ARTICLE IN PRESS

International Dairy Journal 16 (2006) 13061314

www.elsevier.com/locate/idairyj

Review

Antioxidative peptides derived from milk proteins

Anne Pihlanto
MTT Agrifood Research Finland, Food Research, FIN-31600 Jokioinen, Finland
Received 12 September 2005; accepted 3 May 2006

Abstract
Antioxidants may function by preventing the formation of radicals or by scavenging radicals or hydrogen peroxide and other
peroxides. Milk contains several antioxidant factors, like vitamins and enzymes. Possible antioxidant activity of milk proteins and
hydrolysates has also been shown. Peptides generated from the digestion of milk proteins are reported to have antioxidative activities.
Milk-derived antioxidative peptides are composed of 511 amino acids including hydrophobic amino acids, proline, histidine, tyrosine or
tryptophan in the sequence. The structureactivity relationship or the antioxidant mechanism of peptides is not fully understood.
Antioxidant activity of the hydrolysates seems to be inherent to the characteristic amino acid sequences of peptides derived, depending
on the protease specicity. The results suggest that the hydrolysates from milk proteins could be used as natural antioxidants in
enhancing antioxidant properties of functional foods and in preventing oxidation reaction in food processing. Further studies are needed
to elucidate the role of antioxidative peptides in the protective function in humans.
r 2006 Elsevier Ltd. All rights reserved.
Keywords: Antioxidant activity; Milk protein; Hydrolysates; Peptides

Contents
1.
2.

3.

4.
5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Methods to analyse antioxidative activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Activities and properties of antioxidative peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Enzymatic hydrolysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Application in foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Health effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction
The importance of oxidation in the body and in
foodstuffs has been widely recognized. Oxidative metabolism is essential for the survival of cells. A side effect of this
Tel.: +358 3 41883276; fax: +358 3 41883244.

E-mail address: anne.pihlanto@mtt..

0958-6946/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.idairyj.2006.06.005

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dependence is the production of free radicals and other

reactive oxygen species that cause oxidative changes. There
is increasing evidence for the involvement of such species in
a variety of normal in vivo regulatory systems. When an
excess of free radicals is formed, they can overwhelm
protective enzymes like superoxide dismutase, catalase and
peroxidase, and cause destructive and lethal cellular effects
(e.g. apoptosis) by oxidizing membrane lipids, cellular

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A. Pihlanto / International Dairy Journal 16 (2006) 13061314

proteins, DNA and enzymes, thus shutting down cellular

respiration. It is well-known that lipid peroxidation
occurring in food products causes deteriorations in food
quality, for example rancid avour, unacceptable taste and
shortening of shelf life. In addition, it has been recognized
that oxidative stress plays a signicant role in a number of
age specic diseases. The factors involved in these diseases
are the lipid peroxides and low molecular weight compounds produced during the late stage of the oxidative
reaction. For example, many studies have shown increased
oxidative damage to all the major classes of biomolecules
in the brains of Alzheimers patients (Halliwell, 2001; Liu,
Raina, Smith, Sayre, & Perry, 2003). Other pathological
conditions associated with evidence of signicant free
radical mediated injury include atherosclerosis, diabetes,
rheumatoid arthritis (Abuja & Albertini, 2001; Halliwell,
2000; Halliwell & Whiteman, 2004; Hoelzl et al., 2005).
Furthermore, cancer is probably a consequence of oxidative DNA-damage (Collins, 2005). To prevent foods from
undergoing deterioration and to provide protection against
serious diseases, it is important to inhibit the peroxidation
of lipids and formation of free radicals occurring in the
living body and foodstuffs. Lipid oxidation is inhibited by
antioxidative agents. Articial antioxidants (BHA, BHT
and n-propyl gallate) exhibit strong antioxidant activity
against several oxidation systems. However, because
articial antioxidants pose potential risks in vivo, their
use in foodstuffs is restricted or prohibited in some
countries. Safer antioxidants from natural sources, mainly
nonprotein compounds from plants, have therefore been
developed. Some proteins from certain foods have been
reported to have the ability to scavenge active oxygen
species (Okada & Okada, 1998). Several food protein
hydrolysates have been found to exhibit antioxidant
activity (Chen, Muramoto, & Yamauchi, 1995; Chen,
Muramoto, Yamauchi, Fujimoto, & Nokihara, 1998;
Davalos, Miguel, Bartolome, & Lopez-Fandino, 2004;
Saiga, Tanabe, & Nishimura, 2003). In addition, several
amino acids, for example tyrosine, methionine, histidine,
tryptophan and proline, have been shown to act as
antioxidants, although in specic cases (e.g. high concentration) they may also act as pro-oxidants (Jung, Kim, &
Kim, 1995; Marcuse, 1960).
Beyond the presence of valuable macro- and micronutrients, milk contains antioxidant factors. These are
represented by naturally occurring vitamins (i.e. E and
C), beta-carotene, and enzymatic systems, mainly superoxide dismutase, catalase and glutathione peroxidase
(Lindmark-Mansson & Akesson, 2000). In addition,
possible antioxidant activity of milk whey has also been
suggested. It would include chelation of transition metals
by serum albumin and lactoferrin, an iron-binding
glycoprotein, as well as free radical scavenging activity by
amino acids, such as tyrosine and cysteine (Colbert
& Decker, 1991; Meucci, Mordente, & Martorana,
1991; Shinmoto, Dosako, & Nakajima, 1992; Taylor &
Richardson, 1980).

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The following chapters will describe the antioxidative

peptides derived from milk proteins and the production
and potential applications in food.
2. Chemistry
2.1. Methods to analyse antioxidative activity
A broad variety of in vitro techniques has been
developed for the detection of antioxidants on the basis
of different antioxidative mechanism under variable conditions reecting the multifunctional properties of antioxidants in both physiological and food-related antioxidation
processes. These methods are based on the direct interaction with reactive molecules or their reactivity with metal
ions, and the effects are monitored by chemical measurements. Conicting results have been obtained for antioxidative capacity and activity in different assays due to, for
example, the presence of interacting components, the mode
of initiation of oxidation or the analytical method for
measuring oxidation.
Radical scavenging is the main mechanism by which
antioxidants act in foods. The radical scavenging assays
primarily operate by direct measurement of hydrogen
donation or electron transfer from the potential antioxidant to a free radical in simple lipid free systems. The
ability to scavenge specic radicals may be targeted as, for
example, hydroxyl radical, superoxide radical or nitric
oxide radical. The electron transfer antioxidant capacity is
commonly quantied by the Trolox equivalent antioxidant
capacity (TEAC) and the ferric reducing antioxidant power
(FRAP) assays. The TEAC assay relies on the reduction of
coloured cation radical of 2,20 -azinobis(3-ethylbenzothiazoline-6-sulphonate) (ABTS+) and the antioxidant capacity is quantied as the concentration of water solublevitamin E analogue, Trolox, that produces the same effect
as the sample (Miller & Rice-Evans, 1996). The FRAP
assay measures antioxidant capacity by the reduction of the
ferric tripyridyltriazine complex to the blue ferrous
complex (Benzie & Strain, 1999). Scavenging by a stable
chromogen radical, such as 1,1-diphenyl-2-picrylhydrazyl
radical (DPPHd), quantied spectrophotometrically in
organic media is extensively used for comparison of
homologous series of antioxidants. The decrease in colour
has been correlated to a dose response curve with a
standard antioxidant as in the TEAC assay (Arnao, 2000).
These methods may be useful for screening antioxidants,
but antioxidant effectiveness in foods should be studied by
other methods, since their activity in foods is dependent on
a variety of factors including polarity, solubility, and
metal-chelating activity.
Methods based on stable radicals have been criticized
because these stable radicals are foreign to biological
systems, in contrast to the short-lived radicals, like the
hydroxyl and peroxylradicals occurring as reaction intermediates in oxidative processes. Assays based on these
radicals have been accordingly developed such as the

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A. Pihlanto / International Dairy Journal 16 (2006) 13061314

oxygen radical absorbance capacity (ORAC) assay based

on quenching of uorescence from the protein b-phycoerythrin by radicals (Cao, Soc, & Prior, 1997). Peroxyl
radicals, which are intermediates in lipid oxidation, may
be generated by azo compounds such as lipophilic
a,a-azobisisobutyronitrile (AIBN) and 2,20 -azobis(2,4-dimethlylvaleronitrile) (AMVN) and the hydrophilic
2,20 azobis(2-amidinopropane) dihydrochloride (AAPH).
These initiators have been found to be useful in the study
of the kinetics of radical scavenging in various systems
because radicals are generated at a reproducible and
constant rate depending on temperature (Niki, 1990).
These azo compounds are also articial and not natural
to food or other biological systems. The most widely used
antioxidant assay has been the total radical trapping
antioxidant parameter (TRAP) method developed by
Wayner, Burton, Ingold, and Locke (1985). It is based on
measurements of induction times in the oxidation of a lipid
dispersion exposed to a free radical source with a constant
rate of free radical production in aerobic conditions. 2,20 Azobis(2-amidinopropane) (ABAP) has been extensively
employed as a source of alkylperoxyl free radicals and the
decrease in oxygen concentration is used to measure the
oxidation rate. This procedure has been modied by
employing other radical sources and other techniques to
monitor the process (Lissi, Salim-Hanna, Pascual, & del
Castillo, 1995).
Evaluation of the ability to inhibit or halt lipid oxidation
in model systems is based on measuring changes in the
concentration of compounds being oxidized, on depletion
of oxygen or on formation of oxidation products.
Quantication of the loss of reactants (oxygen, unsaturated
fatty acids), formation of free radicals, and formation of
oxidation products may be the most appropriate marker
depending on the stage of oxidation. The depletion of
oxygen and the electron spin resonance (ESR) spectroscopy detection of radical, either directly or indirectly by
spin trapping, can be used to follow the initial steps during
oxidation (Andersen & Skibsted, 2002). Standard methods
for measurement of lipid oxidation as peroxide value
(POV, formation of lipid hydroperoxides), thiobarbituric
acid reactive substances (TBARS, formation of secondary
oxidation products) among others are used. The TBARS
test assay is based on the measurement of malondialdehyde
(MDA) formed as a consequence of lipid peroxidation and
the test can be conducted with subcellular membrane
preparations or intact cells. The test is unspecic and does
not only measure the formation of malondialdehyde, but
also other oxo-compounds. Prevention of MDA formation
can be used to assess antioxidant properties (for a review
see Antolovich, Prenzler, Patsalides, McDonald, & Robards, 2002; Becker, Nissen, & Skibsted, 2004; Decker,
Warner, Richards, & Shahidi, 2005; Hoelzl et al., 2005).
Since various methods are used to test the antioxidant
activity the results are expressed in a variety of ways which
makes comparison difcult. A general major problem
associated with the use of these chemical analytical

methods is that they are conducted under non physiological conditions. Accordingly, the results cannot be extrapolated to the in vivo situation. A step closer to human
situation is the use of intact cells which can be challenged
with reactive oxygen species (ROS) generating chemicals or
radiation in the absence and presence of putative antioxidants. The extracellular release of superoxide or
intracellular ROS and superoxide production, for example,
can be measured by a plate reader assay with cells in situ or
by ow cytometric analysis. Biomarkers, such as 8hydroxy-20 -deoxyguanosine, of oxidative damage in cellular DNA have also been developed (Loft & Mller, 2002;
Halliwell & Whiteman, 2004; Hoelzl et al., 2005).
2.2. Activities and properties of antioxidative peptides
Peptides generated from the digestion of various proteins
are reported to have antioxidative activities. For example,
soybean proteins are known to contain antioxidative
peptides. Soy peptides are composed of 316 amino acids
including hydrophobic amino acids, valine or leucine, at
the N-terminal positions, and proline, histidine or tyrosine
in the sequence. Studies with peptides containing histidine
have demonstrated that these peptides can act as metal-ion
chelators, active-oxygen quencher, and hydroxyradical
scavenger. The ability of protein hydrolysates to inhibit
deleterious changes caused by lipid oxidation appears to be
related to the nature and composition of the different
peptide fractions produced, depending on the protease
specicity. For example, the antioxidant activity of a soy
protein hydrolysaste was attributed to those peptides with
a LeuLeuProHisHis sequence (Chen, Muramoto,
Yamauchi, & Nokihara, 1996; Chen et al., 1998).
Caseins have been shown to provide antioxidant activity
against TBARS in both Fe/ascorbate induced peroxidation
of arachidonic derived liposomes and model linoleic acid
systems (Cervato, Cazzola, & Cestaro, 1999; Wong &
Kitts, 2003). Laakso (1984) reported that milk casein
inhibit lipoxygenase-catalyzed lipid autoxidation. Quenching of free radicals by oxidation of amino acid residues in
casein was proposed as the explanation. However, free
amino acids could not substitute for casein as the
antioxidant, suggesting that the primary structure of casein
molecules played a role. Suetsuna, Ukeda, and Ochi (2000)
isolated and identied free radical scavenging activity from
peptic digest of casein. The peptide, TyrPheTyrPro
GluLeu, was found to possess a potent superoxide anion
radical scavenging activity (Table 1). The C-terminal
dipeptide GluLeu sequence proved to be important for
the activity.
Caseins have polar domains that contain phosphorylated
serine residues, and their characteristic sequences, SerP
SerPSerPGluGlu, are effective cation chelators that
form complexes with calcium, iron and zinc. Thus,
phosphorylated caseins and/or their peptides in the
aqueous phase could be a source of natural chelators to
control lipid oxidation in food emulsions by binding and

b-Lactoglobulin
(b-lg)

Fermentation with
Lactobacillus delbrueckii
subsp. bulgaricus
Corolase PP
TrpTyrSerLeuAlaMetAlaAlaSerAspIle
MetHisIleArgLeu
TyrValGluGluLeu

AlaArgHisProHisProHisLeuSerPheMet

b-lg f(1929)
b-lg f(145149)
b-lg f(4246)

k-cn f(96106)

b-cn f(177183)
b-cn f(169176)
b-cn f(170176)

AlaValProTyrProGlnArg
LysValLeuProValProGluLys
ValLeuProValProGluLys
Caseinophosphopeptides

Milk

b-cn f(98105)

ValLysGluAlaMetAlaProLys

Trypsin

Radical scavenging activity

(ORAC)

DPPH radical scavenging

activity

Inhibition of TBARS
formation
Free radical scavenging activity

Radical scavenging activity

Superoxide anion
Hydroxyl radical
DPPH radical
Inhibition of enzymatic and
non enzymatic lipid
peroxidation
DPPH radical scavenging
activity

as1-cn f(144149)

TyrPheTyrProGluLeu

Pepsin

Antioxidative activity

Fragment

Casein (cn)

Peptide

Treatment

Protein source

Table 1
Antioxidative peptides derived from milk proteins

HernandezLedesma,
Davalos,
Bartolome, and
Amigo (2005a)

Diaz and Decker

(2004)
Kitts and Weiler
(2003)
Kudoh et al.
(2001)

Rival et al.
(2001b)

Suetsuna et al.
(2000)

Reference

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A. Pihlanto / International Dairy Journal 16 (2006) 13061314

partitioning transition metals away from the emulsion

droplet. Casein hydrolysates have been shown to be more
effective inhibitors of lipid oxidation than the enriched
caseinophosphopeptides at equal phosphorous content.
Antioxidant properties might, therefore not be uniquely
attributed to chelating metals by phosphoseryl residues but
also to scavenging of free radicals (Diaz, Dunn, McClements, & Decker, 2003). The antioxidant mechanism of
caseinophosphopeptides (CPP) and casein hydrolysates has
further been studied in phosphatidylcholine liposome
model systems (Diaz & Decker, 2004). Both samples were
effective inhibitors of TBARS development when oxidation
was promoted by ferric/ascorbate. In addition, high
amounts of CPP were observed to be pro-oxidants,
whereas casein hydrolysates were only antioxidative.
Casein hydrolysates and low molecular weight casein
hydrolysates had better peroxyl radical scavenging activities than enriched CPP, which might be explained by a
comparison of their amino acid content. Casein hydrolysates were reported to have higher concentration of
histidine, lysine, proline and tyrosine than CPP, and all
these amino acids have been previously found to act as free
radical scavengers. In the presence of AAPH, which
generates peroxyl radicals that result in deterioration of
lipids by a free radical chain reaction, CPP appears to act
as a chain-breaking antioxidant, reducing the peroxidation
of liposome lipid. A similar nding was obtained with the
quenching of a hydrophilic free radical, ABTS (Kitts &
Weiler, 2003). Casein calcium peptides, molecular mass
about 3 kDa, have been shown to possess strong antioxidant activity with the b-carotene bleaching method, and
they also showed scavenging activity against radicals such
as superoxide, DPPH and hydroxyl radicals. The activity
was dose dependent (Sakanaka, Tachibana, Ishihara, &
Juneja, 2005).
The effects of various types of bovine casein on the
activity of lipoxygenase in vitro have been evaluated and
the domain within the protein responsible for the
inhibitory activity identied (Rival, Boeriu, & Wichers,
2001a; Rival, Fornaroli, Boeriu, & Wichers, 2001b). They
found that a tryptic b-casein digest and tryptic and
subtilisin digests of whole casein retained their inhibitory
properties. The highest inhibition of linoleic acid oxidation
was observed in the fraction containing b-casein f(169176)
with a trace amount of b-casein f(3348). The effect was
lower than that of the unfractionated digest but higher
than the fraction containing the undigested casein. The
structure-function relationship between the amino acid
sequence, antioxidant capacity and effectiveness were
assayed with synthetic peptides using three different
methods (Rival et al., 2001b). Most of the casein-derived
peptides were antioxidants and presumably oxidized by
free radical intermediate(s) formed during linoleic acid
oxidation. A structurally related compound, carnosine (the
dipeptide b-Ala-His), was shown to exhibit both antioxidant and lipoxygenase inhibitory properties (Decker &
Faraji, 1990). b-Casein f(177183) was a strong antiox-

idant, but almost inactive against enzymatically induced

linoleic acid oxidation. Other compounds, such as vitamin
E, are reported to be antioxidants without being signicant
inhibitors of linoleic acid oxidation (Auerbach, Kiely, &
Cornicelli, 1992). Rival et al. (2001b) concluded that
casein-derived peptides inhibited enzymatic and nonenzymatic lipid peroxidation, most likely by being a
preferred target over fatty acid free radicals. Indirect
evidence suggested that proteins/peptides can be oxidized
during the process, according to a site- or sequence-specic
mechanism.
Hernandez-Ledesma, Davalos, Bartolome, and Amigo
(2005) identied several peptides in the b-lactoglobulin A
hydrolysate by corolase PP. One of the peptides,
TrpTyrSerLeuAlaMetAlaAlaSerAspIle, possessed higher radical scavenging activity than butylated
hydroxyanisole (BHA). The ORAC-FL value of this
peptide was lower than that of tryptophan. The peptides,
MetHisIleArgLeu and TyrValGluGluLeu showed
radical scavenging activity that was attributed to the
presence of methionine and tyrosine, although the antioxidant activity of these amino acids on their own was
higher. The results with equimolar amino acid mixtures
suggested that peptide conformation can lead to both
synergistic and antagonistic effects in comparison with
those exerted by the amino acid mixture.
Several models obtained by multivariate analysis methods, i.e., stepwise linear regression, principal component
analysis and discriminant analysis, were found to be useful
to predict the antioxidant ability of various peptide
fractions on the basis of peptide size and amino acid
composition. The statistical models may be helpful in
designing antioxidant peptides with desired properties
(Pena-Ramos, Xiong, & Arteaga, 2004).
Neither the structureactivity relationship nor the
antioxidant mechanism of peptides is fully understood.
Saito et al. (2003) have screened 40 peptides structurally
related to LeuLeuProHisHis, which is an antioxidative
peptide isolated from soybean protein digests, and
ProHisHis was identied as the active centre. The
antioxidative properties of peptides varies depending
on their structure and assay system. Several unique
tripeptides were identied such as TyrHisTyr, Xaa
XaaTrp/Tyr and XaaXaaCys(SH) that had a
strong synergistic effect with phenolic antioxidants,
a high radical scavenging activity, and a high peroxynitrite
scavenging activity, respectively. The antioxidant activities
of tryptophan and tyrosine may be explained by the
special capability of phenolic and indolic groups to
serve as hydrogen donors. The phenoxyl and indolyl
radicals are much more stable and have longer lifetimes
than simple peroxy radical, so any reverse reaction or the
propagation of the radical-mediated peroxidizing
chain reaction are inhibited. As shown in Table 1,
antioxidative peptides so far identied from milk proteins
contain one or more residues of histidine, proline, tyrosine
and tryptophan.

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3. Technologies
3.1. Enzymatic hydrolysis
Casein and casein tryptic hydrolysates were proposed to
act as free radical scavengers when they were observed to
inhibit lipoxygenase conversion of linoleic acid in the
corresponding monohydroxyperoxide (Rival et al., 2001a).
Furthermore, casein and casein hydrolysates protected
linoleic acid from oxidation in the presence of the peroxyl
radical generator AAPH and showed free radical scavenging activity in a linoleic hydroperoxide-activated ferryl
haemoglobin system.
It has been demonstrated in model systems that the
antioxidant properties of whey proteins can be increased
through fractionation or hydrolysis with certain enzymes,
as some peptides and fractions possess stronger antioxidant
activity than others. Tong, Sasaki, McClements, and
Decker (2000), tested the antioxidant activity of high
molecular mass components of whey in an emulsion
system. The results indicated that high molecular mass
proteins were more effective than low molecular mass
polypeptides in inhibiting lipid peroxide production. PenaRamos et al. (2004) found that native and hydrolyzed whey
protein fraction 445 kDa showed a higher TBARS
inhibition compared with lower molecular mass fractions
and hydrolysate mixtures. In contrast, commercial whey
protein hydrolysates showed a higher inhibitory effect in
most of the lower molecular mass fractions. Colbert and
Decker (1991) reported an inhibition of copper-catalysed
liposome oxidation for whey protein fractions with Mr
o0.5 kDa. Hydrolysis of heat-treated whey protein isolate
by commercial crude enzymes, especially protease F,
produced antioxidant activity, in terms of inhibition of
TBARS formation in an iron-catalyzed liposomal system
by as much as 65% depending on the proteases and
hydrolysis conditions used (Pena-Ramos & Xiong, 2001).
The results furthermore suggested that the hydrolytic
components with antioxidant activity probably were
peptides with relatively low molecular mass (o10 kDa),
as well as certain particular free amino acids. Despite the
antioxidative activity demonstrated by the hydrolysates,
none of them was as effective as propyl gallate (0.01%).
This might be due to the possibility that hydrolysates may
contain both antioxidative and pro-oxidative components
making them a less efcient antioxidant system against
lipid oxidation. In general, the ability of whey protein
isolate fractions to delay lipid oxidation was found to be
related to the prevalence of histidine and hydrophobic
amino acids. Hernandez-Ledesma, Davalos et al. (2005)
investigated the antioxidant activity of hydrolysates from
whey proteins a-lactalbumin and b-lactoglobulin A by
commercial proteases (pepsin, trypsin, chymotrypsin,
thermolysin and coroloase PP). Corolase PP was the most
appropriate enzyme to produce hydrolysates having radical
scavenging activity (ORAC-FL values) and the 3 kDa
permeate fraction was mainly responsible for the antiox-

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idant activity found in the whole hydrolysates. Generally,

no relationship was found between the degree of hydrolysis
and the antioxidative activity for different whey protein
hydrolysates. These results indicated that the antioxidant
activity of the hydrolysates was inherent to the characteristic amino acid sequences of peptides derived, depending
on the protease specicity.
3.2. Fermentation
It has been shown that some lactobacilli possess
antioxidative activity and are able to reduce risk of ROS
accumulation during the ingestion of food (Kaizu, Sasaki,
Nakajima, & Suzuki, 1993; Peuhkuri et al., 1996). Lactic
acid bacteria are able to degrade the superoxide anion and
hydrogen peroxide (Korpela, Lahteenmaki, Sievi, Saxelin,
& Vapaatalo, 1997; Kullisaar et al., 2002). To date, only a
few antioxidant peptides have been identied in fermented
dairy products. A k-casein derived peptide with DPPH
radical scavenging activity has been found in milk
fermented with Lactobacillus delbrueckii ssp. bulgaricus
(Kudoh, Matsuda, Igoshi, & Oki, 2001). Moreover,
Hernandez-Ledesma, Miralles, Amigo, Ramos, and Recio
(2005) found a moderate ABTS radical scavenging capacity
in commercial fermented milk from Europe. Further
studies of this radical scavenging activity in different
HPLC fractions showed low TEAC values.
3.3. Application in foods
The antioxidant activity demonstrated by whey protein
hydrolysates suggest that, besides modifying physiological
properties of food products, these protein hydrolysates
have potential to enhance product stability by preventing
oxidative deterioration. A feasible application of
peptides or hydrolysates as antioxidants is their addition
to muscle foods, since they are subjected to rapid oxidative
reactions.
Enriched CPP, casein hydrolysates and low molecular
weight casein hydrolysates have proved to be inhibitors of
TBARS formation in cooked ground beef. The ability to
inhibit TBARS formation was highest with enriched CPP,
even though the hydrolysates had better antioxidant
activities. As cooking increases the catalytic activity of
iron, these results suggest that the stronger chelating
activity of enriched CPP may make them more effective
antioxidants in cooked muscle foods (Diaz & Decker,
2004). Casein calcium peptides showed strong antioxidant
activity against lipid oxidation in ground beef homogenates. Incorporation of casein calcium peptides (2%)
inhibited about 70% of lipid oxidation in homogenates.
This indicates that peptides may be as useful in meat
processing as other antioxidants, helping to prevent
formation of an off-avour in meat products, thereby
increasing shelf life (Sakanaka et al., 2005).
Whey proteins are already being utilized as functional
ingredients in meat products. Whey hydrolysates may be

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A. Pihlanto / International Dairy Journal 16 (2006) 13061314

also potential antioxidants in meat products. Pena-Ramos

and Xiong (2003) evaluated the antioxidant activity of
selected whey hydrolysates in cooked meat pork patties.
The results indicated that at an application level of 2% the
whey protein isolates and their hydrolytic products not
only reduced the cooking loss but also suppressed lipid
oxidation in cooked pork patties during refrigerated
storage. Notably, hydrolysis with protamex improved the
capability of whey protein to inhibit early-stage lipid
oxidation (formation of hydroperoxides or conjugated
dienes) as well as to retard propagation of the oxidation
process (degradation of hydroperoxides leading to TBARS
formation). Thus, partially hydrolyzed whey proteins seem
to be particularly attractive ingredients for quality improvement in processed muscle foods.
4. Health effects
Earlier human studies focused predominantly on the
effects of vitamins (A, C, E) and carotenoids, more recently
the effects of fruit juices (from grapes, kiwi) and beverages
(soy milk, tea, coffee), vegetables (tomato products,
berries, Brussels sprouts) and other components of the
human diet (coenzyme Q10, polyunsaturated fatty acids).
Some of the analytical methods which can be used for the
detection of antioxidative activity under in vitro conditions, can also be employed to assess protective effects in
human intervention studies. Today, strong efforts are made
to elucidate the different parameters of oxidative DNAdamage, cancer and other age-related diseases (Hoelzl
et al., 2005).
The results of some animal and human studies suggest
that fermented milk products do have an antioxidative
effect. A fermented milk product was found to exert an
antiperoxidative action on rats fed a vitamin-E decient
diet; the lactic acid bacteria and whey proteins, particularly
b-lactoglobulin, in the product contributed partly to the
observed antiperoxidative action (Zommara, Tougo,
Sakanao, & Imaizumi, 1998). Kullisaar et al. (2003) noted
that the consumption of fermented goat milk improved
anti-atherogenicity in healthy subjects by prolonging the
resistance of the lipoprotein fraction to oxidation, lowering
the levels of peroxidized lipoproteins, oxidized LDL, 8isoprostanes and the glutathione redox ration, and enhancing total antioxidative activity. The active components
behind these activities were not identied. However, the
release of various bioactive peptides from milk proteins
through microbial proteolysis has been shown (Gobbetti,
Minervini, & Rizzello, 2004; Korhonen & Pihlanto, 2003).
Accordingly, further studies are needed to elucidate the
role of antioxidative peptides in the protective function of
fermented milk products.
5. Conclusion
Some casein and whey protein-derived antioxidative
peptides have been identied. Further research about the

structureactivity relationship of peptides and synergisitic

and antagonistic affects among amino acids and other
antioxidative compounds should be carried out. Molecular
studies are needed to assess the mechanisms by which the
antioxidative peptides exert their activities. The activities
have, so far, only been tested in simple in vitro screening
trials and no evidence is available at present which proves
that they are also effective in humans. It is anticipated that
in the near future such targets will be related to various
lifestyle-related disease groups, such as cardiovascular
diseases, cancers, osteoporosis, stress and obesity. Physiologically active peptides derived from milk proteins offer a
promising approach to prevent, control and even treat such
disease conditions through a regulated diet.

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