to yield the phenoxyl radical in competition with electron transfer. Such information has not been available for oxidation of lipids, but a kinetic study of methyl linoleate quenching of triplet-riboflavin clearly indicates that lipid radicals are formed by hydrogen atom transfer (Huvaere et al., 2010). Density functional calculations confirmed that electron transfer is endergonic, while hydrogen atom transfer is exergonic.
Iron(II)/iron(III) catalysis of protein oxidation by hydrogen peroxide becomes site specific through coordination of iron(II) to lysine side chain to yield protein carbonyls, which are often used as marker of oxidation of meat proteins (Stadtman, 1990). Protein oxidation is also important for protein functionality as in bread. The gluten network in wheat bread dough is damaged by reduction of the disulfide bridges by glutathione, and bromate and other oxidants have been used for flour improvement (cf. Fig. 1.12). Bromate is now being replaced by ascorbate. Notably, ascorbate is a reductant, but is oxidized enzymatically in the dough by oxygen to yield dehydroascorbate, which is the actual oxidant protecting the gluten disulfide bridges (Grosh and Weiser, 1999).
Other oxidoreductases like laccase, a multicopper enzyme that catalyzes formation of phenolic radicals in lignin and from tyrosin in proteins may be used to oxidatively modify protein functionality (Steffensen et al., 2008).
Fig. 1.12 Gluten network (R-S-S-R) in bread depends on oxidation of cystein residues to cross-linking cystin as mediated by glutathione. Reducing conditions in the dough have been avoided by using bromate as flour improvement. Bromate is now being replaced by ascorbate which, through
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experiments or in human intervention studies. For food systems this has become even clearer as it now appears that antioxidants protecting lipids do not necessarily protect proteins, at least in meat (Lund et al., 2007).
Radical scavenging alone does not constitute a good antioxidant. Carotenoids are not chain-breaking antioxidants but are, besides being quenchers of singlet oxygen, efficient radical scavengers through three mechanisms (Galano, 2007):
R· Car ! Rÿ Car· 1.20
R· Car ! [R ÿ Car]· 1.21
R· Car ! RH Car(ÿH)· 1.22
Carotenoids may regenerate each other from their radical cations:
Car1· Car2! Car1 Car2· 1.23
for which reaction the following hierarchy has been established (Mortensen et al., 2001):
lycopene > -carotene > zeaxanthin > lutein >> canthaxanthin > astaxanthin 1.24 with lycopene being the most efficient radical scavenger. Notably astaxanthin, which is the least efficient radical scavenger among the carotenoids considered and with a moderate tendency of forming radical cations, is often found to have very positive effects on oxidative stability of food lipids (Jensen et al., 1998). In Fig. 1.13 Antioxidant evaluation strategy as proposed by Becker et al. (2004). The final
evaluation of antioxidant protection of food and beverages depends on storage experiments, while the final evaluation of health effects of antioxidants depends on human intervention studies. Quantification of radical scavenging capacity or reducing
efficiency alone only provides guidelines for the final evaluation.
Understanding oxidation processes in foods 25
salmon muscles, astaxanthin was thus found to be of equal importance as -tocopherol as antioxidant in protecting the highly unsaturated lipids. Astaxanthin is among the least reducing carotenoids and a very poor radical scavenger as shown in experiments establishing the ordering of carotenoids in eq. 1.24.
Further theoretical calculations confirmed that astaxanthin is positioned low in the antioxidant hierarchy of carotenoids (Galano, 2007). Still astaxanthin has been found to be superior to the more reducing carotenoids as an antioxidant in protecting liposomes as models for cell membranes (Naguib, 2000).
Carotenoids should apparently not only be considered as an electron donor:
Car O2·ÿ! Car· O22ÿ 1.25
but also as an electron acceptor:
Car O2·ÿ! Car·ÿ O2 1.26
Quantum mechanical calculations have provided a two-dimensional ranking of carotenoids and also of antioxidants like vitamin A, C and E according to their antiradical capacity (MartõÂnez et al., 2008). Based on a combination of ionization energy and electron affinity, an electron acceptance index, Ra, and an electron donating index, Rd, were defined relative to fluor and sodium, respectively (see Table 1.5). An electron acceptor/donor classification is shown in Table 1.6 with examples of compounds affecting oxidative processes in biological system. Compounds which are both good electron donors (Rdlow) and good electron acceptors (Rahigh) are the best antiradical compounds since they easily donate and accept an electron (corresponding to reactions of eq. 1.25 and eq. 1.26). -carotene is such a good antiradical compound. Compounds with the opposite properties, i.e. Rdhigh and bad electron donors and Ralow and bad electron acceptors like ascorbic acid, are poor radical scavengers. The best antioxidants are compounds with low Raas bad electron acceptors but which are good electron donors corresponding to a low Rd. -tocopherol is such a compound. The last group is exemplified by astaxanthin: high Ra as a good electron acceptor and high Rdas a poor electron donor, and such compounds are now termed antireductants (MartõÂnez et al., 2008). Notably, Raand Rdare not inversely proportional to each other and the novelty lies accordingly in the two-dimensional classification.
Among the carotenoids, the highly red-coloured like astaxanthin are the best antireductants, but the worst antioxidants. In contrast, the more colourless antioxidants are the best electron donors and the best antioxidants like -tocopherol. This two dimensional ordering of antioxidants seems to explain the often unexpected positive effect of the highly coloured carotenoids. Even astaxanthin does not scavenge phenoxyl radicals (Table 1.5), astaxanthin as an antireductant may prevent the formation of the more reactive ROS from the less reactive. A proper combination of antioxidants like -tocopherol and anti-reductants like astaxanthin as seen for salmon, may explain the positive effects seen for lipid stability (Jensen et al., 1998). Ascorbic acid has in radical scavenging assays been found to be a rather poor antioxidant compared with 26 Oxidation in foods and beverages and antioxidant applications
Table 1.5 Physico-chemical properties of carotenoids in comparison with -tocopherol and ascorbic acida
Rd Ra 1O2quenching Phenoxyl Relative tendencyd Eë Car·+/diadzein2ÿ
k2(l molÿ1sÿ1)b scavenging to form car·+ V vs. NHEe k2(l molÿ1sÿ1)f Relative ratec
-carotene 1.40 0.46 4.6 109 1 1 0.84 5.8 109
zeaxanthin 1.44 0.49 6.8 109 0.79 10 0.85 8.3 109
canthaxanthin 1.93 0.82 11.2 109 ~0 0.95 5.7 1010
astaxanthin 2.10 0.94 9.9 109 ~0 3 0.97 9.2 1010
lycopene 6.9 109 1.66 8 0.81
-tocopherol 0.31 0.15 2.7 107 0.80
ascorbate 1.29 0.11 0.22
aRd !ÿ/!ÿNaand Ra !/!Frelative to sodium and fluor, respectively. !ÿ= (3I + A)2/16 (I-A) and != (I + 3A)2/16 (I-A) is based on I, the (vertical) ionization energy, and A, the (vertical) electron affinity. From Martinez et al. (2008).
bSecond-order rate constant at 25 ëC. From Min and Boff (2002).
cIn di-tert-butyl peroxide/benzene (7/3, v/v) at 20 ëC, phenoxyl radical generated photochemically. From Mortensen and Skibsted (1997).
dLaser flash photolysis in CHCl3(490 nm, 120 fs pulses). From Han et al. (2002).
eStandard reduction potential of one-electron oxidized antioxidant, for car·+determined in CH2Cl2, for oxidized -tocopherol in DMF, and for the ascorbyl radical in water. From Han et al. (2002) and Jùrgensen and Skibsted (1998).
fBimolecular regeneration of carotenoid by the isoflavonoid dianion in methanol/chloroform (1/10) at 25 ëC. From Han et al. (2010).
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flavonoids like quercetin and the tea polyphenols (Wolfe and Liu, 2007) in agreement with the two-dimensional classification. Still ascorbic acid may prevent oxidation reaction but rather through oxygen depletion or through regeneration of phenolic antioxidants. The highly coloured anthocyanins have been classified as relatively good electron donors (Rdmoderately high) and bad electron acceptors (Ralow) resulting in antioxidant properties between vitamin C and vitamin E (Table 1.6). However, solvents effect seems significant due to their positive charge (MartõÂnez, 2009). It should further be noted (Table 1.5) that the most efficient singlet-quenchers are found among the good antireductants, while the best antioxidants are the best phenoxyl scavengers and best reductors.
Antioxidant synergism between / -carotene and tocopherols/tocotrienols has been observed in red palm oil (Schroeder et al., 2006). Since -tocopherol and -tocotrienol were shown to regenerate the carotenes from their radical cations, rather than the opposite, and in agreement with their respective standard reduction potentials as seen in Table 1.5, it was concluded that the carotenoids were oxidized sacrificially, in effect protecting the tocopherols/tocotrienols as the better (chain-breaking) antioxidants. The two-dimensional classification of the actual compounds now available (Table 1.5) seems to confirm this previous conclusion with the carotenes being the best radical scavengers and the tocopherols/tocotrienols the best antioxidants. This important type of antioxidant synergism in model systems depends on differences in reaction rate and may be classified as a kinetic effect. For systems where the more efficient chain-breaking antioxidants are regenerated by the antioxidant less efficient as chain breakers but being more reducing, the synergism is rather the result of thermodynamic control. Such examples may be found in the interaction between -tocopherol and plant phenols. Although the BDE predicts the tocopherols to be more reducing than the plant phenols, as seen for quercetin and the tea polyphenols in Table 1.3, solvent effects including pH are very important for the redox potential of plant phenols, which in solution may become the more reducing as seen for quercetin as compared to -tocopherol (Pedrielli and Skibsted, 2002).
Table 1.6 Electron donor/acceptor classification of potential radical scavengersa Ra Low electron acceptor index High electron acceptor index
Rd (bad acceptors) (good acceptors)
Low electron donation index Good radical scavengers Best radical scavengers
(good donors) Good antioxidants Example: -carotene
Example: Vitamin E
High electron donation index Poor radical scavengers Good radical scavengers (bad donors) Example: Vitamin C Good antireductants
Example: Astaxanthin
aRaand Rdare relative to fluor and sodium, respectively, as defined in Table 1.5. Based on Martinez et al. (2008).
28 Oxidation in foods and beverages and antioxidant applications
For lipid systems of increasing structural organization, antioxidant synergism may be caused by compartmentalization. For the less investigated interaction between carotenoids and plant phenols, clear antioxidant synergism has been observed and assigned to regeneration of carotenoids as active radical scavengers in the lipid phase by plant phenols at the lipid/water interface (Han et al., 2007).
For isoflavonoids in combination with carotenoid radical cations, regeneration rate at the diffusion limit was observed especially for astaxanthin as seen in Table 1.5. According to the two-dimensional classification of Table 1.5, astaxanthin has the highest electron accepting index among the carotenoids and accordingly should act as an antireductant in the lipid phase. Notably, the regeneration reaction for the astaxanthin radical cation by the moderately reducing isoflavonoid daidzein is faster for astaxanthin than for -carotene in agreement with their reduction potentials, Table 1.5 (Han et al., 2010).