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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
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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
Inhibition of TBARS
formation
Free 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)
Rival et al.
(2001b)
Suetsuna et al.
(2000)
Reference
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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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References
Abuja, P., & Albertini, R. (2001). Methods for monitoring oxidative
stress, lipid peroxidation and oxidation resistance of lipoproteins.
Clinica Chimica Acta, 306, 117.
Andersen, M., & Skibsted, L. (2002). Detection of early events in lipid
oxidation by electron spin resonance spectroscopy. European Journal
of Lipid Science and Technology, 104, 6568.
Antolovich, M., Prenzler, P. D., Patsalides, E., McDonald, S., & Robards,
K. (2002). Methods for testing antioxidant activity. The Analyst, 127,
183198.
Arnao, M. (2000). Some methodological problems in the determination of
antioxidant activity using chromogen radicals: A practical case. Trends
in Food Science & Technology, 11, 419421.
Auerbach, B. J., Kiely, J. S., & Cornicelli, J. A. A. (1992). A
spectrophotometric microtiter-based assay for the detection of
hydroperoxy derivatives of linoleic acid. Analytical Biochemistry,
201, 375380.
Becker, E. M., Nissen, L. R., & Skibsted, L. H. (2004). Antioxidant
evaluation protocols: Food quality or health effects. European Food
Research and Technology, 219, 561571.
Benzie, I., & Strain, J. (1999). Ferric reducing/antioxidant power assay:
Direct measure of total antioxidant activity of biological uids and
modied version for simultaneous measurement of total antioxidant
power and ascorbic acid concentration. Methods in Enzymology, 299,
1527.
Cao, G., Soc, E., & Prior, R. (1997). Antioxidant and prooxidant
behaviour of avonoids: Structureactivity relationships. Free Radical
Biology and Medicine, 22, 749760.
Cervato, G., Cazzola, R., & Cestaro, B. (1999). Studies on the antioxidant
activity of milk caseins. International Journal of Food Science and
Nutrition, 50, 291294.
Chen, H. M., Muramoto, K., & Yamauchi, F. (1995). Structural analysis
of antioxidative peptides from soybean b-conglycinin. Journal of
Agricultural and Food Chemistry, 43, 574578.
Chen, H. M., Muramoto, K., Yamauchi, F., Fujimoto, K., & Nokihara,
K. (1998). Antioxidative properties of histidine-containing peptides
designed from peptide fragments found in the digests of a soybean
protein. Journal of Agricultural and Food Chemistry, 46, 4953.
Chen, H. M., Muramoto, K., Yamauchi, F., & Nokihara, K. (1996).
Antioxidant activity of designed peptides based on the antioxidative
peptide derived from digests of a soybean peptide. Journal of
Agricultural and Food Chemistry, 44, 26192623.
Colbert, L. B., & Decker, E. A. (1991). Antioxidant activity of an
ultraltration permeate from acid whey. Journal of Food Science, 56,
12491250.
Collins, A. (2005). Antioxidant intervention as a route to cancer
prevention. European Journal of Cancer, 41, 19231930.
ARTICLE IN PRESS
A. Pihlanto / International Dairy Journal 16 (2006) 13061314
Davalos, A., Miguel, M., Bartolome, B., & Lopez-Fandino, R. (2004).
Antioxidant activity of peptides derived from egg white proteins by
enzymatic hydrolysis. Journal of Food Protection, 67, 19391944.
Decker, E. A., & Faraji, H. (1990). Inhibition of lipid oxidation by
carnosine. Journal of the American Oil Chemists Society, 67, 650652.
Decker, E. A., Warner, K., Richards, M. P., & Shahidi, F. (2005).
Measuring antioxidant effectiveness in food. Journal of Agricultural
and Food Chemistry, 53, 43034310.
Diaz, M., & Decker, E. A. (2004). Antioxidant mechanisms of caseinophosphopeptides and casein hydrolysates and their application in
ground beef. Journal of Agricultural and Food Chemistry, 52,
82088213.
Diaz, M., Dunn, C. M., McClements, D. J., & Decker, E. A. (2003). Use
of caseinophosphopeptides as natural antioxidants in oil-inwater emulsions. Journal of Agricultural and Food Chemistry, 51,
23652370.
Gobbetti, M., Minervini, F., & Rizzello, C. (2004). Angiotensin Iconverting-enzyme-inhibitory and antimicrobial bioactive peptides.
Society of Dairy Technology, 57, 172187.
Halliwell, B. (2000). Lipid peroxidation, antioxidants and cardiovascular
disease: How should we move forward? Cardiovascular Research, 47,
410418.
Halliwell, B. (2001). Role of free radicals in the neurodegenerative
diseases: Therapeutic implications for antioxidant treatment. Drugs
Aging, 18, 479484.
Halliwell, B., & Whiteman, M. (2004). Measuring reactive species and
oxidative damage in vivo and in cell culture: How should you do it and
what do the results mean? British Journal of Pharmacology, 142,
231255.
Hernandez-Ledesma, B., Davalos, A., Bartolome, B., & Amigo, L. (2005).
Preparation of antioxidant enzymatic hydrolysates from a-lactalbumin
and b-lactoglobulin. Identication of active peptides by HPLC-MS/
MS. Journal of Agricultural and Food Chemistry, 53, 588593.
Hernandez-Ledesma, B., Miralles, B., Amigo, L., Ramos, M., & Recio, I.
(2005). Identication of antioxidant and ACE-inhibitory peptides in
fermented milk. Journal of the Science of Food and Agriculture, 85,
10411048.
Hoelzl, C., Bichler, J., Ferk, F., Simic, T., Nersesyan, A., Elbling, L., et al.
(2005). Methods for the detection of antioxidants which prevent age
related diseases: A critical review with particular emphasis on human
intervention studies. Journal of Physiology and Pharmacology, 56,
4964.
Jung, M. Y., Kim, S. K., & Kim, S. Y. (1995). Riboavin-sensitized
photooxidation of ascorbic acid: Kinetics and amino acid effects. Food
Chemistry, 53, 397403.
Kaizu, H., Sasaki, M., Nakajima, H., & Suzuki, Y. (1993). Effect of
antioxidative lactic acid bacteria on rats fed a diet decient in vitamin
E. Journal of Dairy Science, 76, 24932499.
Kitts, D. D., & Weiler, K. (2003). Bioactive proteins and peptides from
food sources. Applications of bioprocesses used in isolation and
recovery. Current Pharmaceutical Design, 9, 13091323.
Korhonen, H. J. T., & Pihlanto, A. (2003). Food-derived bioactive
peptides - opportunities for designing future foods. Current Pharmaceutical Design, 9, 12971308.
Korpela, R., Lahteenmaki, T., Sievi, E., Saxelin, M., & Vapaatalo, H.
(1997). Lactobacillus rhamnosus GG shows antioxidative properties in
vascular endothelial cell culture. Milchwissenschaft, 52, 503505.
Kudoh, Y., Matsuda, S., Igoshi, K., & Oki, T. (2001). Antioxidative
peptide from milk fermented with Lactobacillus delbrueckii subsp.
bulgaricus IFO13953. Nippon Shokuhin Kagaku Kaishi, 48, 4455.
Kullisaar, T., Songisepp, E., Mikelsaar, M., Zilmer, K., Vihalemm, T., &
Zilmer, M. (2003). Antioxidative probiotic fermented goats milk
decreases oxidative stress-mediated atherogenicity in human subjects.
British Journal of Nutrition, 90, 449456.
Kullisaar, T., Zilmer, M., Mikelsaar, M., Vihalemm, T., Annuk, H.,
Kairane, C., et al. (2002). Two antioxidative lactobacilli strains as
promising probiotics. International Journal of Food Microbiology, 72,
215224.
1313
ARTICLE IN PRESS
1314
Wayner, D., Burton, G., Ingold, K., & Locke, S. (1985). Quantitative
measurement of the total peroxyl radical-trapping antioxidant
capability of human blood plasma by controlled lipid peroxidation.
FEBS Letters, 187, 3337.
Wong, P. Y. Y., & Kitts, D. D. (2003). Chemistry of buttermilk solid
antioxidant activity. Journal of Dairy Science, 86, 15411547.