Antioxidant Peptides
5. Action Mechanisms of Antioxidant Peptides
5.1. Chemical Reactions
The action modes of antioxidant peptides have been extensively investigated and they are illustrated in Figure 3.3 . In general, antioxidant peptides are capable of acting as a radical scavenger, a proton donor, and metal - ion chelator. Antioxidant peptides are generally short peptides (2 - 10 amino acid resi- dues), and the amino acid sequence is a determinant factor of the effi cacy. Furthermore, the presence of certain particular amino acid residues, notably his- tidine, tyrosine, tryptophan, methionine, cysteine, and proline, is signifi cantly correlated with radical quenching activity of peptides (Saito et al. 2003 ;
Figure 3.3. Schematic representation of chemical and physical mechanisms of antioxidant peptides to inhibit oxidative pro- cesses. (1) Metal chelation; (2) radical scavenging; (3) physical hindrance (shielding; repulsion).
Leu - Leu - Pro - His - His showed a strong inhibition of lipid peroxidation, and the His - His segment played an essential role in the noted antioxidant activity of the peptide. The activity of the dipeptide His - His was further improved by the addition of leucine or proline to the N - terminus (Chen et al. 1996 ). Peptides with the Pro - His - His sequence had the greatest syn- ergism with lipid - soluble antioxidants, for example, tocopherols and butylated hydroxyanisole (BHA).
These peptides have the ability to chelate transi- tional metal ions and quench active oxygen species, the order of amino acids along the peptide chain, the
chain length, and the predominance of particular amino acids in the peptides affect the peptides ’ ability to inhibit lipid oxidation. Table 3.2 lists some examples of numerous antioxidant peptides that have been reported in the literature. These peptides are either derived from enzymatic or microbial hydrolysis or synthesized chemically.
Chen et al. (1995) isolated six peptides with anti- oxidant activity from enzyme hydrolyzed soy β - conglycinin. The peptide with the sequence of
Figure 3.4. Gel fi ltration of whey protein hydrolysate (top) and ESR spectrum (bottom) of • OH signals for samples containing different peptide fractions (I – IV). Adapted from Peng et al. 2008 .
carnosine in muscle tissue has a strong Fe 2+ and Cu 2+
binding power, which is attributed to the histidine residue (Boldyrev and Johnson 2002 ). Amino acid residues containing phosphorylated hydroxyl side chain groups (serine and threonine) and carboxyl groups (glutamic acid and aspartic acid) are also metal - ion binders. Because transitional metal ions promote lipid hydroperoxide decomposition, metal binding by peptides would partially inhibit the prop- agation of lipid peroxidation that forms reactive oxygen radicals.
Binding of metal ions by peptides may also change the redox cycling capacity, which is impor- tant for some metal - catalyzed oxidation. Using fer- ritin (a serum polypeptide) as an example, the less reactive ferric ion (Fe 3+ ) is localized in the polypep- tide ’ s cavity, where it nucleates and aggregates to form ferric hydroxide core unable to be converted to the more reactive ferrous species (Fe 2+ ) (Arosio and Levi 2002 ). The disruption of iron redox equi- librium leads to a diminishment of free Fe 2+ , thereby preventing the decomposition of hydroperoxides.
5.2. Physical Interactions
In lipid droplet and emulsion systems, peptides could inhibit lipid peroxidation by serving as a physical barrier, that is, a membrane (Figure 3.3 ).
Because peptides are surface active components, they can partition at the oil - water interface, forming a thick membrane or coating to prevent direct contact of lipids and radicals and other oxidizing including • OH. The overall antioxidant activity is
attributed to the collective effects of these chemical actions.
Saito et al. (2003) constructed a comprehensive tripeptide library using a solid - phase synthesis pro- cedure, and the antioxidant activity of the resulting peptides was evaluated. Of the 108 peptides contain- ing either histidine or tyrosine residues, two tyro- sine - containing tripeptides showed higher activity than those of two His - containing tripeptides in the peroxidation of linoleic acid. Tyr - His - Tyr showed strong synergistic effects with phenolic antioxi- dants. Of the 114 synthesized tripeptides containing proline or histidine, Pro - His - His exhibited the great- est antioxidant activity. Substitution of other amino acid residues for either the N - terminus or C - terminus of the tripeptide did not signifi cantly alter their anti- oxidative activity. Furthermore, cysteine - containing tripeptides showed a strong peroxynitrite - scavenging activity. Tripeptides containing tryptophan or tyrosine residues at the C - terminus had strong radical - scavenging activities, but weak peroxynitrite - scavenging activity.
Sequestering metal - ion catalysts is another important mechanism by which peptides inhibit oxi- dative processes (Wang and Xiong 2005 ; Nguyen et al. 2006 ). In cells and living tissue, transitional metal ions act as initiators to produce radical and nonradical oxygen species; and • OH generated in a Fenton - like process is a classic example. Stabilization of metal prooxidants through sequestering inhibits the radical production. The endogenous dipeptide
Table 3.2. Examples of exogenous peptides that exhibit antioxidant activity in vitro and in model systems.
Source Sequence Reference
β - Conglycinin (soy) Leu - Leu - Pro - His - His Chen et al. 1995
Albumin (rice) Asp - His - His - Gln Wei et al. 2007
Yellowfi sh sole (fermented) Arg - Pro - Asp - Phe - Asp - Leu - Glu - Pro - Pro - Tyr Jun et al. 2004 Mussel (fermented) His - Phe - Gly - Asp - Pro - Phe - His Rajapakse et al. 2005
Milk (fermented) Val - Leu - Pro - Val - Pro - Gln - Lys Rival et al. 2000
β - Lactoglobulin (milk) Trp - Tyr - Ser - Leu - Ala - Met - Ala Hernandez - Ledesma et al. 2007
Synthetic (albumin) Asp - Ala - His - Lys Bar - Or et al. 2001
Synthetic Phe - His - Lys - Ala - Leu - Tyr Nguyen et al. 2006
Synthetic Lys - Arg - Glu - Ser Navab et al. 2005
Synthetic Pro - His - His Saito et al. 2003
with automation. There are continuing efforts to develop effi cient, selective column chromatography methods that can replace batch operations for desalt- ing. Polarity - based solvent extraction is also a feasible technique to separate and isolate antioxi- dant peptides. For peptides with different positive and negative charge distributions, an ion - exchange column can be useful. A membrane system to produce and separate antioxidant peptides from whey protein has been described by Cheison et al.
(2007) . In this system, an enzymatic membrane reactor fi tted with either a 10 - or 3 - kDa nominal molecular weight cutoff tangential fl ow fi lter mem- brane is reportedly successful in obtaining active peptide fractions with high product recovery rates.
6.2. Microbial Fermentation
The production of antioxidant peptides through direct microbial fermentation rather than using puri- fi ed enzymes is an integral part of healthy food production in many countries. Natto and tempeh are fermented soybean products that contain antioxidant peptides by the action of fungal proteases (Sheih et al. 2000 ; Iwa et al. 2002 ). The type, amount, and activity of the peptides produced depend on the specifi c cultures used, and Rhizopus spp. are some of the commonly used cultures. Douchi, also a soybean product fermented by fungal cultures (e.g., Aspergillus spp.), contains antioxidant peptides released by microbial enzymes. As reported by Wang et al. (2008) , the rats that were fed antioxidant douchi extracts showed signifi cantly elevated anti- oxidant enzyme activity in liver and kidney, when compared with control rats. Lipid peroxidation in both organs of douchi - fed rats was also sup- pressed. Furthermore, antioxidant peptides can be produced from fermented meats. A hepta - peptide with the sequence of His - Phe - Gly - Asp - Pro - Phe - His and having a strong radical - scavenging activity was isolated from fermented marine blue mussel ( Mytilus edulis ) (Rajapakse et al. 2005 ).
Many strains of lactic acid bacteria (LAB) are known for their antioxidant activity. LAB - fermented goat milk has been shown to lower oxidative stress and alleviate atherogenicity in humans (Kullisaar compounds. It was reported that the formation of a
thick protein membrane (whey protein) barrier instead of a nonprotein fi lm (Tween 20) resulted in an improved oxidative stability of emulsifi ed Menhaden oil (Donnelly et al. 1998 ). Furthermore, under acidic conditions, protonated amino groups could repel cationic prooxidants (for example, Fe 2+
and Cu 2+ ) thereby inhibiting the initiation of lipid peroxidation (Kellerby et al. 2006 ). It is not clear whether lipid micelles in the digestive tract and in the body circulation system are protected by anti- oxidant peptides through such physical hindrances.
The effi cacy of the protective fat globule membrane could be further enhanced by the construction of a multilayer interface where cationic protein mem- brane (the inner layer) formed at low pH may be reinforced by the deposit of another layer of an anionic polymer, for example, a polysaccharide (Pallandre et al. 2007 ).
6. Preparation of Antioxidant Peptides