Food Chemistry 135 (2012) 12451252

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Food Chemistry
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Analytical Methods

ACE inhibitory peptides and antioxidant peptides derived from in vitro

digestion hydrolysate of hen egg white lysozyme
Shengqi Rao a, Jun Sun b, Yuntao Liu b, Huawei Zeng c, Yujie Su b, Yanjun Yang b,
a
School of Food Science and Engineering, Yangzhou University, Jiangsu, Yangzhou 225127, China
b
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Jiangsu, Wuxi 214122, China
c
Angel Yeast Co. Ltd., Hubei, Yichang 443003, China

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

Article history: Lysozyme from hen egg white is a well-known antimicrobial protein with high ratio of hydrophobic and
Received 24 October 2011 positively charged amino acid residues. In order to explore functional bioactivities of enzymatic hydrol-
Received in revised form 12 April 2012 ysates of lysozyme, the protein was subjected to a simulated gastrointestinal digestion and the resulting
Accepted 11 May 2012
hydrolysate (LPH2) showed a strong competitive angiotensin I-converting enzyme (ACE) inhibitory activ-
Available online 19 May 2012
ity (IC50 = 12.6 lg/ml) and a remarkable antioxidant activity. The LPH2 was fractionated using a 3 kDa
cut-off membrane and the obtained permeate LPH23 kDa was analysed by MALDI-TOF-TOF MS. Using
Keywords:
this technology, 38 different peptides were identied and some of these peptides were well t with struc-
Angiotensin I-converting enzyme
ACE inhibitory peptide
ture requirements of ACE inhibitory peptides and/or antioxidant peptides. The ndings from this study
Antioxidant peptide suggest that the protein containing high proportion of hydrophobic and positively charged residues have
Lysozyme the potential to generate multifunctional peptides, and these peptides would be benecial ingredient to
Gastrointestinal enzymes be used in functional foods.
MALDI-TOF-TOF Crown Copyright 2012 Published by Elsevier Ltd. All rights reserved.

1. Introduction
Hypertension is a major risk factor for cardiovascular and end
stage renal diseases. It can affect people of all ages and endangers
approximately one billion worldwide (Murray & FitzGerald, 2007).
Since angiotensin I-converting enzyme (ACE) (EC 3.4.15.1) plays a
critical physiological role in raising blood pressure, the inhibition
of ACE activity can lead to an overall antihypertensive effect.
Endogenous and exogenous reactive oxygen species and free radi-
cals also have been implicated in the occurrence of hypertension
and other degenerative diseases. The amount of these reactive
species is controlled by endogenous antioxidants until it reaches
a level when the antioxidants are overwhelmed, a state known
as oxidative stress. Exogenous dietary antioxidants can decrease
the contribution of exercise-induced oxidative stress and improve
the animals physiological condition (Yu et al., 2006). Although
having obvious functional effects for improving human health,
synthetic ACE inhibitors and antioxidants are reported to have
various undesirable side effects (Murray & FitzGerald, 2007) and
potential health hazards (Barlow & Schlatter, 2010). Recently, pro-
tein hydrolysates and constituent peptides with ACE inhibitory
activities or antioxidant activities have received considerable
Corresponding author. Tel./fax: +86 510 85863566.
E-mail addresses: raoshengqi2001@yahoo.com.cn (S. Rao), yangyj@jiangnan.
edu.cn (Y. Yang).
interest from the food industry because of the consumers concern
over the safety of the synthetic counterpart.
Some enzymatic hydrolysates of food staffs or proteins, such as
axseed protein (Udenigwe & Aluko, 2010), camel milk casein
(Salami et al., 2011), protein isolate from pumpkin oil cake (Vastag,
Popovic, Popovic, Krimer, & Pericin, 2011) and others, have been
reported to exert both ACE inhibitory and antioxidant activities.
The combination of ACE inhibitory and antioxidant activities in
protein hydrolysates or peptides could be very helpful for the con-
trol of cardiovascular diseases by synergies of different regulatory
mechanisms. However, most of reported enzymatic hydrolysates
of various food protein sources show low bioactivities. Although
the active peptides puried from these hydrolysates exhibit potent
inhibitory potencies, it is very difcult for their commercialisation
because of low yield, high cost, and multilink of separation and
purication processes. Therefore, an effective enzymatic hydroly-
sate of a protein or total proteins in food stuff with potent ACE
inhibitory and antioxidant activities is greatly desirable, because
it can directly be used as food additive or drug against hyperten-
sion while do not need for further peptide purication.
Biological activities of protein hydrolysates are related to the
amino acid composition, size and conguration of peptides. For in-
stance, ACE prefers inhibitors or substrates containing hydropho-
bic amino acid residues at each of the three C-terminal positions
(Murray & FitzGerald, 2007; Rao et al., 2012). Regarding the antiox-
idant activity, the presence of certain hydrophobic amino acids

0308-8146/$ - see front matter Crown Copyright 2012 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.05.059
1246 S. Rao et al. / Food Chemistry 135 (2012) 12451252
(His, Trp, Tyr, Phe, Met, Leu, Gly or Pro) and basic amino acids (Arg
or Lys) has been reported to enhance the scavenging activities of
peptides (Sarmadi & Ismail, 2010). Overall, proteins with high con-
tent of hydrophobic and basic amino acid residues should be of
interest to produce dual-functional peptides with ACE inhibitory
and antioxidative activities. In this respect, several major proteins,
including ovalbumin, ovotransferrin, ovomucoid, lysozyme and
ovomucin in hen egg white, were analysed using on-line software
(ProtScale). It was found that lysozyme possesses high proportion
of hydrophobic (43.4%) and basic (13.7%) amino acid residues,
and it may be the potential to prepare ACE inhibitory peptides or
antioxidant peptides.
Based on the above rationale, the objectives of the present re-
search are: (1) examine the in vitro ACE inhibitory and antioxidant
activities of the hydrolysate obtained by gastrointestinal prote-
ases; (2) identify and characterise the peptides generated in the
hydrolysate.
2. Materials and methods
2.1. Materials
Angiotensin I-converting enzyme (EC 3.4.15.1; 5.5 U/mg pro-
tein) from rabbit lung, pepsin (E.C. 3.4.23.1; P2500 U/mg protein)
from hog stomach, a-chymotrypsin (E.C. 3.4.21.1; P40 U/mg pro-
tein) from bovine pancreas, trypsin (E.C. 3.4.21.3; 10,000 BAEE U/
mg protein) from New Zealand-sourced pancreas and 1,1-diphe-
nyl-2-pycryl-hydrazyl (DPPH) were purchased from Sigma Chemi-
cal Co. (St. Louis, MO). Hippurylhistidylleucine (HHL) was
obtained from the peptide Institute (Osaka, Japan). Carboxymethyl
chitosan (CM-CTS) was purchased from Sigma. Fresh hen eggs
were bought from local market. All other reagents, unless other-
wise specied, were of analytical or guaranteed reagent grade.
2.2. Preparation of superparamagnetic carboxymethyl chitosan (CM-
CTS) nanoparticles
Superparamagnetic CM-CTS nanoparticles, that is Fe3O4 nano-
particles with CM-CTS coating, were prepared using a chemical
coprecipitating method (Sun, Su, Rao, & Yang, 2011). The average
particle size and morphology of the obtained functionalised nano-
particles were examined using transmission electron microscope
(TEM, JEOL JEM-2100 (HR)). The aqueous dispersion of the particles
was drop-cast onto a carbon-coated copper grid and the grid was
air-dried at room temperature before loading into the microscope.
2.3. Purication of lysozyme in hen egg white by superparamagnetic
nanoparticles
Egg white was isolated from fresh eggs and diluted to 50% (V/V)
with phosphate buffer solution (PBS, 20 mM, pH 9.0). The diluted
egg white was kept stirring for 6 h in an ice bath, centrifuged at
10000g for 20 min at 4 C, and then collected the supernatant.
40 ml of the supernatant was mixed with 100 mg of superparamag-
netic nanoparticles and kept continuous stirring for 30 min at
ambient temperature (about 25 C) for absorbing of the aimed lyso-
zyme. Subsequently, the superparamagnetic nanoparticles with
target lysozymes were precipitated using a magnet to remove
unabsorbed hybrid proteins in the supernatant. After that, the lyso-
zymes from the magnetite nanoparticles was eluted with PBS
(20 mM, pH 5.0) containing 0.5 M NaCl. The puried lysozyme solu-
tion was dialysed against PBS (10 mM, pH 7.5) to remove salts and
then lyophilised for subsequent enzymatic hydrolysis analysis. The
samples during purication process were subjected to 12% sodium
dodecyl sulphatepolyacrylamide gel electrophoresis (SDSPAGE)
analysis.
2.4. In vitro digestion of puried lysozyme
The simulated human gastrointestinal digestion process was car-
ried out according to the method of Herregods et al. (2011) with
some modications. For this purpose, ve hundred milligram of
lysozyme was suspended in 50 ml of HCl solution (pH 2.0) to make
a 1% (w/v) slurry. After heating at 80 C in a water bath for 15 min,
the protein slurry was cooled to 37 C. Subsequently, the digestion
in the stomach was simulated by adding pepsin in a 1/100 (w/w) en-
zyme/substrate ratio and incubating for 2 h at 37 C and pH 2.0. The
further small intestine phase was estimated by adding a-chymo-
trypsin and typsin (each 0.5%) and incubating for 4 h at 37 C and
pH 7.5. Hydrolysis was conducted in a shaking bath (thermally con-
trolled incubator) under constant stirring (150 rpm). All samples
were boiled for 10 min to inactivate the enzymes and centrifuged
at 10,000g for 20 min at 4 C, and the resulting supernatant was l-
tered, lyophilised, and stored at 20 C for further experiments.
2.5. Ultraltration
Ultraltration was performed with a 3 kDa cut-off membrane,
using an amicon ultra-15 centrifugal lter device (Millipore Corpo-
ration) for a volume up to 15 ml. Fifty milligram of hydrolysate
sample was dissolved in 10 ml of distilled water. The lter device
with sample was centrifuged at 4000g for 1 h at 4 C, and the
resulting permeate was kept at -20 C until further analysis.
2.6. Assay of ACE inhibitory activity
ACE inhibitory activity of the samples was measured in vitro fol-
lowing the spectrophotometric assay described by Cushman and
Cheung (1971) with some modications as explained below. A
10100 ll of sample solution was mixed with 80 ll of a 5 mM
HHL borate buffer containing 100 mM borate and 300 mM NaCl
(pH 8.3), and then added to 190 ll volume by using 100 mM bo-
rate. The borate buffer (100 mM, pH 8.3) was used instead of an
ACE solution for blank determination. The above mixture was pre-
incubated at 37 C for 5 min before adding 10 ll of ACE solution
(3 mU). The reaction mixture was incubated for 30 min at the same
temperature and terminated by an addition of 1 N HCl (200 ll).
The released hippuric acid was extracted with 1.2 ml ethyl acetate.
After centrifugation (4500g, 5 min), 0.8 ml of the upper layer was
transferred to a test tube and vacuum-dried at 80 C for 30 min
by using a centrifugal concentrator. The hippuric acid was redis-
solved in 0.8 ml of deionised water, and absorbance was measured
at 228 nm using spectrophotometer. Triplicate tests were per-
formed for each sample. The concentration of each peptide re-
quired to inhibit 50% of ACE activity was dened as the IC50 value.
To clarify the ACE-inhibition mode, two different peptide con-
centrations (13.3 and 26.5 lg/ml) of the hydrolysate LPH2 were
added to each reaction mixture described as above. The enzyme
activities were measured at different substrate concentrations
(0.5, 1, 2, and 3.5 mM). The inhibition kinetics of ACE in the pres-
ence of inhibitory peptides was determined with a Lineveaver
Burk plot.
2.7. Determination of antioxidant activities
2.7.1. DPPH radical scavenging activity
The scavenging activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radical was measured according to the method of Shimada, Fujika-
wa, Yahara, and Nakamura (1992) with a slight modication. An ali-
quot of 0.5 ml of sample solution was added with 0.5 ml of 0.2 mM
DPPH in 95% ethanol. The mixture was incubated for 30 min in the
darkness at room temperature. The absorbance of the resulting
solution was measured at 517 nm with a spectrophotometer. A
 S. Rao et al. / Food Chemistry 135 (2012) 12451252
standard with glutathion was also made in the similar manner for
comparison. The ethanol was used as a control. The radical scaveng-
ing capacity of the tested samples was measured as a decrease in the
absorbance of DPPH radical and was calculated by using the follow-
ing equation:
Scavenging activity % Acontrol  Asample  100=Acontrol
All determinations were conducted in triplicate.
2.7.2. Reducing power assay
The reducing power was measured according to the method of
Oyaizu (1986) with several modication. An aliquot of 1 ml sample
(0.2 M PBS, pH 6.6) was mixed with 1 ml of 1% potassium ferric
cyanide solution. The mixture was incubated at 50 C for 30 min
followed by the addition of 1 ml 10% (w/v) TCA. One ml of the incu-
bation mixture was added with 1 ml of distilled water and 0.2 ml of
0.1% (w/v) ferric chloride in test tubes. After a 10 min reaction time,
the absorbance of resulting solution was read at 700 nm. Higher
absorbance suggested stronger reducing power. A standard with
glutathion was also made in the similar manner for comparison.
2.7.3. Lipid peroxidation inhibition assay
The lipid peroxidation inhibition activity of the samples was
measured in a linoleic acid emulsion system according to the
methods of Osawa and Namiki (1985). Briey, a sample of the
hydrolysate was dissolved in 2.5 ml of 50 mM phosphate buffer
(pH 7.0) and added to a mixture of linoleic acid (32.5 ll) and 95%
ethanol (2.5 ml), and then the nal volume was adjusted to
6.25 ml with distilled water. Sample was replaced with vitamin E
for comparative purposes. The mixture was incubated in a sealed
tube at 42 C in a dark room, and the degree of linoleic acid oxida-
tion was evaluated by measuring the ferric thiocyanate method
according to Mitsuda, Yasumoto, and Iwami (1996). In short, an ali-
quot (0.05 ml) of reaction mixture was mixed with 75% ethanol
(2.35 ml) followed by the addition of 30% ammonium thiocyanate
(0.05 ml) and 20 mM ferrous chloride solution (0.05 ml) in 3.5%
HCl. After 3 min, the degree of colour development, representing
the linoleic acid oxidation, was measured at 500 nm.
2.8. Analysis by online RP-HPLC-MS/MS
The hydrolysate was subjected to ultraperformance liquid chro-
matography (UPLC) for peptide separation in an Acquity UPLC BEH
C18 column (2.1  120 mm) (Waters Corporation, Milford, MA).
The elution was made in linear gradient mode from 0.1% formic
acid to 40% acetonitrile containing 0.1% formic acid (20 min) at a
ow rate of 0.3 ml/min. Identication of the sequence of peptides
present in the fraction exerting remarkable ACE inhibitory activity
was performed by matrix-assisted laser desorption/ionisation
time-of-ight/time-of-ight mass spectrometry (MALDI-TOF-TOF
MS). Both MS and MS/MS data were acquired with a Waters Synapt
Mass Quadrupole Time-of-Flight Mass Spectrometer (Waters Cor-
poration, Milford, MA). Spectra were recorded over the mass/
charge (m/z) ranges of 1001500 in both MS and MS/MS modes.
Peptide fragmentation in the MS and MS/MS mode was respec-
tively achieved by collision-induced dissociation (CID) using atmo-
spheric air as the collision gas, and the collision energies were set
at 6 eV and 20 eV for MS and MS/MS, respectively. The signal
threshold to perform auto-MS/MS in the data-dependent acquisi-
tion was 20 counts/s in the total ion current, and the precursor ions
were isolated within a range of m/z 3.0. Instrumental control and
data analysis were performed using MassLynx software version
4.1 (Micromass UK Ltd., Wythenshawe, Manchester, UK). Both
the peptide sequencing module of the software and manual
calculations were used to process the MS/MS data and to perform
1247
peptide sequencing. The peptide sequences were matched to the
published sequence of the lysozyme derived from protein data in
UniProtKB.
2.9. Peptide synthesis
The identied peptides in part were chemically synthesised by
GenScript Corporation (Nan Jin, China). The purity (all above 99%)
and sequence of these peptides were veried by analytical RP-
HPLC-MS/MS. ACE inhibitory activities of these chemically pep-
tides were determined as described above and expressed as IC50.
3. Results and discussion
3.1. Purication of lysozyme from hen egg white by
superparamagnetic nanoparticles
Since low content (about 3.5%) of lysozyme in egg white total
protein (You, Udenigwe, Aluko, & Wu, 2010), the classical method
for lysozyme purication refers to a combination of precipitation,
centrifugation, dialysis, ultraltration and chromatography. It
makes the purication process time-consuming and high-cost for
large-scale production. CM-CTS have good tolerable pH and good
solubility, which may be due to the abundant of COOH and
NH2 groups in it. Therefore, CM-CTS can act as an ion-exchange
material for carboxyl groups once modied onto the magnetic
Fe3O4 nanoparticles. The resultant magnetic separation technology
arose from Fe3O4 nanoparticles with CM-CTS provides an easy and
rapid way to purify the aimed lysozyme from HEW. Typical TEM
micrograph of prepared Fe3O4 nanoparticles with CM-CTS coating
using a chemical coprecipitating method is shown in Fig. 1(A),
and the nanoparticles are spherical in shape with an average size
of about 15 nm. It is reported that magnetic particles less than
25 nm will exert superparamagnetism (Sun et al., 2011), and hence
our prepared Fe3O4 (PEG + CM-CTS) nanoparticles have superpara-
magnetic properties. Taking advantage of this character, about
28 mg lysozyme with 95% purity was obtained from 1 g HEW after
one-step purication by superparamagnetic nanoparticles
(Fig. 1B).
3.2. ACE inhibitory activities of lysozyme hydrolysates
To verify whether the active peptides were produced efciently
by gastrointestinal digestion, the high pure lysozyme was sub-
jected to the two stage hydrolysis at simulated physiological con-
ditions. After digestion, the hydrolysates of lysozyme showed
potent ACE inhibitory activities. The peptic hydrolysate (LPH1)
had an IC50 value of 160.2 lg/ml (Table 1). Further hydrolysis with
a-chymotrypsin and trypsin (LPH2) decreased signicantly IC50
values to 12.6 lg/ml (Table 1), this result implied that intestine en-
zymes should be essential for releasing more active peptides
responsible for ACE inhibitory potency. The IC50 values were seen
in the range from 160 and 3770 lg/ml in a variety of other proteins
or foods enzymatic hydrolysates (Herregods et al., 2011; Majum-
der & Wu, 2010; Segura-Campos, Chel-Guerrero, & Betancur-
Ancona, 2011; Udenigwe & Aluko, 2010; Vastag et al., 2011). In
contrast, the ACE inhibitory power from lysozyme hydrolysate ob-
tained by gastrointestinal enzymes was very strong.
As shown in Fig. 2, the inhibition mode of the LPH2 hydrolysate
was estimated by a LineweaverBurk plot and found to be compet-
itive. The Ki value of the hydrolysate was calculated to be 13.4 lg/
ml, which was smaller than that of puried peptides (RYPSYG and
DERF) from bovine casein hydrolysate prepared by AS1.398 neutral
protease (Jiang, Tian, Brodkorb, & Huo, 2010). The competitive
inhibitors are able to enter the ACE protein molecule, interact with
1248 S. Rao et al. / Food Chemistry 135 (2012) 12451252

Fig. 1. Purication of lysozyme from hen egg white by superparamagnetic CM-CTS nanoparticles. (A) Size distribution of superparamagnetic CM-CTS nanoparticles; (B) 12%
SDSPAGE analysis of purication of lysozyme. Lane 1, natural HEW solution; Lanes 2 and 4, the supernatants after adsorption; Lanes 3 and 5, eluted samples.

the active sites and prevent substrate binding, thus resulting in the
inhibition of ACE activity.
In order to exert an antihypertensive effect in vivo, the ACE
inhibitory peptides not only have to be resistant to gastrointestinal
degradation but also have to be absorbed into the bloodstream in
their intact form. Subsequently, small ACE inhibitory peptides
(26 amino acids) are the most interesting ones, because they
are most likely to be absorbed into the bloodstream (Verstraete,
Vermeirssen, Van Camp, Decroos, & Van Wijmelbeke, 2003). To
pinpoint peptides exhibiting a potential ACE inhibitory effect, the
hydrolysates LPH1 and LPH2 were respectively subjected to ultra-
ltration through a 3 kDa cut-off membrane. After membrane sep-
aration, the ltrate of the LPH1 (LPH13 kDa) showed a lower IC50
value of 75.2 lg/ml than that of the original hydrolysate
(IC50 = 160.2 lg/ml) (Table 1). This result well agrees with many
reports that the inhibitory activity of small molecular-weight
(MW) peptides is generally stronger than that of the larger pep-
tides (Segura-Campos et al., 2011). But the ltrate LPH23 kDa re-
vealed an increased IC50 value (28.6 lg/ml) compared to that of the
original hydrolysate LPH2 (IC50 = 12.6 lg/ml) (Table 1). This phe-
nomenon indicated the larger MW peptides in the nal hydroly-
sate also greatly contribute to the inhibitory potency against
ACE. Similarly, the high MW peptide (21 AA) from milk proteins
fermented with Lactobacillus (Lb.) helveticus NCC 2765 (Robert,
Razaname, Mutter, & Juillerat, 2004) also has been found to possess
strong ACE inhibitory potencies. As for high MW active peptides,
the structureactivity relationship is expected to further research.
3.3. Antioxidant activity of lysozyme hydrolysates
The nal hydrolysate LPH2 and its permeate LPH23 kDa after
ultraltration were also evaluated for their antioxidant activities.
Many in vitro determination techniques of antioxidant activity
have been developed because of the difference of antioxidant
mechanisms. The response of antioxidants depends on factors such
as the solvent and substrate used in the test and afnity between
substrate and antioxidant. Accordingly, it is better to use different
assays based on different mechanisms to assess the antioxidant
potency.
The use of the DPPH assay to assess the scavenging activity of
substances has been widely reported in the literature. As shown
in Table 1, the LPH2 (25.7%) and LPH23 kDa (65.3%) showed sig-
nicant DPPH radical-scavenging activity at concentration of
2.0 mg/ml. The low molecular weight fraction LPH23 kDa had
higher scavenging activity. This value was less than and close to
that of the glutathion at the same concentration and comparable
to that of the enzymatic hydrolysate of alfalfa leaf protein (Xie,
Huang, Xu, & Jin, 2008). Obviously, the LPH23 kDa had the ability
to quench the DPPH radical, which suggested that the LPH23 kDa
possibly contained some substrates which were electron donors
and could react with free radicals to convert them to more stable
products and terminate the radical chain reaction.
The reducing power of the LPH2 and LPH23 kDa were also gi-
ven in Table 1. Similar to the result from DPPH assay, the LPH2
3 kDa revealed more strong reducing power when compared to
LPH2. When the peptide concentration of LPH23 kDa was at
2 mg/ml, the reducing power was 0.29. The value was lower than
and close to that of glutathion, while the concentration of LPH2
3 kDa was ten times that of glutathion. The rapeseed protein
hydrolysates prepared with alcalase have been reported to exhibit
notable reducing power which was 0.51 at 2.00 mg/ml (Pan, Jiang,
& Pan, 2011), and this value is slightly higher than that present in
this study. There has a better correlation between antioxidant
activity and the reducing power. Samples with higher reducing
power have better potencies to donate electron and free radicals
to form stable substances, thus interrupting the free radical chain

Table 1
ACE inhibitory and antioxidative activities of hydrolysate from lysozyme by gastrointestinal proteases.

Sample or control ACE inhibitory activity (lg/ml) Antioxidant activity

DPPH radical % (mg/ml) Reducing power A700 nm (mg/ml) Lipid peroxidation inhibition % (mg/ml)
LPH1 160.2 2.5a
LPH13 kDa 75.2 2.7b
LPH2 12.6 1.2c 25.7 0.22a (2.0) 0.16 0.03a (2.0) 76.4 0.21a (0.5)
LPH23 kDa 28. 6 0.8d 63.2 0.16b (2.0) 0.29 0.07b (2.0) 85.2 0.18b (0.5)
Glutathion 74.6 0.09c (2.0) 0.35 0.02c (0.2)
Vitamin E 78.4 0.29a (0.05)

Results are the mean values of triplicate analyses standard deviation. ad Means within the same column without a common letter differ signicantly (P < 0.05) with Tukeys
test. The value in the bracket indicates the concentration (mg/ml) of sample or control.
 S. Rao et al. / Food Chemistry 135 (2012) 12451252 1249

10 LPH2 In order to further determine the antioxidant activity in another

Control system, the samples were characterised for their antioxidant activ-
1/Abs 228 nm 8 13.3 g/ml ities by measuring their abilities to protect linoleic acid against
oxidation. At the end of the 7-day lipid peroxidation, the inhibition
26.5 g/ml
6 rate of LPH2 and LPH23 kDa were 76.4% and 85.2% respectively,
which were closer to that of the positive control, vitamin E, a
4 well-known lipid-soluble natural antioxidant (Table 1). The result
agreed with the free radical-scavenging activity and reducing
2 power analysed above.

0 3.4. Identication and characteristics of peptides in the LPH23 kDa

-1 -0.5-2 0 0.5 1 1.5 2 2.5 3
The sequence of the peptides in the LPH23 kDa was investi-
1/[S] (mM) gated in this work by the method of UPLC-MALDI-TOF-TOF MS.
The obtained peptide separation total ion chromatography (TIC)
Fig. 2. LineweaverBurk plot for determination of inhibitory mode of LPH2 on ACE.
prole was shown in Fig. 3(A). To obtain precursor ions in the sam-
ACE inhibitory was determined in the presence or absence of LPH2 as described in
the text using HHL as the enzyme substrate. ples, the cone voltage and collision energy in data-dependent MS
experiments were set into 30 V and 6 eV, respectively, and the
resultant averaged MS spectra of the LPH23 kDa were seen in
reactions (Juntachote & Berghofer, 2005). The assay result of reduc- Fig. 3(B). In subsequent MS/MS experiments, the collision energy
ing power demonstrated that LPH23 kDa possessed a moderate was changed into 20 eV and the cone voltage (30 V) was kept con-
ability to donate electron, which was involved in the antioxidant stant, to get more fragments for analysis of amino acid composi-
activity. tion. Fig. 3(C) illustrated the MS/MS spectrum of the m/z 718.29

Fig. 3. Identication of peptide sequence. (A) TIC chromatogram corresponding to the separation of the LPH23 kDa using a UPLC coupled to a Waters Synapt Mass
Quadrupole Time-of-Flight Mass Spectrometer; (B) Averaged MS spectra of the LPH23 kDa corresponding to the TIC chromatogram; (C) MS/MS spectrum of the ion m/z
718.29 relevant to the peak for 5.87 min retention time in the TIC.
1250 S. Rao et al. / Food Chemistry 135 (2012) 12451252

Table 2
Identication of peptides present in the LPH23 kDa by MALDI-TOF-TOF.

Sequence RT Obsd massb(qa) Position Sequence RT Obsd massb(qa) Position

NR 1.05 289.15 (1) f(4445) or f(113114) SSDITA 5.26 593.26 (1) f(8590)
MK 1.36 278.16 (1) f(1213) ESNF 5.39 496.19 (1) f(3538)
MKR 1.38 434.25 (1) f(1214) ITASVN 5.63 604.32 (1) f(8893)
AMK 1.64 349.19 (1) f(1113) KVF 5.84 393.24 (1) f(13)
NTQAT 2.01 534.23 (1) f(3943) HGLDNY 5.87 718.29 (1) f(1522)
NTQATNR 2.25 804.36 (1) f(3945) WIR 6.04 474.27 (1) f(123125)
TPGSR 2.35 517.26 (1) f(6973) KIVSDGNGMNA 6.35 1106.5 (1) f(97107)
QINSR 3.17 617.32 (1) f(5761) YSLGN 6.43 553.25 (1) f(2327)
LSSD 3.17 421.19 (1) f(8487) LSSDITA 7.37 706.35 (1) f(8490)
RGY 3.21 359.20 (1) f(2123) GIL 8.74 302.20 (1) f(5456)
GTDVQ 3.38 519.24 (1) f(117121) VAW 8.80 375.19 (1) f(109111)
SLGN 3.55 390.18 (1) f(2427) IVS 9.25 318.17 (1) f(98100)
CS 3.79 209.06 (1) f(8081) SLGNW 9.86 576.26 (1) f(2428)
KIVSD 4.15 561.33 (1) f(97101) CNIPCSAL 10.2 818.41 (1) f(7683)
AKF 4.27 365.21 (1) f(3234) YGIL 10.71 465.26 (1) f(5356)
GSTDY 4.33 542.22 (1) f(4953) WW 11.16 391.20 (1) f(6263)
GTDVQA 4.50 590.26 (1) f(117122) YSLGNW 11.57 739.33 (1) f(2328)
NTGGSTDY 4.78 872.30 (1) f(4653) GTDVQAWIR 11.98 1045.5 (1) f(117125)
HGLDNYR 4.91 873.40 (1) f(1521) WVAW 12.49 561.25 (1) f(108111)

RT means retention time in the TIC prole. The sequence of lysozyme was as follows: KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQI
NSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRL (The sequence comes from protein data UniProtKB, and the record
number is P00698.
a
Charge state of the precursor ion.
b
Molecular ion mass observed in the MALDI-TOF-TOF system calculated in Daltons (Da).

relevant to the peak for 5.87 min retention time in the TIC, and the
corresponding peptide sequence was identied as HGLDNY, com-
pared to the amino acid sequence of the lysozyme and character-
ised by the peptide sequencing module of the MassLynx 4.1
software. Using this analytical method, 38 peptides in the LPH2
3 kDa were identied in this study and listed in Table 2, and their
observed and calculated mass matched as well as their sequence.
Blast sequence similarity searches revealed above 98% homology
of the identied sequences with lysozyme.
Pepsin is an endopeptidase acting at stomach level which
hydrolyses peptide bonds within protein sequences randomly to
produce relatively large peptides. Savoie, Gauthier, Marin, and Pou-
liot (2005) report that hydrolysis with pepsin often generates pep-
tides containing Phe, Tyr, or Leu in N-terminal position. This is the
case of different peptides possessing these amino acids in such a
position, such as peptide LSSD, YSLGN, LSSDITA, YGIL, and YSLGNW
in the LPH23 kDa (Table 2). Furthermore, pepsin also frequently
cleaves at Phe, Tyr, Trp, or Leu in C-terminal region (Kageyama,
2002), and this cleavage character is likewise feasible for a-chymo-
trypsin (Folk & Schirmer, 1965). In these respects, some peptides
having these amino acids at the C-terminus have been identied
in this work such as RGY, GIL, VAW, KVF, and others (Table 2).
On the other hand, peptides having a C-terminal Arg or Lys fre-
quently appear in the identied sequences and it would be charac-
teristic of a trypsin action (Gray & Cooper, 1971), as in cases of
AMK, TPGSR, and WIR (Table 2). Overall, these digestive proteases
are suitable to generate peptides that contain aromatic (Phe, Tyr
and Trp), basic (Arg and Lys) or Leu residues at C-terminus (Rao,
Xu, Su, Sun, & Yang, 2011). Interestingly, these residues at C-termi-
nus or within the sequences have great contribution to the ACE
inhibitory or antioxidant activity of the peptides (Murray &
FitzGerald, 2007; Sarmadi & Ismail, 2010).
The relationships between the structure and activity level of
various inhibitory peptides indicate that binding to ACE is strongly
inuenced by the C-terminal tripeptide sequence of the substrate.
The hydrophobic residues in the C-terminal tripeptide may well
interact with subsites at the active site of ACE (Ondetti & Cushman,
1982). Notably, ACE prefers to have substrate or competitive inhib-
itors that contain aromatic residues such as Phe, Tyr, and Trp or ali-
phatic residues Leu at the nal position of C-terminus (Cheung,
Wang, Ondetti, Sabo, & Cushman, 1980). Activity data also suggests
that the positive charge on the guanidine or e-amino group of C-
terminal Arg or Lys side chains, respectively, contribute substan-
tially to their inhibitory activity against ACE. Similarly, the pres-
ence of aromatic, positively charged, and hydrophobic residues in
the peptide are important in contributing to the radical-scavenging
capacity of peptides through donating hydrogen to reactive oxygen
species. Considering the high proportion of hydrophobic and basic
residues in the lysozyme sequence, enzymatic characteristics of
gastrointestinal enzymes and structureactivity relationships of
bioactive peptides, the simulated gastrointestinal digestion of lyso-
zyme was conducted to produce functional peptides in this study.
As expected and shown in Table 2, the C-terminal residues of many
peptides generated from lysozyme by gastrointestinal enzymes are
well t for the C-terminal residues requirements of ACE inhibitory
peptides or antioxidant peptides according to the structureactiv-
ity relationship described as above.
To further verify bioactivities of generated peptides, we synthe-
sized eight tripeptides among the identied peptides from the
LPH23 kDa. As shown in Table 3, all tripeptides showed high
ACE inhibitory activities, with IC50 values of below 100 lM. These
sequences all contained an aromatic, basic residue or Leu at the C-
terminus, which devoted to the inhibitory potency against ACE.
The sequences VAW yielded the strongest bioactivities with an
IC50 value of 2.8 lM. This peptide has a branched-chain aliphatic
residue (Val) at the N-terminus and an aromatic residue (Trp) at
the C-terminus. It was reported that the peptides with this struc-
ture possessed potent ACE inhibitory potency (Cheung et al.,
1980). Additionally, it is important to highlight the fact that the
positively charged residue (Lys/Arg) at the middle position, has a
positive inuence for peptide-enzyme binding and therefore rein-
forces the inhibitory potency of the tripeptides (Majumder & Wu,
2010). This kind of active peptides, such as AKF (IC50 = 6.5 lM)
and MKR (IC50 = 25.7 lM), also showed potent ACE inhibitory
activities.
Davalos, Miguel, Bartolome, and Lopez-Fandino (2004) report
that Tyr and Trp show the highest antioxidant activity among various
natural amino acids. Moreover, Tyr and Trp are generally accepted as
antioxidants contributing to the activities of the identied peptides
(Chen, Muramoto, & Yamauchi, 1995). The peptides RGY, WIR and
 S. Rao et al. / Food Chemistry 135 (2012) 12451252
Table 3
ACE inhibitory or/and antioxidant activities of synthetic peptides.
Peptide ACE inhibitory Peptide or
Activity IC50 (lM) Control
KVF 14.0 RGY
MKR 25.7 WIR
AMK 94.2 VAW
AKF 6.5 Glutathion
GIL 53.8 Vitamin E
Results for ACE inhibitory activity are the mean values of triplicate analyses. Results for antioxidant activity are the mean values of triplicate analyses standard deviation.
AC
Means within the same column without a common letter differ signicantly (P < 0.05) with Tukeys test.
a
Absorbance at 700 nm for a peptide or a control tested at concentration of 5 mM.
b
The lipid peroxidation inhibition activity in a linoleic acid emulsion system for a peptide or a control at concentration of 0.5 mM or 0.1 mM, respectively.
VAW contain these two residues, and hence their antioxidant activi-
ties were determined in this study. As expected, these peptides all
showed strong antioxidant effects (Table 3). Notably, the peptide
WIR revealed the highest reducing power with a value of 1.32 at
5 mM, and the values of RGY and VAW was close to that of glutathion
at the same concentration. However, the lipid peroxidation inhibition
of these peptides doesnot show signicant difference. The inhibitions
of the peptides were all above 75% at 0.5 mM, which were comparable
to that of vitamin E (78.4%) at 0.1 mM. This result suggested that these
peptides have signicant protective effect on peroxidation of linoleic
acid, and thus possessed potent antioxidant activities. It was worthy
to highlight that hydrophobic (Gly, Ile, Ala and Val) and basic (Arg)
residues within the sequences also played important roles in the anti-
oxidant activities of these peptides.
4. Conclusion
In the present study, the in vitro digestion hydrolysate (LPH2) of
hen egg white lysozyme was found to exert a potent dual activity
as they showed both in vitro ACE inhibitory and antioxidant activ-
ities. Moreover, conduction of ultraltration signicantly improved
the antioxidant activity of the LPH2, and 38 peptides in the ltra-
tion LPH23 kDa were identied using the analytical technique
MALDI-TOF-TOF. Of special interest were fragments KVF, MKR,
AMK, AKF, RGY, WIR, VAW and GIL found in the LPH23 kDa, and
they showed great ACE inhibitory activities. Furthermore, the pep-
tides RGY, WIR and VAW were also found to exhibit strong antiox-
idant activities. The results reported in this work suggest that
physiological digestion of lysozyme may promote the generation
of peptides with ACE inhibitory and antioxidant activities. Never-
theless, further work using animal models will be needed to con-
clude if these sequences and the intact lysozyme may have a
physiological role in blood pressure regulation. Experiments using
spontaneously hypertensive rats are currently in progress.
Acknowledgments
This work has received nancial support from the National 863
Programs of China (2007AA10Z330), the Doctoral Research Funds
of Jiangnan University (No. JUDCF11018), and the Graduate Educa-
tion Innovation Project in Jiangsu Province (CXZZ11_0489).
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