ORI GI NAL PAPER

Antioxidant activity of ovine casein hydrolysates: identication

of active peptides by HPLCMS/MS
Jose A

ngel Gomez-Ruiz Ivan Lopez-Exposito

Anne Pihlanto Mercedes Ramos Isidra Recio
Received: 25 August 2007 / Revised: 20 December 2007 / Accepted: 28 December 2007 / Published online: 5 February 2008
Springer-Verlag 2008
Abstract This paper shows the potential role of different
ovine casein fractions and their hydrolysates to exert
antioxidant activity. ABTS
+
decolorization assay was used
to evaluate the antioxidant activity of the casein fractions
(b-, j- and a
s
-caseins) before and after their hydrolysis by
pepsin, trypsin and chymotrypsin. Although the antioxidant
activity increased in all the fractions after hydrolysis, the
effect was particularly remarkable in the j-casein fraction,
which increased its antioxidant activity almost threefold.
Further assays in a linoleic acid oxidation system showed
that j-casein hydrolysate inhibited lipid peroxidation.
Analysis of the ovine j-casein hydrolysate by RP-HPLC
MS/MS allowed the identication of 12 peptide sequences
with potential antioxidant properties. One of the most
abundant peptides, the fragment HPHPHLSF [f(98105)]
was chemically synthesized. Results showed that this j-
casein-derived peptide was a potent inhibitor of linoleic
acid oxidation with an activity similar to that obtained with
the synthetic antioxidant BHT. Although other peptides
might also contribute, HPHPHLSF was the one most likely
to be responsible for the activity found in the j-casein
hydrolysate.
Keywords Antioxidant peptides Gastrointestinal
enzymes Mass spectrometry Ovine casein hydrolysates
Ovine j-casein
Introduction
The importance of oxidation in the body and in foodstuffs
has been widely recognized. Oxidative metabolism is
essential for the survival of cells. As a result of this metab-
olism free radicals are formed, which can overwhelm
protective enzymes such as superoxide dismutase, catalase
and peroxidase, causing destructive and lethal cellular
effects by oxidizing membrane lipids, cellular proteins,
DNA and enzymes. Oxidative stress plays a signicant role
in a number of age-specic diseases such as diabetes, cancer
and atherosclerosis [1]. In foods, it is recognized that lipid
peroxidation causes deteriorations in food quality, unac-
ceptable taste and shortening of self-life. Currently,
synthetic antioxidants such as BHA and BHT are commonly
used in food. However, the potential adverse effects of these
synthetic additives have stimulated their replacement by
natural antioxidants derived from dietary sources [2].
One practical approach to solve this problem is the use
of safer antioxidants from natural sources, mainly non-
protein compounds from plants. However, some proteins
from certain foods have also been reported to have the
ability to scavenge active oxygen species [3]. In addition to
food proteins, some food-protein hydrolysates have been
found to exhibit antioxidant activity [46]. Among them,
milk protein hydrolysates and milk-derived peptides
obtained by fermentation have recently started to be
J. A

. Gomez-Ruiz I. Lopez-Exposito M. Ramos

I. Recio (&)
Instituto de Fermentaciones Industriales,
Consejo Superior de Investigaciones Cienticas (CSIC),
Juan de la Cierva 3, 28006 Madrid, Spain
e-mail: recio@i.csic.es;
Jose-Angel.Gomez-Ruiz@ec.europa.eu
J. A

. Gomez-Ruiz
Food Safety and Quality Unit, Institute for Reference Materials
and Measurements (IRMM), Retieseweg, 2440 Geel, Belgium
A. Pihlanto
Biotechnology and Food Research, MTT Agrifood Research
Finland, 31600 Jokioinen, Finland
1 3
Eur Food Res Technol (2008) 227:10611067
DOI 10.1007/s00217-008-0820-3
investigated (for a recent review see [7]). The utilization of
protein hydrolysates or peptides to improve the antioxidant
activity in foods presents additional advantages over other
natural antioxidants, since they also confer nutritional
value, as well as, desired functional properties. Antioxidant
peptides can be obtained by enzymatic hydrolysis of whey
proteins or puried b-lactoglobulin, the major whey protein
[810]. Regarding the casein fraction of milk, there are also
antioxidant peptides identied from the major casein
fractions, i.e., b-casein [11, 12] and a
s1
-casein [13].
However, the identication of antioxidant peptides from
minor caseins (j- or a
s2
-casein) is a more labor-intensive
task and, to the best of our knowledge, there is scarcely one
report that shows antioxidant activity in a bovine j-casein
derived peptide [14].
Research on bioactive peptides is mainly focused on
bovine milk proteins, paying less attention to caseins from
other mammalian species [15]. However, given the high
homology among the sequences of bovine, ovine and
caprine milk proteins, it would be predictable that bioactive
peptides released from bovine proteins could also be con-
cealed within the sequences of ovine and caprine milk
proteins. Although in the world production, ovine milk
production ranks third, dairy sheep farming is a vital part of
the national economy of many countries, especially in the
Mediterranean and Middle East region due to the produc-
tion of cheese.
The aim of this work was to evaluate the antioxidant
activity of different ovine casein fractions hydrolyzed with
several gastrointestinal enzymes, mimicking, to a certain
extent, what caseins could undergo in vivo when milk is
consumed. Gastrointestinal enzymes were food grade and
commercially available for production purposes. Antioxi-
dant activity of the casein fractions was evaluated before
and after hydrolysis by the ABTS
+
decolorization assay. j-
Casein fraction showed the greatest improvement in terms
of antioxidant activity following enzymatic hydrolysis.
Peptides present in this hydrolysate were identied by
HPLCMS/MS. One of these peptides, with amino acid
sequence HPHPHLSF, was chemically synthesized and its
antioxidant activity evaluated in a linoleic acid oxidation
system.
Materials and methods
Enzymatic hydrolysis
Ovine fractions were isolated by cation-exchange using an
FPLC apparatus (GE Healthcare, Uppsala, Sweden) on a
prepacked Mono S SR 5/5 column (GE Healthcare, Upp-
sala, Sweden) following the method described by Gomez-
Ruiz et al. [16]. The different casein fractions were
collected, dialyzed with Spectra/Por

dialysis membranes
with 6,0008,000 Da cut-off for 48 h at 4 C, and lyoph-
ilized. These fractions were analyzed by capillary
electrophoresis (CE) as previously described [17].
Casein fractions (a
s
-, b-, j- and isoelectric casein) were
dissolved at 3 g/L in 0.01 M HCl at pH 2.0. The fractions
were rst hydrolysed with pepsin A (570 U/mg protein, EC
3.4.23.1) for 2 h at 37 C and then, after raising the pH to 8
with NaOH 0.1 M, with trypsin (44 U mg/protein, EC
3.4.21.4) and chymotrypsin (10,500 U/mg protein, EC
3.4.21.1) for 4 h at 37 C. The enzyme:substrate ratio was
1:25 (w/w) for the treatment with pepsin and 1:200 (w/w)
for the treatment with trypsin and chymotrypsin. The
reaction was stopped by heating the solution at 95 C for
30 min. The solution was centrifuged at 35,000g, and the
supernatant collected, lyophilized and stored at -20 C
previous to its use.
2,2
0
-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid,
ABTS
+
) decolorization assay
The assay was carried out according to Re et al. [18].
Samples (20 lL) were combined with 2 mL of ABTS
+
reagent and the absorbance at 734 nm monitored for
20 min at 30 C using a Lambda Bio 20 UV/Vis spectro-
photometer (PerkinElmer, Cambridge, UK). Readings
taken after 15 min were used to calculate antioxidant
activity. Trolox, the water-soluble analog of vitamin E, was
used as a reference standard. A standard curve was pre-
pared by measuring the percent inhibition values at
different concentrations of Trolox. Inhibition values were
calculated as follows (Eq. 1):
%Inhibition 100A
t0sample
A
t5sample
=A
t0sample

A
t0solvent
A
t5solvent
=A
t0solvent
:
1
The Trolox equivalent antioxidant capacity (TEAC) of
each sample represents the concentration of Trolox with
the same antioxidant capacity as the extract. Samples were
prepared by triplicate and the activity of each was
measured in duplicate.
Assay in a linoleic acid oxidation system
Linoleic acid was oxidized in a linoleic acid model system
to measure the antioxidant activity of j-casein hydrolysate
and synthetic peptide HPHPHLSF following a modied
version of the method proposed by Mendis et al. [19].
Briey, 375 lL of 0.1 M sodium phosphate buffer (pH 7.0)
and 375 lL of 50 mM linoleic acid in ethanol were mixed
and 500 lL of the test sample (pH 7.0) was added. BHT
1062 Eur Food Res Technol (2008) 227:10611067
1 3
(Sigma, St. Louis, MO) was used instead of test sample as
positive control and water as a negative control. The mixed
solution was incubated in the dark at 60 C. The degree of
linoleic acid oxidation was measured at 24-h intervals
using the ferric thiocyanate method described in Osawa
and Namiki [20]. An aliquot (0.1 mL) of the reaction
mixture was mixed with 75% ethanol (4.5 mL) followed by
the addition of 30% ammonium thiocyanate (0.1 mL), HCl
of 1.0 N (0.2 mL) and 20 mM of ferrous chloride solution
in 3.5% HCl (0.1 mL). After 5 min incubation, the color
development, which represents the linoleic acid oxidation,
was measured at 500 nm. Antioxidant activity (%) was
calculated as follows (Eq. 2):
Abs control 500nm
Abs sample 500nm=Abs control 500nm 100:
2
Samples were prepared by triplicate and the activity of
each was measured in duplicate.
Analysis by on-line RP-HPLCESI-MS
RP-HPLC separation of the j-casein hydrolysate was
performed on a HP Agilent 1100 HPLC System (Agilent
Technologies, Waldbron, Germany) connected on-line to
an Esquire-LC quadrupole ion trap (Bruker Daltonik,
Bremen, Germany) as previously described [21]. Solvent
A was a mixture of water:TFA, 1,000:0.37, and B was a
mixture of acetonitrile:TFA, 1,000:0.27. Peptides were
eluted with a linear gradient of solvent B in A increasing
from 0 to 45% in 60 min, and from 45 to 70% in 5 min
at a ow rate of 0.8 mL/min. The ow was split post
UV detector by connecting a T-piece (Valco, Houston,
TX, USA) with a 75 lm ID peek outlet tube, of an
adjusted length, to give a ow of approximately 20 lL/
min which was directed into the mass spectrometer via
the electrospray interface. Spectra were recorded over the
mass/charge (m/z) range 1001,500. Precursor ions were
fragmented with a voltage ramp going from 0.35 to
1.4 V. Using Data Analysis
TM
(version 3.0; Bruker
Daltoniks), the m/z spectral data were processed and
transformed to spectra representing mass values. Bio-
Tools (version 2.1; Bruker Daltoniks) was used to
process the MS (n) spectra and perform peptide
sequencing.
Peptide synthesis
HPHPHLSF was synthesized by GenScript Corporation
(Piscataway, NJ, USA). Genscript also veried the purity
of the peptide by analytical RP-HPLCMS.
Results and discussion
Radical scavenging activity
Firstly, the antioxidant activity of each ovine casein frac-
tion was evaluated by the ABTS
+
method. Results showed
that the a
s
-casein fraction was the most active with a TEAC
value of 1.63 mg/mL, i.e., a concentration of 1 mg/mL of
a
s
-casein was able to scavenge the same amount of radicals
as a solution of 1.63 mg/mL of Trolox (Fig. 1a). On the
contrary, intact j-casein showed the lowest activity with a
TEAC value of 0.53 mg/mL, and the whole ovine casein
reached a TEAC value of 0.85 mg/mL (Fig. 1a). The non-
hydrolysed b-casein fraction revealed an antioxidant
capacity similar to that of Trolox. A study on the antioxi-
dant activity of different amino acids assessed by the
ABTS
+
assay, reported that Cys was the most active amino
acid followed by Trp, Tyr and His. The remaining amino
acids analyzed did not exhibit antioxidant activity by the
ABTS
+
method [22]. When the primary structure of the
different ovine caseins is compared, it can be observed that
a
s2
-casein is the fraction containing the highest number
(22) of the four amino acids mentioned above. a
s1
-casein
and j-casein possess 17 residues belonging to this group of
amino acids with antioxidant activity while b-casein pos-
sesses only 9. Based on the primary structure, j-casein
could be expected to exhibit a similar activity to the a
s
-
casein fraction (that included a
s1
-casein, as major compo-
nent, and a
s2
-casein) and higher than that of b-casein.
However, it was found that j-casein had weak antioxidant
activity (TEAC = 0.53 mg/mL), even lower than that of
the b-casein fraction (TEAC = 1.06), which possesses
only nine potentially active amino acid residues. Conse-
quently, these results indicated that not only the primary
structure of the proteins but also other factors, such as
protein conformation, are important to exert antioxidant
activity by the ABTS
+
assay.
Hydrolysis of the different casein fractions with gas-
trointestinal enzymes increased the antioxidant activity in
all cases (Fig. 1a). Although the a
s
-casein fraction was still
the most active (TEAC value of 1.76 mg/mL), the highest
increase in activity after hydrolysis was found for the j-
casein fraction. The antioxidant activity improved almost
threefold reaching a TEAC value of 1.5 mg/mL, similar to
that obtained when the whole casein was hydrolysed.
Consequently, the enzymatic hydrolysis of j-casein
released antioxidant peptides that were inactive when
included within the intact protein. Other authors have
reported that accessibility to the oxidantantioxidant test
systems is greater for small peptides and amino acids than
for large peptides and proteins [23]. The development of
antioxidant activity following enzymatic hydrolysis of
caseins is in line with previous reports [11]. Short peptides
Eur Food Res Technol (2008) 227:10611067 1063
1 3
exhibiting antioxidant activity have been isolated and
characterized from a
s1
-casein [13] and b-casein [11, 12, 24,
25], both from cow milk. However, to our knowledge, this
is the rst time that antioxidant activity in peptides derived
from ovine j-casein has been reported.
Inhibition of lipid peroxidation
Peptides may act by different antioxidant mechanisms
under variable conditions, reecting the multifunctional
properties of these compounds in both physiological and
food-related oxidation processes. It is, therefore, important
to evaluate the antioxidant activity by different methods.
With the aim of evaluating the capacity of ovine j-casein
hydrolysate to inhibit lipid peroxidation (which may be of
importance in food processes), an assay in a linoleic acid
oxidation system was performed. Figure 1 displays the
results of antioxidant activity of the j-casein hydrolysate
(Fig. 1b) compared with that of BHT (Fig. 1c). It should be
noted that the antioxidant capacity of the j-casein hydro-
lysate was dose-dependant. It can be seen that the
maximum activity obtained corresponded to the concen-
tration of 1.71 mg/mL after 2 days of incubation, although
lower concentrations also displayed a notable antioxidant
activity. For instance, at a concentration of 0.22 mg/ml and
after 3 days of incubation at 60 C the hydrolysate exhib-
ited an antioxidant activity of 82.4%, comparable to the
activity found with the same concentration of BHT.
Despite BHT can be active at lower concentrations than
those used in this study, protein hydrolysates could be used
as food ingredients in much higher proportions than these
antioxidant additives.
Analysis and characterization of antioxidant peptides
by RP-HPLCMS/MS
The next step was to identify the peptide or peptides
responsible for the antioxidant activity detected in the
j-casein hydrolysate. Since the hydrolysate is produced by
the action of different enzymes, the presence of numerous
peptides was expected. To sequence major peptides
contained in the j-casein hydrolysate, it was subjected to
RP-HPLC coupled on-line to an ion trap mass spectrome-
ter. Figure 2a shows the UV-chromatogram obtained for
the j-casein hydrolysate. In this peak, at least four different
singly charged ions could be identied. Figure 2b
illustrates the MS/MS spectrum of a singly charged ion
with m/z 971.3 and the amino acid sequence of the
identied peptide with the major fragment ions. It can be
seen that y
00
ions were predominant in the spectrum. In
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
2
)
l
m
/
g
m
(
C
A
E
T
-casein -casein -casein
s
(A)
Time (days)
(C)
0
20
40
60
80
100
0 1.0 2.0 3.0
Time (days)
(B)
0
20
40
60
80
100
0 1.0 2.0 3.0
A
n
t
i
o
x
i
d
a
n
t

a
c
t
i
v
i
t
y

%
A
n
t
i
o
x
i
d
a
n
t

a
c
t
i
v
i
t
y

%
casein
Fig. 1 a Antioxidant activity of the different ovine casein fractions
before (open square) and after (lled square) the hydrolysis with
pepsin, trypsin and chymotrypsin measured by the ABTS
+
decolor-
ization assay expressed as Trolox equivalent antioxidant capacity
(TEAC). b % Antioxidant activity of the ovine j-casein hydrolysate
in a linoleic acid oxidation system for 3 days at 3.42 mg/mL (open
square), 1.71 mg/mL (grey square), 0.85 mg/mL (dark grey square),
0.22 mg/mL (black square). c % Antioxidant activity of the synthetic
antioxidant BHT in a linoleic acid oxidation system for 3 days at
1,000 lM (i.e., 0.22 mg/mL) (open square), 100 lM (grey square),
10 lM (dark grey square), 1 lM (black square). Vertical bars
indicate mean values SD. The assays were conducted in triplicate
1064 Eur Food Res Technol (2008) 227:10611067
1 3
addition, the most intense ions were originated from the
cleavage of peptide bonds involving the imino acid proline
either at the N- or C-terminal [26]. After sequence inter-
pretation and database searching, the MS/MS spectrum was
matched to ovine j-casein f(98105). Following the strat-
egy of identifying the peptides by matching the MS/MS
spectra to selected peptides with a given mass, a total of 12
sequences could be unambiguously identied in the ovine
j-casein hydrolysate, corresponding to the most abundant
peptides in the hydrolysate (Table 1).
It has been reported that amino acids such as His and
Leu are effective at inhibiting oxidation of fatty acids
tested in a linoleic acid model system [27]. One of the most
abundant peptides of the ovine j-casein hydrolysate cor-
responded to peptide HPHPHLSF [f(98105)] that
comprises three His residues and one of Leu. With this
information and bearing in mind the amino acid sequence
of the other peptides identied (Table 1), inhibition of the
lipid peroxidation might be mostly attributed to the pres-
ence of the peptide HPHPHLSF. In addition, the j-casein
derived peptide presents high homology with the sequence
HPHPHL identied in a digest of a soybean protein that
has been reported to show a high capacity to inhibit lipid
peroxidation [28]. Consequently, the peptide j-casein
f(98105) was chemically synthesized and its antioxidant
activity in a linoleic acid model system evaluated. Figure 3
shows the results of the antioxidant activity of the sequence
HPHPHLSF (Fig. 3a) and the synthetic compound BHT
(Fig. 3b) assayed in a range of concentrations from 1,000
to 1 lM. At these concentrations, HPHPHLSF displayed a
capacity to inhibit lipid peroxidation similar to that of
BHT. As an example, 1 lM concentration of HPHPHLSF
at the third day of assay had an antioxidant percentage of
74.1%, and the same concentration of BHT displayed an
antioxidant percentage of 88.4%. As occurred for the total
ovine j-casein hydrolysate, the antioxidant activity of the
j-casein f(98105) was dose-dependant. As shown in
Fig. 3a, at the third day of assay the highest concentration
of peptide revealed an antioxidant activity of 43.7%, con-
siderably lower than when the concentration was
diminished 10
3
times, for which an activity of 74.1% was
measured.
The strong activity shown by the peptide HPHPHLSF
must be related to the presence within its sequence of the
fragments HPH, HL and HPHL. These fragments have
been reported to possess a strong antioxidant activity [28].
15 20 25 30 35 40 45
Time (min)
0
200
400
600
800
A
b
s

(
U
A
)
(B)
(A)
9 . 3 5 3
0 . 3 0 5
1 . 0 0 6
2 . 9 1 7
2 . 7 3 7
2 . 4 3 8
3 . 3 5 9
0 . 0
5 . 0
0 . 1
5 . 1
0 . 2
5.
0 1 x
. s n e t n I
0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 z / m
b
3
H -
2
O
y
4
y
5
y
6
y
7
y
6
H -
2
O
7
3
b s n o i
y s n o i
F S L H P H P H
5 6 4
] H + M [
+
H -
2
O
] H + M [
+
3 . 1 7 9
MS/MS
Fig. 2 a UV-chromatogram of the j-casein hydrolysate by pepsin,
trypsin and chymotrypsin. b Tandem mass spectrum of ion m/z 971.3.
Following sequence interpretation and database searching, the MS/
MS spectrum was matched to ovine j-casein f(98105). The sequence
of this peptide is displayed with the fragment ions observed in the
spectrum. Fragments ions are labeled according to the nomenclature
proposed by Roespstorff and Folhman [31]. For clarity, only the b and
y
00
fragment ions are labeled
Eur Food Res Technol (2008) 227:10611067 1065
1 3
Furthermore, Kudoh et al. [14] identied a bovine j-casein
antioxidant peptide obtained by fermentation of milk with
Lactobacillus delbrueckii subsp bulgaricus IFO13953 that
corresponds to the sequence ARHPHPHLSFM and thus,
includes the peptide identied in this study. Although the
structure-activity relationship of antioxidative His-con-
taining peptides has not yet been well dened, the activity
must be attributed to the hydrogen donating ability, lipid
peroxyl radical trapping, and/or the metal ion-chelating
ability of the imidazole group [29]. In addition, the active
sequence does contain not only several His residues, but
also hydrophobic amino acids (Leu, Pro and Phe) that have
been reported to be important to protect against linoleic
acid oxidation [19].
The structure of some of the identied peptides in the
j-casein hydrolysate may anticipate the presence of other
biological activities. For instance, peptide SRYPSY
[f(3338)] had previously been identied as an opioid
agonist, and peptide YIPIQ [f(2529)] is included within
the sequence of a well-known opioid peptide (YIP-
IQYVLSR) sharing ve amino acids at the N-terminal
(the region involved in the interaction with the opioid
receptor) [30].
Conclusion
The results show for the rst time the antioxidant activity
of ovine milk casein fractions before and after their
hydrolysis with gastrointestinal enzymes. The enzymatic
hydrolysis enhanced the antioxidant activity in all fractions
but especially in the j-casein fraction. A peptide identied
in the j-casein hydrolysate as HPHPHLSF [f(98105)]
appears to be the main responsible for this activity,
showing a similar ability to inhibit lipid peroxidation to
that of the synthetic antioxidant BHT. Despite the need for
further research, ovine caseins could be considered as
suitable natural antioxidants to prevent oxidation reactions
in food processing and become ingredients of functional
foods.
Acknowledgments This study has received nancial support from
the projects AGL2005-03381 and CM-S0505-AGR-0153 nanced by
Ministerio Educacion y Ciencia and Comunidad de Madrid,
respectively.
Table 1 Peptide sequences
identied by on-line HPLC
ESI-MS/MS in the ovine j-
casein hydrolysed with pepsin,
trypsin and chymotrypsin
a
Monoisotopic mass value
Observed mass Calculated mass
a
Molecular ion (m/z)
selected for MS/MS (charge)
Fragment Sequence
523.2 523.2 524.2 (1) f(1821) FDDK
632.4 632.3 633.4 (1) f(2529) YIPIQ
647.5 647.3 648.5 (1) f(6166) YAKPVA
771.3 771.3 772.3 (1) f(3338) SRYPSY
822.3 822.3 823.3 (1) f(1217) EKDERF
835.3 835.4 836.3 (1) f(1824) FDDKIAK
970.4 970.4 971.4 (1) f(98105) HPHPHLSF
973.3 973.5 974.3 (1) f(4350) YQQRPAVL
998.4 998.5 999.4 (1) f(6775) VRSPAQTLQ
1068.4 1068.5 1069.4 (1) f(130139) PTVHSTPTTE
1221.5 1221.7 1222.5 (1) f(7686) WQVLPNAVPAK
1267.4 1267.6 1268.6 (1) f(5160) INNQFLPYPY
A
n
t
i
o
x
i
d
a
n
t
a
c
t
i
v
i
t
y
(
%
)
0
0 2
0 4
0 6
0 8
100
0 . 3 0 . 2 0 . 1 0
Time (days)
Time (days)
A
n
t
i
o
x
i
d
a
n
t

a
c
t
i
v
i
t
y

%
(B)
0
0 2
0 4
0 6
0 8
100
0 . 3 0 . 2 0 . 1 0
(A)
Fig. 3 Percentage of antioxidant activity of the synthetic peptide
HPHPHLSF (a) and synthetic compound BHT (b) in a linoleic acid
oxidation system for 3 days tested at 1,000 lM (open square),
100 lM (grey square), 10 lM (dark grey square) and 1 lM (black
square). Vertical bars indicate mean values SD. The assays were
conducted in triplicate
1066 Eur Food Res Technol (2008) 227:10611067
1 3
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