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Food Chemistry
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Analytical Methods
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 interest from the food industry because of the consumers concern
over the safety of the synthetic counterpart.
Hypertension is a major risk factor for cardiovascular and end Some enzymatic hydrolysates of food staffs or proteins, such as
stage renal diseases. It can affect people of all ages and endangers axseed protein (Udenigwe & Aluko, 2010), camel milk casein
approximately one billion worldwide (Murray & FitzGerald, 2007). (Salami et al., 2011), protein isolate from pumpkin oil cake (Vastag,
Since angiotensin I-converting enzyme (ACE) (EC 3.4.15.1) plays a Popovic, Popovic, Krimer, & Pericin, 2011) and others, have been
critical physiological role in raising blood pressure, the inhibition reported to exert both ACE inhibitory and antioxidant activities.
of ACE activity can lead to an overall antihypertensive effect. The combination of ACE inhibitory and antioxidant activities in
Endogenous and exogenous reactive oxygen species and free radi- protein hydrolysates or peptides could be very helpful for the con-
cals also have been implicated in the occurrence of hypertension trol of cardiovascular diseases by synergies of different regulatory
and other degenerative diseases. The amount of these reactive mechanisms. However, most of reported enzymatic hydrolysates
species is controlled by endogenous antioxidants until it reaches of various food protein sources show low bioactivities. Although
a level when the antioxidants are overwhelmed, a state known the active peptides puried from these hydrolysates exhibit potent
as oxidative stress. Exogenous dietary antioxidants can decrease inhibitory potencies, it is very difcult for their commercialisation
the contribution of exercise-induced oxidative stress and improve because of low yield, high cost, and multilink of separation and
the animals physiological condition (Yu et al., 2006). Although purication processes. Therefore, an effective enzymatic hydroly-
having obvious functional effects for improving human health, sate of a protein or total proteins in food stuff with potent ACE
synthetic ACE inhibitors and antioxidants are reported to have inhibitory and antioxidant activities is greatly desirable, because
various undesirable side effects (Murray & FitzGerald, 2007) and it can directly be used as food additive or drug against hyperten-
potential health hazards (Barlow & Schlatter, 2010). Recently, pro- sion while do not need for further peptide purication.
tein hydrolysates and constituent peptides with ACE inhibitory Biological activities of protein hydrolysates are related to the
activities or antioxidant activities have received considerable amino acid composition, size and conguration of peptides. For in-
stance, ACE prefers inhibitors or substrates containing hydropho-
Corresponding author. Tel./fax: +86 510 85863566.
bic amino acid residues at each of the three C-terminal positions
E-mail addresses: raoshengqi2001@yahoo.com.cn (S. Rao), yangyj@jiangnan. (Murray & FitzGerald, 2007; Rao et al., 2012). Regarding the antiox-
edu.cn (Y. Yang). 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 2.4. In vitro digestion of puried lysozyme
or Lys) has been reported to enhance the scavenging activities of
peptides (Sarmadi & Ismail, 2010). Overall, proteins with high con- The simulated human gastrointestinal digestion process was car-
tent of hydrophobic and basic amino acid residues should be of ried out according to the method of Herregods et al. (2011) with
interest to produce dual-functional peptides with ACE inhibitory some modications. For this purpose, ve hundred milligram of
and antioxidative activities. In this respect, several major proteins, lysozyme was suspended in 50 ml of HCl solution (pH 2.0) to make
including ovalbumin, ovotransferrin, ovomucoid, lysozyme and a 1% (w/v) slurry. After heating at 80 C in a water bath for 15 min,
ovomucin in hen egg white, were analysed using on-line software the protein slurry was cooled to 37 C. Subsequently, the digestion
(ProtScale). It was found that lysozyme possesses high proportion in the stomach was simulated by adding pepsin in a 1/100 (w/w) en-
of hydrophobic (43.4%) and basic (13.7%) amino acid residues, zyme/substrate ratio and incubating for 2 h at 37 C and pH 2.0. The
and it may be the potential to prepare ACE inhibitory peptides or further small intestine phase was estimated by adding a-chymo-
antioxidant peptides. trypsin and typsin (each 0.5%) and incubating for 4 h at 37 C and
Based on the above rationale, the objectives of the present re- pH 7.5. Hydrolysis was conducted in a shaking bath (thermally con-
search are: (1) examine the in vitro ACE inhibitory and antioxidant trolled incubator) under constant stirring (150 rpm). All samples
activities of the hydrolysate obtained by gastrointestinal prote- were boiled for 10 min to inactivate the enzymes and centrifuged
ases; (2) identify and characterise the peptides generated in the at 10,000g for 20 min at 4 C, and the resulting supernatant was l-
hydrolysate. tered, lyophilised, and stored at 20 C for further experiments.
standard with glutathion was also made in the similar manner for peptide sequencing. The peptide sequences were matched to the
comparison. The ethanol was used as a control. The radical scaveng- published sequence of the lysozyme derived from protein data in
ing capacity of the tested samples was measured as a decrease in the UniProtKB.
absorbance of DPPH radical and was calculated by using the follow-
ing equation: 2.9. Peptide synthesis
Scavenging activity % Acontrol Asample 100=Acontrol
The identied peptides in part were chemically synthesised by
All determinations were conducted in triplicate. GenScript Corporation (Nan Jin, China). The purity (all above 99%)
and sequence of these peptides were veried by analytical RP-
2.7.2. Reducing power assay HPLC-MS/MS. ACE inhibitory activities of these chemically pep-
The reducing power was measured according to the method of tides were determined as described above and expressed as IC50.
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 3. Results and discussion
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- 3.1. Purication of lysozyme from hen egg white by
bation mixture was added with 1 ml of distilled water and 0.2 ml of superparamagnetic nanoparticles
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 Since low content (about 3.5%) of lysozyme in egg white total
absorbance suggested stronger reducing power. A standard with protein (You, Udenigwe, Aluko, & Wu, 2010), the classical method
glutathion was also made in the similar manner for comparison. for lysozyme purication refers to a combination of precipitation,
centrifugation, dialysis, ultraltration and chromatography. It
2.7.3. Lipid peroxidation inhibition assay makes the purication process time-consuming and high-cost for
The lipid peroxidation inhibition activity of the samples was large-scale production. CM-CTS have good tolerable pH and good
measured in a linoleic acid emulsion system according to the solubility, which may be due to the abundant of COOH and
methods of Osawa and Namiki (1985). Briey, a sample of the NH2 groups in it. Therefore, CM-CTS can act as an ion-exchange
hydrolysate was dissolved in 2.5 ml of 50 mM phosphate buffer material for carboxyl groups once modied onto the magnetic
(pH 7.0) and added to a mixture of linoleic acid (32.5 ll) and 95% Fe3O4 nanoparticles. The resultant magnetic separation technology
ethanol (2.5 ml), and then the nal volume was adjusted to arose from Fe3O4 nanoparticles with CM-CTS provides an easy and
6.25 ml with distilled water. Sample was replaced with vitamin E rapid way to purify the aimed lysozyme from HEW. Typical TEM
for comparative purposes. The mixture was incubated in a sealed micrograph of prepared Fe3O4 nanoparticles with CM-CTS coating
tube at 42 C in a dark room, and the degree of linoleic acid oxida- using a chemical coprecipitating method is shown in Fig. 1(A),
tion was evaluated by measuring the ferric thiocyanate method and the nanoparticles are spherical in shape with an average size
according to Mitsuda, Yasumoto, and Iwami (1996). In short, an ali- of about 15 nm. It is reported that magnetic particles less than
quot (0.05 ml) of reaction mixture was mixed with 75% ethanol 25 nm will exert superparamagnetism (Sun et al., 2011), and hence
(2.35 ml) followed by the addition of 30% ammonium thiocyanate our prepared Fe3O4 (PEG + CM-CTS) nanoparticles have superpara-
(0.05 ml) and 20 mM ferrous chloride solution (0.05 ml) in 3.5% magnetic properties. Taking advantage of this character, about
HCl. After 3 min, the degree of colour development, representing 28 mg lysozyme with 95% purity was obtained from 1 g HEW after
the linoleic acid oxidation, was measured at 500 nm. one-step purication by superparamagnetic nanoparticles
(Fig. 1B).
2.8. Analysis by online RP-HPLC-MS/MS
3.2. ACE inhibitory activities of lysozyme hydrolysates
The hydrolysate was subjected to ultraperformance liquid chro-
matography (UPLC) for peptide separation in an Acquity UPLC BEH To verify whether the active peptides were produced efciently
C18 column (2.1 120 mm) (Waters Corporation, Milford, MA). by gastrointestinal digestion, the high pure lysozyme was sub-
The elution was made in linear gradient mode from 0.1% formic jected to the two stage hydrolysis at simulated physiological con-
acid to 40% acetonitrile containing 0.1% formic acid (20 min) at a ditions. After digestion, the hydrolysates of lysozyme showed
ow rate of 0.3 ml/min. Identication of the sequence of peptides potent ACE inhibitory activities. The peptic hydrolysate (LPH1)
present in the fraction exerting remarkable ACE inhibitory activity had an IC50 value of 160.2 lg/ml (Table 1). Further hydrolysis with
was performed by matrix-assisted laser desorption/ionisation a-chymotrypsin and trypsin (LPH2) decreased signicantly IC50
time-of-ight/time-of-ight mass spectrometry (MALDI-TOF-TOF values to 12.6 lg/ml (Table 1), this result implied that intestine en-
MS). Both MS and MS/MS data were acquired with a Waters Synapt zymes should be essential for releasing more active peptides
Mass Quadrupole Time-of-Flight Mass Spectrometer (Waters Cor- responsible for ACE inhibitory potency. The IC50 values were seen
poration, Milford, MA). Spectra were recorded over the mass/ in the range from 160 and 3770 lg/ml in a variety of other proteins
charge (m/z) ranges of 1001500 in both MS and MS/MS modes. or foods enzymatic hydrolysates (Herregods et al., 2011; Majum-
Peptide fragmentation in the MS and MS/MS mode was respec- der & Wu, 2010; Segura-Campos, Chel-Guerrero, & Betancur-
tively achieved by collision-induced dissociation (CID) using atmo- Ancona, 2011; Udenigwe & Aluko, 2010; Vastag et al., 2011). In
spheric air as the collision gas, and the collision energies were set contrast, the ACE inhibitory power from lysozyme hydrolysate ob-
at 6 eV and 20 eV for MS and MS/MS, respectively. The signal tained by gastrointestinal enzymes was very strong.
threshold to perform auto-MS/MS in the data-dependent acquisi- As shown in Fig. 2, the inhibition mode of the LPH2 hydrolysate
tion was 20 counts/s in the total ion current, and the precursor ions was estimated by a LineweaverBurk plot and found to be compet-
were isolated within a range of m/z 3.0. Instrumental control and itive. The Ki value of the hydrolysate was calculated to be 13.4 lg/
data analysis were performed using MassLynx software version ml, which was smaller than that of puried peptides (RYPSYG and
4.1 (Micromass UK Ltd., Wythenshawe, Manchester, UK). Both DERF) from bovine casein hydrolysate prepared by AS1.398 neutral
the peptide sequencing module of the software and manual protease (Jiang, Tian, Brodkorb, & Huo, 2010). The competitive
calculations were used to process the MS/MS data and to perform 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 have been developed because of the difference of antioxidant
inhibition of ACE activity. mechanisms. The response of antioxidants depends on factors such
In order to exert an antihypertensive effect in vivo, the ACE as the solvent and substrate used in the test and afnity between
inhibitory peptides not only have to be resistant to gastrointestinal substrate and antioxidant. Accordingly, it is better to use different
degradation but also have to be absorbed into the bloodstream in assays based on different mechanisms to assess the antioxidant
their intact form. Subsequently, small ACE inhibitory peptides potency.
(26 amino acids) are the most interesting ones, because they The use of the DPPH assay to assess the scavenging activity of
are most likely to be absorbed into the bloodstream (Verstraete, substances has been widely reported in the literature. As shown
Vermeirssen, Van Camp, Decroos, & Van Wijmelbeke, 2003). To in Table 1, the LPH2 (25.7%) and LPH23 kDa (65.3%) showed sig-
pinpoint peptides exhibiting a potential ACE inhibitory effect, the nicant DPPH radical-scavenging activity at concentration of
hydrolysates LPH1 and LPH2 were respectively subjected to ultra- 2.0 mg/ml. The low molecular weight fraction LPH23 kDa had
ltration through a 3 kDa cut-off membrane. After membrane sep- higher scavenging activity. This value was less than and close to
aration, the ltrate of the LPH1 (LPH13 kDa) showed a lower IC50 that of the glutathion at the same concentration and comparable
value of 75.2 lg/ml than that of the original hydrolysate to that of the enzymatic hydrolysate of alfalfa leaf protein (Xie,
(IC50 = 160.2 lg/ml) (Table 1). This result well agrees with many Huang, Xu, & Jin, 2008). Obviously, the LPH23 kDa had the ability
reports that the inhibitory activity of small molecular-weight to quench the DPPH radical, which suggested that the LPH23 kDa
(MW) peptides is generally stronger than that of the larger pep- possibly contained some substrates which were electron donors
tides (Segura-Campos et al., 2011). But the ltrate LPH23 kDa re- and could react with free radicals to convert them to more stable
vealed an increased IC50 value (28.6 lg/ml) compared to that of the products and terminate the radical chain reaction.
original hydrolysate LPH2 (IC50 = 12.6 lg/ml) (Table 1). This phe- The reducing power of the LPH2 and LPH23 kDa were also gi-
nomenon indicated the larger MW peptides in the nal hydroly- ven in Table 1. Similar to the result from DPPH assay, the LPH2
sate also greatly contribute to the inhibitory potency against 3 kDa revealed more strong reducing power when compared to
ACE. Similarly, the high MW peptide (21 AA) from milk proteins LPH2. When the peptide concentration of LPH23 kDa was at
fermented with Lactobacillus (Lb.) helveticus NCC 2765 (Robert, 2 mg/ml, the reducing power was 0.29. The value was lower than
Razaname, Mutter, & Juillerat, 2004) also has been found to possess and close to that of glutathion, while the concentration of LPH2
strong ACE inhibitory potencies. As for high MW active peptides, 3 kDa was ten times that of glutathion. The rapeseed protein
the structureactivity relationship is expected to further research. hydrolysates prepared with alcalase have been reported to exhibit
notable reducing power which was 0.51 at 2.00 mg/ml (Pan, Jiang,
3.3. Antioxidant activity of lysozyme hydrolysates & Pan, 2011), and this value is slightly higher than that present in
this study. There has a better correlation between antioxidant
The nal hydrolysate LPH2 and its permeate LPH23 kDa after activity and the reducing power. Samples with higher reducing
ultraltration were also evaluated for their antioxidant activities. power have better potencies to donate electron and free radicals
Many in vitro determination techniques of antioxidant activity to form stable substances, thus interrupting the free radical chain
Table 1
ACE inhibitory and antioxidative activities of hydrolysate from lysozyme by gastrointestinal proteases.
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
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.
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 Wang, Ondetti, Sabo, & Cushman, 1980). Activity data also suggests
corresponding peptide sequence was identied as HGLDNY, com- that the positive charge on the guanidine or e-amino group of C-
pared to the amino acid sequence of the lysozyme and character- terminal Arg or Lys side chains, respectively, contribute substan-
ised by the peptide sequencing module of the MassLynx 4.1 tially to their inhibitory activity against ACE. Similarly, the pres-
software. Using this analytical method, 38 peptides in the LPH2 ence of aromatic, positively charged, and hydrophobic residues in
3 kDa were identied in this study and listed in Table 2, and their the peptide are important in contributing to the radical-scavenging
observed and calculated mass matched as well as their sequence. capacity of peptides through donating hydrogen to reactive oxygen
Blast sequence similarity searches revealed above 98% homology species. Considering the high proportion of hydrophobic and basic
of the identied sequences with lysozyme. residues in the lysozyme sequence, enzymatic characteristics of
Pepsin is an endopeptidase acting at stomach level which gastrointestinal enzymes and structureactivity relationships of
hydrolyses peptide bonds within protein sequences randomly to bioactive peptides, the simulated gastrointestinal digestion of lyso-
produce relatively large peptides. Savoie, Gauthier, Marin, and Pou- zyme was conducted to produce functional peptides in this study.
liot (2005) report that hydrolysis with pepsin often generates pep- As expected and shown in Table 2, the C-terminal residues of many
tides containing Phe, Tyr, or Leu in N-terminal position. This is the peptides generated from lysozyme by gastrointestinal enzymes are
case of different peptides possessing these amino acids in such a well t for the C-terminal residues requirements of ACE inhibitory
position, such as peptide LSSD, YSLGN, LSSDITA, YGIL, and YSLGNW peptides or antioxidant peptides according to the structureactiv-
in the LPH23 kDa (Table 2). Furthermore, pepsin also frequently ity relationship described as above.
cleaves at Phe, Tyr, Trp, or Leu in C-terminal region (Kageyama, To further verify bioactivities of generated peptides, we synthe-
2002), and this cleavage character is likewise feasible for a-chymo- sized eight tripeptides among the identied peptides from the
trypsin (Folk & Schirmer, 1965). In these respects, some peptides LPH23 kDa. As shown in Table 3, all tripeptides showed high
having these amino acids at the C-terminus have been identied ACE inhibitory activities, with IC50 values of below 100 lM. These
in this work such as RGY, GIL, VAW, KVF, and others (Table 2). sequences all contained an aromatic, basic residue or Leu at the C-
On the other hand, peptides having a C-terminal Arg or Lys fre- terminus, which devoted to the inhibitory potency against ACE.
quently appear in the identied sequences and it would be charac- The sequences VAW yielded the strongest bioactivities with an
teristic of a trypsin action (Gray & Cooper, 1971), as in cases of IC50 value of 2.8 lM. This peptide has a branched-chain aliphatic
AMK, TPGSR, and WIR (Table 2). Overall, these digestive proteases residue (Val) at the N-terminus and an aromatic residue (Trp) at
are suitable to generate peptides that contain aromatic (Phe, Tyr the C-terminus. It was reported that the peptides with this struc-
and Trp), basic (Arg and Lys) or Leu residues at C-terminus (Rao, ture possessed potent ACE inhibitory potency (Cheung et al.,
Xu, Su, Sun, & Yang, 2011). Interestingly, these residues at C-termi- 1980). Additionally, it is important to highlight the fact that the
nus or within the sequences have great contribution to the ACE positively charged residue (Lys/Arg) at the middle position, has a
inhibitory or antioxidant activity of the peptides (Murray & positive inuence for peptide-enzyme binding and therefore rein-
FitzGerald, 2007; Sarmadi & Ismail, 2010). forces the inhibitory potency of the tripeptides (Majumder & Wu,
The relationships between the structure and activity level of 2010). This kind of active peptides, such as AKF (IC50 = 6.5 lM)
various inhibitory peptides indicate that binding to ACE is strongly and MKR (IC50 = 25.7 lM), also showed potent ACE inhibitory
inuenced by the C-terminal tripeptide sequence of the substrate. activities.
The hydrophobic residues in the C-terminal tripeptide may well Davalos, Miguel, Bartolome, and Lopez-Fandino (2004) report
interact with subsites at the active site of ACE (Ondetti & Cushman, that Tyr and Trp show the highest antioxidant activity among various
1982). Notably, ACE prefers to have substrate or competitive inhib- natural amino acids. Moreover, Tyr and Trp are generally accepted as
itors that contain aromatic residues such as Phe, Tyr, and Trp or ali- antioxidants contributing to the activities of the identied peptides
phatic residues Leu at the nal position of C-terminus (Cheung, (Chen, Muramoto, & Yamauchi, 1995). The peptides RGY, WIR and
S. Rao et al. / Food Chemistry 135 (2012) 12451252 1251
Table 3
ACE inhibitory or/and antioxidant activities of synthetic peptides.
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- Cushman, D. W., & Cheung, H. S. (1971). Spectrophotometric assay and properties of
the angiotensin-converting enzyme of rabbit lung. Biochemical Pharmacology,
ties were determined in this study. As expected, these peptides all
20(7), 16371648.
showed strong antioxidant effects (Table 3). Notably, the peptide Davalos, A., Miguel, M., Bartolome, B., & Lopez-Fandino, R. (2004). Antioxidant
WIR revealed the highest reducing power with a value of 1.32 at activity of peptides derived from egg white proteins by enzymatic hydrolysis.
5 mM, and the values of RGY and VAW was close to that of glutathion Journal of Food Protection, 67(9), 19391944.
Folk, J. E., & Schirmer, E. W. (1965). Chymotrypsin C. I. Isolation of the zymogen and
at the same concentration. However, the lipid peroxidation inhibition the active enzyme: Preliminary structure and specicity studies. Journal of
of these peptides doesnot show signicant difference. The inhibitions Biological Chemistry, 240, 181192.
of the peptides were all above 75% at 0.5 mM, which were comparable Gray, G. M., & Cooper, H. L. (1971). Protein digestion and absorption.
Gastroenterology, 61(4), 535544.
to that of vitamin E (78.4%) at 0.1 mM. This result suggested that these Herregods, G., Van Camp, J., Morel, N., Ghesquiere, B., Gevaert, K., Vercruysse, L.,
peptides have signicant protective effect on peroxidation of linoleic et al. (2011). Angiotensin I-converting enzyme inhibitory activity of gelatin
acid, and thus possessed potent antioxidant activities. It was worthy hydrolysates and identication of bioactive peptides. Journal of Agricultural and
Food Chemistry, 59(2), 552558.
to highlight that hydrophobic (Gly, Ile, Ala and Val) and basic (Arg) Jiang, Z. M., Tian, B., Brodkorb, A., & Huo, G. C. (2010). Production, analysis and
residues within the sequences also played important roles in the anti- in vivo evaluation of novel angiotensin-I-converting enzyme inhibitory peptides
oxidant activities of these peptides. from bovine casein. Food Chemistry, 123(3), 779786.
Juntachote, T., & Berghofer, E. (2005). Antioxidative properties and stability of
ethanolic extracts of Holy basil and Galangal. Food Chemistry, 92(2), 193202.
Kageyama, T. (2002). Pepsinogens, progastricsins, and prochymosins: structure,
4. Conclusion function, evolution, and development. Cellular and Molecular Life Sciences, 59(2),
288306.
In the present study, the in vitro digestion hydrolysate (LPH2) of Majumder, K., & Wu, J. P. (2010). A new approach for identication of novel
antihypertensive peptides from egg proteins by QSAR and bioinformatics. Food
hen egg white lysozyme was found to exert a potent dual activity Research International, 43(5), 13711378.
as they showed both in vitro ACE inhibitory and antioxidant activ- Mitsuda, H., Yasumoto, K., & Iwami, K. (1996). Antioxidative action of indole
ities. Moreover, conduction of ultraltration signicantly improved compounds during the autoxidation of linoleic acid. Eiyo to Shokuryo, 19,
210214.
the antioxidant activity of the LPH2, and 38 peptides in the ltra-
Murray, B. A., & FitzGerald, R. J. (2007). Angiotensin converting enzyme inhibitory
tion LPH23 kDa were identied using the analytical technique peptides derived from food proteins: biochemistry, bioactivity and production.
MALDI-TOF-TOF. Of special interest were fragments KVF, MKR, Current Pharmaceutical Design, 13(8), 773791.
Ondetti, M. A., & Cushman, D. W. (1982). Enzymes of the renin-angiotensin system
AMK, AKF, RGY, WIR, VAW and GIL found in the LPH23 kDa, and
and their inhibitors. Annual Review of Biochemistry, 51, 283308.
they showed great ACE inhibitory activities. Furthermore, the pep- Osawa, T., & Namiki, M. (1985). Natural antioxidants isolated from eucalyptus leaf
tides RGY, WIR and VAW were also found to exhibit strong antiox- waxes. Journal of Agricultural and Food Chemistry, 33(5), 777780.
idant activities. The results reported in this work suggest that Oyaizu, M. (1986). Studies on products of browning reactions: Antioxidative
activities of browning products of browning reaction prepared from
physiological digestion of lysozyme may promote the generation glucosamine. Japanese Journal of Nutrition, 44, 307315.
of peptides with ACE inhibitory and antioxidant activities. Never- Pan, M., Jiang, T. S., & Pan, J. L. (2011). Antioxidant activities of rapeseed protein
theless, further work using animal models will be needed to con- hydrolysates. Food and Bioprocess Technology, 4(7), 11441152.
Rao, S. Q., Xu, Z. Z., Su, Y. J., Sun, J., & Yang, Y. J. (2011). Cloning, soluble expression,
clude if these sequences and the intact lysozyme may have a and production of recombinant antihypertensive peptide multimer (AHPM-2)
physiological role in blood pressure regulation. Experiments using in Escherichia coli for bioactivity identication. Protein and Peptide Letters,
spontaneously hypertensive rats are currently in progress. 18(7), 699706.
Rao, S. Q., Liu, S., Ju, T., Xu, W. Q., Mei, G. M., Xu, Y. S., & Yang, Y. J. (2012). Design of
substrate-type ACE inhibitory pentapeptides with an antepenultimate C-
terminal proline for efcient release of inhibitory activity. Biochemical
Acknowledgments
Engineering Journal, 60, 5055.
Robert, M. C., Razaname, A., Mutter, M., & Juillerat, M. A. (2004). Identication of
This work has received nancial support from the National 863 angiotensin-I-converting enzyme inhibitory peptides derived from sodium
Programs of China (2007AA10Z330), the Doctoral Research Funds caseinate hydrolysates produced by Lactobacillus helveticus NCC 2765. Journal of
Agricultural and Food Chemistry, 52(23), 69236931.
of Jiangnan University (No. JUDCF11018), and the Graduate Educa- Salami, M., Moosavi-Movahedi, A. A., Moosavi-Movahedi, F., Ehsani, M. R., Youse,
tion Innovation Project in Jiangsu Province (CXZZ11_0489). R., Farhadi, M., et al. (2011). Biological activity of camel milk casein following
enzymatic digestion. Journal of Dairy Research, 78(4), 471478.
Sarmadi, B. H., & Ismail, A. (2010). Antioxidative peptides from food proteins: A
References review. Peptides, 31(10), 19491956.
Savoie, L., Gauthier, S. F., Marin, J., & Pouliot, Y. (2005). In vitro determination of the
release kinetics of peptides and free amino acids during the digestion of food
Barlow, S., & Schlatter, J. (2010). Risk assessment of carcinogens in food. Toxicology
proteins. Journal of AOAC International, 88(3), 935948.
and Applied Pharmacology, 243(2), 180190.
Segura-Campos, M. R., Chel-Guerrero, L. A., & Betancur-Ancona, D. A. (2011).
Chen, H. M., Muramoto, K., & Yamauchi, F. (1995). Structural analysis of
Purication of angiotensin I-converting enzyme inhibitory peptides from a
antioxidative peptides from soybean b-conglycinin. Journal of Agricultural and
cowpea (Vigna unguiculata) enzymatic hydrolysate. Process Biochemistry, 46(4),
Food Chemistry, 43(3), 574578.
864872.
Cheung, H. S., Wang, F. L., Ondetti, M. A., Sabo, E. F., & Cushman, D. W. (1980).
Shimada, K., Fujikawa, K., Yahara, K., & Nakamura, T. (1992). Antioxidative
Binding of peptide substrates and inhibitors of angiotensin-converting enzyme.
properties of xanthium on the autoxidation of soybean oil in cyclodextrin
Importance of the COOH-terminal dipeptide sequence. Journal of Biological
emulsion. Journal of Agricultural and Food Chemistry, 40(6), 945948.
Chemistry, 255(2), 401407.
1252 S. Rao et al. / Food Chemistry 135 (2012) 12451252
Sun, J., Su, Y. J., Rao, S. Q., & Yang, Y. J. (2011). Separation of lysozyme using Verstraete, W., Vermeirssen, V., Van Camp, J., Decroos, K., & Van Wijmelbeke, L.
superparamagnetic carboxymethyl chitosan nanoparticles. Journal of (2003). The impact of fermentation and in vitro digestion on the formation of
Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, angiotensin I-converting enzyme inhibitory activity from pea and whey protein.
879(23), 21942200. Journal of Dairy Science, 86(2), 429438.
Udenigwe, C. C., & Aluko, R. E. (2010). Antioxidant and angiotensin converting Xie, Z. J., Huang, J. R., Xu, X. M., & Jin, Z. Y. (2008). Antioxidant activity of peptides
enzyme -inhibitory properties of a axseed protein-derived high scher isolated from alfalfa leaf protein hydrolysate. Food Chemistry, 111(2), 370376.
ratio peptide mixture. Journal of Agricultural and Food Chemistry, 58(8), You, S. J., Udenigwe, C. C., Aluko, R. E., & Wu, J. P. (2010). Multifunctional peptides
47624768. from egg white lysozyme. Food Research International, 43(3), 848855.
Vastag, Z., Popovic, L., Popovic, S., Krimer, V., & Pericin, D. (2011). Production of Yu, F. R., Lu, S. Q., Yu, F. H., Feng, S. T., McGuire, P. M., Li, R. D., et al. (2006). Protective
enzymatic hydrolysates with antioxidant and angiotensin-I converting enzyme effects of polysaccharide from Euphorbia kansui (Euphorbiaceae) on the
inhibitory activity from pumpkin oil cake protein isolate. Food Chemistry, swimming exercise-induced oxidative stress in mice. Canadian Journal of
124(4), 13161321. Physiology and Pharmacology, 84(10), 10711079.