Color is a critical factor that consumers use to decide which raw and processed meat products to purchase. This section deals primarily with mechanisms by which heme protein oxidation (e.g., formation of brown pigments) occurs during refrigerated and frozen storage. Autooxidation rate (kox) is a term used to describe Hb and Mb oxidation rates (e.g., metHb and metMb formation).
Sodium chloride (e.g., table salt) accelerated Mb oxidation at post mortem pH values (Andersen et al., 1988). Pink color in uncured, fully-cooked poultry and beef is a separate defect that has been described (Cornforth et al., 1986; Seyfert et al., 2004).
4.5.1 Effect of pH
Soon after death, the pH of muscle drops from around pH 7.4 (physiological pH) to values ranging from 5.5 to 6.8 (post-mortem pH). This is especially noteworthy because Hb and Mb oxidation rates increase as pH is decreased in this pH range (Shikama, 1998; Yin and Faustman, 1993). Rapid Hb and Mb oxidation at low pH is related to the ability of protons (H+) to enter the heme crevice and protonate liganded O2. The neutral superoxide radical (·OOH) then dissociates from the heme crevice resulting in met formation (Fig. 4.2, top). An
Fig. 4.2 Pathways by which hemoglobin and myoglobin autooxidize. Protons facilitate release of neutral superoxide radical (·OOH) from OxyHb. O2and a weakly coordinated water molecule facilitate metHb formation from deoxyHb; the superoxide anion radical (O2·ÿ) is also produced in this reaction. The unprotonated distal histidine (E7) provides hydrogen bonds with liganded O2in OxyHb and water in metHb. Adapted from Brantley
et al. (1993).
82 Oxidation in foods and beverages and antioxidant applications
illustration of how dramatically pH can alter access of protons to the heme crevice is shown in Fig. 4.3. pH reduction from 8.5 to 5.7 caused one of the histidine residues at site CE3 in bovine Hb to swing outward exposing the heme crevice to solvent. Protonation of the distal histidine (E7) at low pH likely causes His(CE3) to swing out away from the porphyrin group. This creates a channel for solvent (e.g., protons and water) to enter the heme crevice accelerating autooxidation (Fig. 4.2). pH reduction from 8.0 to 6.3 also caused structural changes in perch Hb -chains at site CD3 that exposed more of the heme crevice to solvent at the lower pH (Richards et al., 2009).
Lowering the pH from 7.4 to 6.0 dramatically decreases the oxygen affinity of certain fish Hbs (Binotti et al., 1971). This phenomenon in general is termed the `Bohr Effect' which is a decrease in oxygen affinity as the hydrogen ion concentration increases. The Bohr Effect occurs in mammalian Hbs to a slight extent but when the effect is exaggerated (as with trout IV Hb) the term `Root
Fig. 4.3 Gap for solvent entry into a heme crevice of bovine Hb at pH 5.7 and pH 8.5.
The varying sized gap is located between CE3 of the alpha A-chain and the heme-6-propionate. Lowering pH increases exposure of the heme crevice to solvent molecules including protons and water. His(CE3) and the heme group are shown as spheres. The proximal histidine below the heme and the distal histidine above the heme are shown in stick representation. The PDB structures 2QSP and 1G0A were used to prepare the image
shown using PyMOL software.
Heme proteins and oxidation in fresh and processed meats 83
Effect' is used. This creates a large pool of deoxyHb that is in equilibrium with a small pool of oxyHb and ample O2reactant. These conditions facilitate rapid Hb oxidation at pH values near 6 because the relatively high concentration of deoxyHb reacts with copious amounts of O2to produce metHb and superoxide anion radical (·ÿO2) (Fig. 4.2, bottom).
4.5.2 Effect of oxygen partial pressure
If the Root Effect Hbs are disregarded for a moment, low O2partial pressures (PO2) are required for substantial amounts of deoxyMb and deoxyHb to be present in post-mortem muscle. Ground beef turned brown rapidly in O2 -depleted atmospheres (PO2 of 7 mm Hg) compared to normal atmospheric pressure (PO2 of 160 mmHg) (Ledward, 1970). Apparently the O2-depleted atmosphere creates an environment in which a substantial fraction of the Hb and Mb present exist as deoxygenated heme proteins and there still is enough O2
available to promote met formation as described in the lower half of Fig. 4.2.
O2-depleted atmospheres (e.g., low PO2 values) can occur just below the surface of intact muscle since O2from the atmosphere only penetrates 1±4 mm into the tissue (Lawrie, 1974). Consequently it is sometimes observed that the interior of a beef steak contains high levels of met heme protein (brown pigments) while the surface and deep interior contains only reduced heme proteins. The surface will be red in color due to oxy-heme proteins while the deep interior will have purple hues due to the presence of only deoxygenated heme proteins. The deep interior of post mortem muscle is anaerobic.
O2-depleted atmospheres can also occur at the interface of sliced products that are `shingled' and when multiple pieces of muscle are pressed against each other. Stacking of slices or pressing of muscle pieces causes there to be substantial amounts of deoxyHb and deoxyMb that react with the small amount of O2present at the interfaces which facilitates met formation.
Sodium ascorbate (vitamin C) and sodium erythorbate (a synthetic isomer of vitamin C) have O2 scavenging properties. Thus the potential of ascorbate to lower the PO2to a point that accelerates Hb and Mb oxidation should be kept in mind. If all the O2 were to be scavenged by ascorbate, this should inhibit browning since met formation-mediated by deoxygenated heme proteins requires O2as a reactant (Fig. 4.2).
Respiring mitochondria at the surface of muscle can also compete for atmospheric O2which can increase deoxyHb and deoxyMb concentrations at the surface of the muscle. Consumption of O2by mitochondria occurs more readily at elevated pH. This is the reason that beef at elevated pH can appear dark (with purple hues) due to deoxygenation of the heme pigments at the surface.
4.5.3 Autooxidation of different Hbs
Perch Hb and trout IV Hb autooxidized 26-fold and 19-fold faster compared to bovine Hb, respectively at pH 6.3 (Aranda et al., 2009). This was mostly attributed to variation in amino acids surrounding the porphyrin group when 84 Oxidation in foods and beverages and antioxidant applications
comparing the fish and bovine Hbs. The fish Hbs contain isoleucine at site E11 while bovine Hb contains valine. Ile (E11) is closer to the coordinated ligand in the heme pocket compared to valine (Aranda et al., 2009). The larger isoleucine residue displaces ligands (e.g., O2and protonated O2) which increases kox(Fig.
4.2). Mutation studies in sperm whale Mb indicate that Ile at E11 causes very rapid autooxidation compared to the native Val at site E11. The autooxidation rate (kox) for the Val(E11)Ile mutant Mb was 15-fold greater than in the wild-type Mb containing valine at site E11 (Brantley et al., 1993). Fish Hbs also contain threonine at site E10 while bovine Hb contains lysine at E10. The larger lysine can displace solvent from the heme crevice thereby decreasing kox. Fish Hbs also contain glycine at site E14 while bovine Hb contains larger residues.
Larger residues at site E14 should decrease access of solvent to the heme crevice. We observed that kox was significantly greater in a Ala(E14)Gly Mb mutant compared to wild type (unpublished observation). Finally, at site CD3 of the -chains, the gap for solvent entry at pH 5.7 was lowest in bovine Hb (~4 AÊ), intermediate in trout IV (~6 AÊ), and highest in perch Hb (~8 AÊ) which correlated with the koxrates (Aranda et al., 2009).
Certain fish Hbs have low oxygen affinity at post mortem pH values even when exposed to normal PO2(around 160 mmHg). This is termed exaggerated Root and Bohr effects. Trout IV Hb for example is highly oxygenated at pH values near 7.4 while at pH 6.3 (and lower) it is a mixture of mostly deoxyHb and some oxyHb (Binotti et al., 1971). This combination of deoxyHb in the presence of O2promotes rapid autooxidation (Fig. 4.2). Mammalian Hbs have relatively low equilibrium O2dissociation constants at post mortem pH values which is one reason mammalian Hbs are resistant to koxcompared to fish Hbs (Aranda et al., 2009).
Trout IV Hb autooxidized more rapidly compared to trout I Hb at pH 7 and 30 ëC (Fedeli et al., 2001). Trout IV also autooxidized more rapidly compared to trout I at pH 6.3 and 2 ëC (Richards et al., 2005). The rapid koxin trout IV Hb is partly due to the fact that trout IV Hb (a Root Effect Hb) is less oxygenated at these pH values compared to trout I Hb (a non-Root Effect Hb) (Binotti et al., 1971). The ability of residues that can enhance or hinder access of solvent to the heme crevice in each Hb should also be considered.
Rates of autooxidation were around 10-fold higher in Hbs from cold-adapted fish compared to warm-adapted fish while hydrostatic pressure (e.g., deep or shallow dwelling species) did not seem to affect kox (Wilson and Knowles, 1987). A sampling of 27 coastal bottlenose dolphins (Turiops truncatus) exhibited different Hb oxidation rates (Remington et al., 2007). This was attributed to the observation that some dolphins had Hb isoforms that differed compared to the other dolphins examined.
4.5.4 Autooxidation of different Mbs
Oxygen dissociation rates and autooxidation rates were found to be higher in fish Mbs (yellowfin tuna, mackerel and Antarctic teleost) compared to Heme proteins and oxidation in fresh and processed meats 85
mammalian Mbs (sperm whale and horse) (Cashon et al., 1997). Mackerel Mb had low oxygen affinity and high oxygen dissociation rates compared to zebrafish, yellowfin tuna, and Antarctic teleost (Madden et al., 2004). However Antarctic teleost had the highest koxrate among the four fish species indicating factors other than equilibrium oxygen dissociation constants affect autooxidation rates. Mackerel Mb had the most flexible structure based on dynamic simulations. It is interesting to note that aldehydic lipid oxidation products (4-hydroxy-2-nonenal) have been shown to covalently bind to histidine residues of bovine Mb which accelerated metMb formation (Suman et al., 2006).