative potency of ited free radical scavenging activities. The peptide FLPF
peptides containing Leu has been attributed to its long showed the highest DPPH radical scavenging activity and
aliphatic side chain group that conceivably is capable of the IC50 value was 1.51 mM. The IC50 values of peptides LPF
interaction with acyl chains of susceptible fatty acids (Mendis and LLPF were 2.07 and 2.22 mM, respectively. In the ABTS
et al., 2005). Metal chelating amino acid residues Arg were de- radical scavenging activity assay, the peptide LLPF exhibited
tected in the sequences, which have been reported to interact the highest activity with the IC50 value 2.11 mM. However,
with metal ions through their charged groups (Zhang et al., their free radical scavenging activities were significantly low-
1818 JOURNAL OF FUNCTIONAL FOODS 5 ( 2 0 1 3 ) 1 8 1 0 –1 8 2 1

Fig. 4 – Identification of amino acid sequences of peptides in F3 by LC–ESI–Q–TOF MS/MS.


JOURNAL OF FUNCTIONAL FOODS 5 ( 2 01 3 ) 18 1 0–18 2 1 1819

Table 1 – Free radical scavenging activities of synthesized peptides.


Peptide Purity % IC50 (DPPH radical IC50 (ABTS radical
scavenging activity) (mM) scavenging activity) (mM)

Leu-Pro-Phe 98.18 2.07 2.70


Leu-Leu-Pro-Phe 98.04 2.22 2.11
Phe-Leu-Pro-Phe 98.06 1.51 2.83
Ascorbic acid 99.0 0.283 0.367

Control
3 Leu-Pro-Phe
Leu-Leu-Pro-Phe
Phe-Leu-Pro-Phe
Glu-Cys-Gly
Absorbance at 500nm

0
0 1 2 3 4 5 6 7
Incubation time (day)

Fig. 5 – Lipid peroxidation inhibition activity of synthesized peptides at 500 lg/ml.

er than ascorbic acid. In addition, as can be seen from Fig. 5, and FLPF were 2.07, 2.22 and 1.51 mM, respectively. In the ABTS
all three peptides effectively inhibited lipid peroxidation in radical scavenging activity assay, the IC50 values of peptides
the linoleic acid system. At a concentration of 500 lg/ml, LPF, LLPF and FLPF were 2.70, 2.11 and 2.83 mM, respectively.
the peptide LPF showed the highest antioxidant activity, In addition, these peptides effectively inhibited lipid peroxida-
which exhibited 72.84% inhibition of linoleic acid peroxida- tion in the linoleic acid model system. These results suggest
tion. The activities of peptide LLPF and FLPF were 65.13% that hydrolysates from corn gluten meal could be used as nat-
and 68.67%, respectively. However, the activity of the peptides ural antioxidants for enhancing antioxidant properties of
for lipid peroxidation inhibition was lower than that of GSH functional foods and for preventing oxidation reactions in food
(77.59%) (a well known antioxidant peptide). These results processing. Further research should be carried out in order to
that these peptides from corn gluten meal could be used as purify and identify antioxidative peptides in the other frac-
natural antioxidant. tions collected by gel filtration chromatography.

Acknowledgements
4. Conclusions
This work was funded by the Key Projects in the National Sci-
Corn gluten meal can effectively be hydrolyzed by alkaline pro-
ence and Technology Pillar Program during the Twelfth Five-
tease and Flavorzyme to obtain peptides with strong antioxi-
Year Plan Period (2012BAD33B03). Special thanks to Changc-
dant activities. The activity assessment for fractions
hun Center of Mass Spectrometry, Chinese Academy of
separated by ultrafiltration showed that hydrolysates
Sciences.
(Mw < 10 kDa) possessed greater free radical scavenging activ-
ity, metal ion chelating activity and lipid peroxidation inhibi-
tory capacity. The hydrolysates were subsequently
fractionated into 5 fractions by gel filtration chromatography. R E F E R E N C E S

Fraction F3 exhibited the highest antioxidant activities. Three


peptides were identified from fraction F3 using LC-ESI-Q-TOF
MS/MS: Leu-Pro-Phe (375.46 Da), Leu-Leu-Pro-Phe (488.64 Da) Aggarwal, S., Janssen, S., Wadkins, R. M., Harden, J. L., &
Denmeade, S. R. (2005). A combinatorial approach to the
and Phe-Leu-Pro-Phe (522.64 Da). These peptides exhibited
selective capture of circulating malignant epithelial cells by
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