Haemoglobin-mediated lipid oxidation in the fish muscle: A review

Trends in Food Science & Technology 28 (2012) 33e43

Review

Haemoglobinmediated lipid oxidation in the fish muscle: A review Sajid Maqsooda,*, Soottawat Benjakulb and Afaf Kamal-Eldina a

Department of Food Science, Faculty of Food and Agriculture, United Arab Emirates University, Al-Ain 17551, United Arab Emirates (Tel.: D971 552436974; fax: D971 3 7675336; e-mail: [email protected]) b Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Lipid oxidation is a major cause of quality deterioration in muscle-based foods, where flavour, colour, texture and nutritional value can be negatively affected. The presence of haem pigments and trace amounts of metallic ions makes the fish, especially dark flesh fatty fish, prone to lipid oxidation. In contrast to mammalian meat, haemoglobin (Hb) is a major contributor to lipid oxidation in fish and fish products, since the blood is not practically removed prior to processing. Hb is known as an important catalyst of lipid oxidation in fish muscle. Hb can be a source of activated oxygen due to Hb autoxidation, and haem or iron can be released from the protein to promote lipid oxidation. Autoxidation appears to be a critical step in the ability of haem proteins to stimulate lipid oxidation since metHb reacts with peroxides to stimulate formation of compounds capable of initiating and propagating lipid oxidation. Hb-mediated lipid oxidation can be accelerated by reduction in pH and could be due to enhanced autoxidation of Hb at reduced pH. Hb from different fish is known to promote lipid oxidation in fish muscle differently. Thus, the knowledge regarding the pro-oxidative activity of Hb from different fish species can be useful in developing the species-specific antioxidative strategies to retard the lipid oxidation and increase the shelf-life of fish. * Corresponding author. 0924-2244/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tifs.2012.06.009

Introduction Lipid oxidation is one of the key problems associated with the loss of quality of foods (Maqsood & Benjakul, 2011a). Oxidation of unsaturated lipid associated with rancidity and the stability of lipids in the complex food systems have received much attention in the last decade (Kamal-Eldin, 2003). Haem pigments such as Hb and myoglobin (Mb) are believed to be the most important endogenous promoters of lipid oxidation in fish muscle (Chan, Faustman, Yin, & Decker, 1997). Hb has a quaternary structure characteristic of many multi-subunit globular proteins. Hb is made up of four polypeptide chains with each chain containing one haem group. A haem group consists of an iron (Fe) ion (charged atom) held in a heterocyclic ring, known as a porphyrin (Fig. 1a). This porphyrin ring consists of four pyrrole molecules cyclically linked together (by methene bridges) with the iron ion bound in the centre (Fig. 1a) (Steinberg, 2001, pp. 95). The iron ion may be either in the Fe2þ (ferrous) or in the Fe3þ (ferric) state. The process by which ferrous Hb is converted to ferric metHb is called autoxidation (Fig. 1b). Figure 1b explains the scheme of autoxidation and the generation of different radicals which promotes oxidation in the lipids of fish muscle. Superoxide anion radical (O2 $ ) or OOH is liberated in this process, depending on whether deoxy or oxy haem protein undergoes autoxidation (Richards & Hultin, 2002). O2 $ or OOH can readily be converted to hydrogen peroxide (H2O2), which enhances the ability of haem proteins to promote lipid oxidation. Hbs have the ability to decompose preformed lipid hydroperoxides, thereby generating free radicals in what has been postulated to be an important mechanism in the pro-oxidative behaviour of Hbs (Erickson, 2002) (Fig. 1b). The promotion of lipid oxidation by Hb and Mb has been proposed to involve a ferrylHb radical that initiates the oxidation (Everse & Hsia, 1997). The ferrylHb radical is formed by the reaction of metHb with either hydrogen peroxide or lipid hydroperoxides (Fig. 1b). Another pathway of lipid oxidation mediated by Hb includes the action of iron released from the haem protein (Gray, Gomaa, & Buckley, 1996; Morrissey, Sheehy, Galvin, Kerry, & Buckley, 1998). Released iron from Hb has the ability to stimulate lipid oxidation (Tappel, 1955), as it catalyzes the breakdown of preformed lipid hydroperoxides, thereby initiating the production of alkoxyl radicals. These radicals are capable of abstracting a hydrogen atom from polyunsaturated fatty acids with

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Fig. 1. (a) Structure of haemoglobin and (b) schematic diagram of Hb autoxidation and its role in lipid oxidation. LH : lipid; L: lipid radical; LO: lipid alkoxy radical; LOO: lipid peroxy radical; .LOOH: lipid hydroperoxide.

subsequent propagation of lipid oxidation processes (Hargrove, Wilkinson, & Olson, 1996). The catalytic activity of Hb is considered in connection with the quality loss of fish muscle during storage (Maqsood & Benjakul, 2011b). Thus, this review article focuses on describing the pathway or mechanism by which Hb stimulates lipid oxidation in fish muscle and the factors responsible for their pro-oxidative activity which can help in framing the strategies to inhibit lipid oxidation caused by Hb. Mechanism and the factors affecting the Hb autoxidation in fish Hb undergoes spontaneous oxidation (autoxidation) of the iron in its haem groups, forming MetHb, which cannot transport oxygen. Autoxidation of oxygenated Hb (oxyHb) can be written schematically as (Equation (1)):



k Hb Fe2þ O2 / MetHb Fe3þ þ O$ 2

ð1Þ

where k is the observed first-order rate constant (Riggs, 1970). The reaction not only forms MetHb, which is functionally inert with respect to O2 transport, but also a superoxide anion radical O$ 2 , which can lead to further oxidation reactions. O$ 2 dismutates to H2O2, which may react with metHb to form the hypervalent ferrylHb radical known to initiate lipid oxidation (Equation (2)) (Kanner & Harel, 1985) (Fig. 1b).

ð2Þ MetHb Fe3þ þ H2 O2 /Hb$þ Fe4þ ¼ O þ H2 O It is, therefore, important that autoxidation of Hb should be minimized under physiological conditions. The rate of autoxidation of Hb is of lower magnitude than that of free haem groups or that of haem groups that has become

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exposed to the solvent (8 M urea in appropriate buffer) by unfolding of the globin moiety (Sugawara, Matsuoka, Kaino, & Shikama, 1995). Thus, the embedding of the haem group in the globin provides significant protection against autoxidation. The haem is covalently linked to the globin via the proximal histidine residue (His F8) and is wedged into its pocket by a phenylalanine residue (Phe CD1). These two amino acid residues are common to all Hbs mostly having a histidine residue on the distal side of the haem group that is involved in protection against autoxidation (Perutz, 1990). The capacity of Hb to bind with oxygen depends on the presence of non-polypeptide units namely the haem group buried in the hydrophobic pocket of the Hb (Dickerson & Geis, 1986). Autoxidation appears to be a critical step in the ability of haem proteins to stimulate lipid oxidation since metHb reacts with peroxides to stimulate the formation of compounds capable of initiating and propagating lipid oxidation (Everse & Hsia, 1997). The mechanism of autoxidation may involve a nucleophilic displacement of O$ 2 from OxyHb by a water molecule or OH- that enters the haem pocket from the solvent (8 M urea in appropriate buffer) (Shikama, 1990; Tsuruga & Shikama, 1997). The water molecule or OH- (the nucleophile, X ) remains bound to the ferric iron to form aqua or hydroxy-metHb (Equation (3)):

Hb Fe2þ O2 þ X/Hb Fe3þ X þ O$ 2 :

ð3Þ

Autoxidation can also proceed via initial dissociation of the dioxygen followed by binding of the nucleophile with subsequent oxidation of haem iron and formation of superoxide anion radical (Wallace, Houtchens, Maxwell, & Caughey, 1982) (Equations (4)e(6)):

Hb Fe2þ O2 /Hb Fe2þ þ O$ 2 ;

Hb Fe2þ þ X/Hb Fe2þ X;

ð5Þ



Hb Fe2þ X þ O2 /Hb Fe3þ X þ O$ 2 :

ð6Þ

ð4Þ

Poorly oxygenated Hbs autoxidize faster than highly oxygenated Hbs due to the differences in the spin state of the iron atom inside the haem ring (Livingston & Brown, 1981; Maqsood & Benjakul, 2011a). The iron atom of oxyHb (Fe2þ), a 6-coordinated complex with an additional ligand to O2, is in a low spin state and is less susceptible to autoxidation (Richards, Ostdal, & Andersen, 2002a). Hb is monomeric in the oxygenated state and aggregates into oligomers upon deoxygenation (Fago & Weber, 1995). Monomeric oxyHb and isolated a- and b-chains of Hb are more readily oxidized than tetrameric oxyHb (Maqsood & Benjakul, 2011a; Tsuruga, Matsuoka, Hachimori, Sugawara, & Shikama, 1998). The dissociated form of Hb is known to be more pro-oxidative than its native tetramer form (Maqsood & Benjakul, 2011a). The rate of autoxidation of monomeric lamprey (Lampetra fluviatilis) Hb

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was higher than that of hagfish (Myxine glutinosa) Hb, but it was not significantly different from that of the carp (Cyprinus carpio) and yellowfin tuna (Thunnus albacares) with tetrameric Hb (Jensen, 2001). The autoxidation rate of Hb from different fish species is influenced by their habitat in which they reside (Power, 1972). Hbs of fish from sluggish water have less prooxidative Hbs, while those from active streams possess comparatively more pro-oxidative Hbs (Power, 1972; Riggs, 1970). Richards and Hultin (2003) reported that Hb from herring (Clupea harengus) and mackerel (Scomber scombrus) was more pro-oxidative than Hb from trout (Onchorhynchus mykiss). Richards and Hultin (2002) hypothesized that frequent migration of herring and mackerel might have made their Hbs more prone to autoxidation and thus pro-oxidative. However, Maqsood and Benjakul (2011a) showed that active migratory fish like seabass (Lates calcarifer) had less pro-oxidative Hb. Other biological and genetical factors as well as inhabiting environment might also play an essential role in determining the pro-oxidative properties of Hb in the fish (Undeland, Kristinsson, & Hultin, 2004). Wilson and Knowles (1987) found that oxyHb from bottom dwelling fish were more easily autoxidised than those from species living in shallow waters. Autoxidation of Hb from cold-adapted fish was found to be 10-fold faster as compared to warm-water fish (Maqsood & Benjakul, 2011a). A more rapid rate of autoxidation was also detected in Hbs from cold water fish as compared to warm-water fish (pH 7, 20  C) (Wilson & Knowles, 1987). Hbs from tilapia and grouper were found to be more unstable and prone to autoxidation, compared to seabass Hb (Maqsood & Benjakul, 2011a). Even though the carp prefer higher temperatures, with a final temperature preference of 32  C (Maqsood, Singh, Samoon, & Balange, 2010; Maqsood, Singh, Samoon, & Munir, 2011; Pitt, Garside, & Hepburn, 1956), the carp Hb was still found to be more prone to autoxidation than Hagfish (Jensen, 2001). Autoxidation of hagfish Hb attracts special interest, because its Hb lacks the distal His E7 residue and is monomeric in the oxygenated state (Jensen, 2001). Both these characteristics could potentially increase its susceptibility to autoxidation. Hagfish oxyHb was also more resistant to autoxidation than lamprey oxyHb, which is monomeric like hagfish Hb but contains the distal histidine residue (Jensen, 2001). Thus, different Hbs from different fish species showed varied response to autoxidation and promoted lipid oxidation differently. Therefore, the knowledge regarding the autoxidation rate and pro-oxidative activity of Hb from different fish species can be useful in developing speciesspecific antioxidative strategies to retard the lipid oxidation and increase the shelf-life of fish and fish products. Influence of pH on Hb autoxidation and its pro-oxidative activity in fish The autoxidation reaction is enhanced at low pH, while it is reduced at an alkaline pH since the interactions with

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distal histidine become stronger at higher pH (Hargrove, Whitaker, Olson, Vali, & Mathews, 1997). Part of this enhancement of autoxidation at a low pH comes from the increased dissociation of the tetramer to dimers (Tsuruga, Matsuoka, Hachimori, Sugawara, & Shikama, 1998) and possibly full dissociation of fish Hb to monomers at low pH (Manning, Dumoulin, Li, & Manning, 1998). The dissociated form is also more pro-oxidative and has an increased tendency to lose its haem (Benesch & Kwong, 1995). At low pH deoxygenation of Hb is favoured, and this phenomenon may play an important role in oxidation as the pH is decreased (Richards & Hultin, 2000). Moreover, formation of the reactive HOO (peroxy radical) and very reactive HO (hydroxy radical) from superoxide is favoured as the pH is lowered. Both HOO and HO can participate in lipid oxidation (Hultin & Kelleher, 2000). The stability of the haem in the protein is also compromised as the pH is lowered likely due to opening of the haem crevice (Falcioni et al., 1978), which may lead to increased exposure of the haem to fatty acids or its partition into membrane bilayers. Lipid peroxidation catalyzed by trout Hb was found to increase when the pH was lowered from neutrality to 6.0, and levels of deoxyHb and metHb were higher at pH 6.0 than at pH >7 (Richards & Hultin, 2000). The oxygen affinities of Hbs from different species of fish, poultry, and beef at pH values below or at neutrality may be different (Richards, Modra, & Li, 2002b). It was shown that after treatment at pH 3, washed cod muscle became slightly more susceptible to Hb-induced lipid oxidation, while alkaline treatment slightly protected the muscle from trout Hbmediated lipid oxidation (Kristinsson & Hultin, 2004). The same authors demonstrated that exposure of trout Hb to low pH increased its pro-oxidative properties. Richards and Hultin (2000) showed that there was rapid lipid oxidation of washed cod muscle at pH 3.5 catalyzed by trout Hb, while there was a considerable lag phase and a slower rate of oxidation at pH 7.8. Pazos, Medina, and Hultin (2005) reported that in washed cod muscle a decrease in pH from 7.8 to 6.8 had increased the lag phase and decrease the rate of lipid oxidation when stimulated by cod Hb. A further decrease in pH to 3.5 decreased the lag phase and increased the rate further. Not much is known how highly acidic and alkaline conditions can influence the pro-oxidative properties of fish Hb. The behaviour of Hb as a pro-oxidant at highly acidic and alkaline pH is of significant interest to a recently developed process aimed at recovering fish muscle proteins from underutilized fish species and byproducts, many of which are rich in haem proteins (Kristinsson & Hultin, 2004). This process involves extracting fish proteins using a high (w10.5e11.5) or low (w2.5e3.5) pH to solubilize the muscle proteins and selectively recovering the soluble proteins after centrifugation by isoelectric precipitation (pH 5.5). During the low and high pH solubilization step, haemoglobin would have an opportunity to oxidize the lipids

of fish muscle (Kristinsson & Hultin, 2004). Furthermore, haemoglobin may co-precipitate with the muscle protein during isoelectric precipitation and could oxidize the lipids present in the final protein product, which also may result in adverse colour changes. In the physiological range, lowering of pH decreases the oxygen affinity of Hb (Maqsood & Benjakul, 2011a) and increasing the concentration of CO2 also lowers the oxygen affinity. These linkages between the binding of O2 and concentration of Hþ and CO2 are known as Bohr effect (Riggs, 1970). Hb of some fish expressed a large decrease in both oxygen affinity and cooperative binding with oxygen at low pH. This characteristic of Hb is known as the Roots effect (Brittain, 1987). Typically, Bohr effect expresses its role when blood pH drops from about 7.4 to 6.5 (Stryer, 1988). A further decrease in blood pH is known to cause the Root effect (Manning et al., 1998). The oxygenation of certain fish Hbs decreases sharply when pH is reduced from 7.5 to 6.5 at atmospheric oxygen pressure, as shown for trout, seabass, tilapia and grouper Hb (Binotti et al., 1971; Maqsood & Benjakul, 2011a). The relative oxygenation of Hb from Asian seabass, tilapia and grouper decreased with decreasing pH (Maqsood & Benjakul, 2011a). A marked decrease was observed when pH shifted from 7 to 6.5. A similar pattern of oxygen affinity in pH range of 6e8 was found in cod Hb (Pazos et al., 2005) and trout Hb (Richards & Hultin, 2000). Low pH is known to enhance (i) haemin release (Hargrove & Olson, 1996), (ii) solubility of iron released from Hb (Schaefer & Buettner, 1998), (iii) acid-catalyzed Hb autoxidation (Shikama, 1998), and (iv) formation of H2O2 from O2 $ (Halliwell & Gutteridge, 1989), which are known to stimulate lipid oxidation. pH values below neutrality are typical of post-mortem fish muscle and vary with the fish species. As a consequence, different fish can be prone to lipid oxidation at different degrees, depending on the post-mortem pH (Tsuruga et al., 1998). Autoxidation of Hb from seabass, tilapia and grouper at pH 6 and 7 as a function of time is depicted in Fig. 2. Hbs from all fish were less oxygenated at pH 6, compared to pH 7, as indicated by the large differences between the peak and valley (Maqsood & Benjakul, 2011a). The autoxidation rates of all Hbs were monitored by obtaining a spectrum in the wavelength of 500e630 nm. Decrease in both absorbance at 574 nm (peak) and 560 nm (valley) of the Hb is the indication of formation of metHb from oxyHb (Richards & Dettmann, 2003). All Hbs had the greater stability to autoxidation at pH 7 than at pH 6 (Fig. 2). At time 120 h, the visible spectra of metHb were established in both tilapia and grouper Hb at pH 6, but not in case of seabass Hb (Maqsood & Benjakul, 2011a). Maqsood and Benjakul (2011a) also reported that Hbs from Asain seabass, grouper and tilapia catalyzed the lipid oxidation at different degrees in the washed mince prepared from Asain seabass muscle, depending on pH. Lipid oxidation promoted by Hb from these three fish species was more

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Fig. 2. Spectral changes of haemoglobin from Asian seabass (a), tilapia (b) and grouper (c) exposed to pH 6 and 7 as a function of time. The haemoglobin concentration was 6 mM. Source: Maqsood and Benjakul (2011a).

pronounced at pH 6 and 6.5, compared with pH 7, as reflected by higher TBARS, which might be related to the lower degree of oxygenation (Bohr or Root effect) and the increased rates of autoxidation at pH 6, compared with pH 7. It is also known that Hb is dissociated into dimers 10 times more quickly at pH 6.2 than at pH 7.5 (Dumoulin, Manning, Jenkins, Winslow, & Manning, 1997) and dimers undergo autoxidation 16 times faster than tetramers (Griffon et al., 1998). The lowered pro-oxidative activity of Hb with increasing pH was also reported

by Richards and Hultin (2000). Thus, lowering the pH within post-mortem pH from 7.0 to 5.0 caused deoxygenation of the fish Hb and could result in an acceleration of lipid oxidation in the fish muscle. There is a high sequence homology among mammalian and fish Hbs, the oxidation and haemin loss rates among them varies significantly and are known to be highly pH dependent. Aranda et al. (2009) studied the stereochemical mechanisms for the dramatic differences in autooxidation and haemin loss rates of fish versus mammalian Hb by

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determining the crystal structures of perch, trout IV, and bovine Hb at high and low pH. They found fish Hbs autooxidize and release haemin w50- to 100-fold more rapidly than bovine Hb. Higher rates of autooxidation and haemin loss in fish Hbs were studied by determining the first crystal structures of trout IV Hb and perch Hb and compared them to new crystal structures of bovine Hb. Haemin loss and autoxidation rate constants were determined for three Hbs. The structures were determined in a pro-oxidative environment at pH 5.7 and 6.3 and an environment that is not conducive to oxidation at pH 8.0. Perch and Trout Hbs have dramatic 30- to 80-fold higher autoxidation rates at both pH 5.7 and 6.3 compared to bovine Hb under the same conditions. In contrast, the fish Hbs autoxidize only about two times more rapidly than bovine Hb at pH 8.0. All three Hbs exhibit significantly larger rates of autoxidation as the pH was decreased. Perch Hb also has dramatic, w50-fold higher haemin loss rates at pH 5.7 and 6.3 compared with bovine Hb. Thus, the autoxidation of all three Hbs generally show significantly larger rates of haemin loss as pH is decreased. The differences in the amino acid sequence and response toward varied pHs among the fish and bovine Hbs were sufficient to explain the markedly increased rates of autoxidation and haemin loss in the fish Hb compared to those of bovine and presumably other mammalian Hbs. Thus, Hb-medaited lipid oxidation is highly pH dependent, as pH is known to controls various factors which promote autoxidation and thereby pro-oxidative activity of Hb to induce lipid oxidation in fish muscle. Pro-oxidative activity of different forms of fish Hbs Different forms of Hbs are known to stimulate lipid oxidation differently in fish muscle. Pietrzak and Miller (1989) investigated the ability of oxyHb, deoxyHb, and metHb to stimulate lipid oxidation in egg lecithin liposomes. DeoxyHb was around 3.5 times more capable of stimulating lipid oxidation than metHb and oxyHb. DeoxyHb was also found to promote lipid oxidation in washed cod muscle. When pH was reduced from 7.6 to 6.0, oxygenation of trout Hb decreased with a simultaneous increase in lipid oxidation rates in washed cod muscle, suggesting a possible role of deoxyHb as an effective catalyst of lipid oxidation (Richards & Hultin, 2000). Richards et al. (2002a) reported that deoxyHb acted as a stronger oxidation catalyst than oxyHb because it has a haem crevice that is more accessible. The iron atom of the porphyrin group inside the crevice is also kicked out of the plane when deoxygenation occurs (Stryer, 1988). This may allow the iron to more easily interact with lipid hydroperoxides, creating more free radicals to facilitate both Hb autoxidation and lipid oxidation. It was reported that trout Hb stimulated lipid oxidation rapidly at pH 6.0 which was correlated with the increase of deoxyHb content at this pH (Richards, Kelleher, & Hultin, 1998; Richards et al., 2002a). Anodic and cathodic Hbs are also known to stimulate lipid oxidation differently. Richards et al. (2002a) reported

that anodic Hbs from trout initiated lipid oxidation much more rapidly than cathodic counterparts in washed cod muscle during storage at 2  C ( p < 0.01), as indicated by the increase in TBARS. The anodic and cathodic Hbs of rainbow trout are useful in this endeavour since the anodic Hbs are poorly oxygenated at pH values found in post-mortem fish muscle, while the cathodic Hbs are nearly fully oxygenated at post-mortem pH values (Richards et al., 2002a). Because of fewer stabilizing interactions between the porphyrin structure and the globin moiety in deoxyHb as compared to the other Hb species (Antoni & Brunoni, 1971; Livingston & Brown, 1981), anodic Hbs might be expected to be more sensitive to haemin release as compared to the more highly oxygenated cathodic Hbs at the pH used in these studies. Thus, anodic Hbs exhibit the Bohr effect, whereas cathodic Hbs do not. Reduced Hbs (OxyHb and DeoxyHb) are known to be more pro-oxidative than metHb for a number of reasons. (i) Reduced Hbs can autoxidize, whereas metHbs cannot. Superoxide anion radical formed from reduced Hb (oxyHb) autoxidation can dismutate to from hydrogen peroxide which can activate the metHb formed along with superoxide anion radical generated from oxyHb autoxidation (Kanner, German, & Kinsella, 1987; Misra & Fridovich, 1972). If metHb is the initial reactant, there is no source of haem-bound oxygen that can be released as superoxide anion radical and reduced to hydrogen peroxide to activate the haem protein. (ii) Reduced Hbs but not metHbs can act as Fenton reagents, producing the hydroxyl radical (Puppo & Halliwell, 1988). (iii) Alkoxyl radicals are formed in reduced Hb systems exposed to tert-butyl hydroperoxide, while haemichromes are formed in metHb systems (Thornalley, Trotta, & Stern, 1983). The term “reduced Hbs” has been used because reduced Hbs comprise of a mixture of oxyHb and deoxyHb. The percentages of deoxyHb were closely related to the development of rancidity (Richards & Hultin, 2000). The lag phase before TBARS development in the washed fish mince also appeared to be related to the percentages of deoxyHb at the respective pH values. Therefore, an effective role of deoxyHb to act as a catalyst of lipid oxidation is demonstrated (Richards & Hultin, 2000). Ever, Hertle, Kiese, and Klein (1976) showed that the rate of Hb autoxidation by hydrogen peroxide was highest under nitrogen and decreased with increasing oxygen pressure. This suggests that deoxyHb reacts with hydrogen peroxide more readily than oxyHb. Paganga, Rice-Evans, Rule, and Leake (1992) showed a sudden, nearly complete conversion of oxyHb to deoxyHb at about the same time as lipoprotein oxidation exponentially increased. FerrylHb formation also began to increase at around the same time as the deoxygenation occurred. The ferryl form of Hb was the active catalyst, but the instantaneous conversion from oxyHb to deoxyHb suggests that the deoxygenated form of Hb may be involved. Oxy/ deoxyHb was found to be active at both levels of preformed lipid peroxides in the linoleic acid substrate (Richards &

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Hultin, 2000). This suggests that some factor other than preformed lipid peroxides control the catalytic activity of reduced Hbs. MetHb was found to be an active catalyst when pH of the washed cod mince was reduced to 6.0, however, metHb retained its activity when pH was further increase to 7.6. Thus, it appears in these studies that metHb catalyzed lipid oxidation by a lipid hydroperoxidedependent mechanism while reduced or deoxyHbs operated via a pH dependent mechanism (Richards & Hultin, 2000). Another way that deoxyHb can affect lipid oxidation reactions is through its ability to increase the autoxidation rate of oxyHb (Rifkind, Zhang, Heim, & Levy, 1987). Thus, different forms of Hbs have different redox properties and affinity towards autoxidation and thus can promote lipid oxidation differently. General overview of Hb-mediated lipid oxidation in fish muscle Hb has been known to be an effective catalyst of lipid oxidation in fish muscle leading to off-odours and flavours. In contrast to mammalian meat, Hb is a major contributor to lipid oxidation in fish and fish products, since the blood is not practically removed prior to processing (Maqsood & Benjakul, 2011b; Richards & Hultin, 2002). Hb found in the blood of fish can accelerate lipid oxidation, which results in development of off-odours (Richards & Hultin, 2002). Terayama and Yamanaka (2000) reported that mechanically bled skipjack tuna meat was highly valued due to its bright red colour and lack of fishy odour as compared with the un-bled fish. Bleeding was also shown to retard lipid oxidation of minced trout muscle during storage at 2  C (Richards & Hultin, 2002). Bleeding was effective in removing the Hb from the muscle of Asian seabass (Maqsood & Benjakul, 2011b). Fig. 3 shows Asian seabass muscle from the bled and unbled seabass, which clearly shows that bleeding was effective in removing most of the Hb from the seabass slice. Maqsood and Benjakul (2011b) demonstrated that the removal of blood, and thus Hb from the Asian Seabass muscle was effective in lowering lipid oxidation as reflected by

Fig. 3. Photograph of the slices from bled (a) and un-bled (b) Asian seabass. Source: Maqsood and Benjakul (2011b).

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lower formation of peroxide value and thiobarbituric acid reactive substances (TBARS) (Fig. 4 a and b). Bleeding was also found to be effective in retarding the formation of secondary volatile lipid oxidation products in Asian seabass slices during iced storage (Maqsood & Benjakul, 2011b). Among different aldehydic compounds, except pentanal, higher amounts were found in un-bled seabass slices compared with bled counterparts. For example, the peak area of heptanal was 4-folds higher in un-bled samples (22.03%), compared with that of bled samples (6.6%) (Maqsood & Benjakul, 2011b). In other study conducted on washed seabass mince added Hb from different fish species (seabass, tilapia and grouper), 2-Ethylfuran, pentenyl furan; furan, 2-pentyl; 1-octen-3-ol; 2-octenal; 2-hexenal and 2,3-octanedione were among the different volatile lipid oxidation compounds generated after 10 days of iced storage (Fig. 5). Control washed seabass mince showed lower formation of volatile lipid oxidation products when compared with samples added with Hb. Washed seabass mince fortified with tilapia and grouper Hb displayed the higher formation of volatile oxidation products when compared to samples treated with seabass Hb (Maqsood & Benjakul, 2011a) (Fig. 5). Moreover, washed fish mince added with tilapia Hb had the highest peroxide value (PV) within the first 2 days and possessed the greater amount of TBARS throughout the storage of 10 days compared with control samples without addition of Hb. Thus, Hb from tilapia was effective in catalyzing lipid oxidation in washed Asian seabass mince contributing to the development of fishy odour (Maqsood & Benjakul, 2011c; 2011d). Perch and trout Hbs were found to stimulate the lipid oxidation in the washed cod muscle effectively (Richards & Dettmann, 2003). Richards and Hultin (2003) studied the pro-oxidative effect of mackerel, herring and trout Hb in washed cod muscle and found that TBARS and rancidity developed more rapidly in Hb from mackerel and herring compared to that of trout. Increasing cod Hb concentrations between 0.5 and 5 mM reduced the lag phase of lipid oxidation in cod microsomes. Increasing concentrations of Hb increased the maximal amount of lipid oxidation as well as the oxidation rate as measured by TBARS in cod microsomes (Pazos et al., 2005). Pollock Hb has also been found to have higher activity in promoting lipid oxidation compared to that of horse mackerel Hb, which on the other hand was found to be more effective than seabass Hb (Maestre, Pazos, & Medina, 2009). Trout Hb exhibits likewise greater pro-oxidant ability than Hb from tilapia, whereas Hb from the pelagic species mackerel and herring is more active than trout Hb (Richards & Hultin, 2003; Richards et al., 2007). Pollock Hb oxidized the lipids of the washed cod mince at the fastest rate and flounder Hb at the slowest rate. Undeland et al. (2004) also reported that at pH 6, all four Hbs (winter flounder, Atlantic Pollock, Atlantic mackerel and menhaden) were highly and equally active as pro-oxidants when tested in washed cod mince. At pH 7.2, pro-oxidation by all Hbs except that from

S. Maqsood et al. / Trends in Food Science & Technology 28 (2012) 33e43

PV (mg of hydroperoxide/kg sample)

40

a

20

B

18

UB

16 14 12 10 8 6 4 2 0 0

3

6

9

12

15

Storage time (days)

b

TBARS (mg MAD/kg sample)

40

B

35

UB

30 25 20 15 10 5 0 0

3

6

9

12

15

Storage time (days)

Fig. 4. Changes in peroxide value (PV) (a) and thiobarbituric acid reactive substances TBARS (b) in the slices from bled (B) and un-bled (UB) Asian seabass during iced storage for 15 days. Source: Maqsood and Benjakul (2011b).

pollock was slowed down, and activity ranked as pollock > mackerel > menhaden > flounder. The capacity of Atlantic pollock, seabass, and horse mackerel Hbs to promote lipid oxidation has been evaluated in liposomes and washed minced horse mackerel muscle by Maestre et al. (2009). The pro-oxidant ability of fish Hbs was SB

TL

GR

C

10 8 6 4 2

1- octen- 3-ol

2- octena l

Nona na l

2,3-oc tanedione

Penteny l furan

Oct anal

2-pentylfuran

2- Hexena l

Heptanal

Hexanal

0

2-ethylfuran

Normalised peak area

12

related with their vulnerability to suffer autoxidation to metHb and release haemin either in spontaneous situation or in the presence of two representative lipid oxidation products, linolein hydroperoxides and trans-2-pentenal. Fish Hbs tested in liposomes and washed fish muscle showed similar effectiveness to promote lipid oxidation in

Fig. 5. Normalized peak area of the volatile lipid oxidation compounds identified by SPME-GCMS technique in washed mince added with 6 mM haemoglobin from Asian seabass, tilapia or grouper at pH 6 stored in ice for 10 days. SB: Asian seabass, TL: tilapia, GR: grouper and C: control (without addition of haemoglobin). Source: Maqsood and Benjakul (2011a).

S. Maqsood et al. / Trends in Food Science & Technology 28 (2012) 33e43

the following order: pollock Hb > horse mackerel Hb > seabass Hb. Pollock Hb showed a more elevated autoxidation rate and spontaneous haemin loss and also faster oxidation to metHb in the presence of hydroperoxides and trans-2-pentenal. Thus, different studies have shown that Hb from different fish species was found to stimulate lipid oxidation in various fish model system, therefore confirming the role of Hb as a potent pro-oxidant in fish. Conclusion and future trends Autoxidation of Hb and thereafter lipid oxidation in fish muscle is the main cause of the development of undesirable odour and unpleasant colour during storage of fish. Apart from the Hb concentration, other factors also play an important role in the Hb-mediated lipid oxidation. The prooxidative activity of fish Hb in vitro has been found to increase following small reductions in pH within the span that naturally occur during post-mortem storage of the fish (e.g., from pH 7 to 6.2). Oxidation/peroxidation induced by the presence of Hb is highly pH dependent. Mechanisms of Hb-mediated oxidation/peroxidation of polyunsaturated fatty acids and role of acidic pH in promoting lipid oxidation are still a matter of dispute. Consequently, the mechanisms observed under physiological conditions in vivo, are profoundly different from those occurring in post-mortem condition of the fish muscle. Moreover, different fish Hbs activated lipid oxidation in different fish lipid systems differently. The role of deoxyHb in promoting lipid oxidation is somehow well documented, however the exact mechanism and evident based research needs to be carried out to demonstrate the confirmed role of deoxyHb as the more potential pro-oxidant in fish muscle, compared to oxy and metHb. Also, there is need to find out the role of Hb in promoting oxidation of different lipid fractions (triglycerides and phospholipids) present in the fish muscle. At pH values of relevance for post-mortem fish muscle, both metHb and oxyHb have been shown to be major initiators of lipid oxidation and peroxidation. However, perferrylHb, which has been suggested to be a main candidate as an initiator of lipid oxidation/peroxidation both at physiological pH and at more acidic pH values, has not yet been proven to be involved in lipid oxidation/peroxidation processes. Therefore, there is a need to demonstrate the role of perferrylHb in the lipid oxidation of fish muscle. References Antoni, E., & Brunoni, M. (1971). Hemoglobin and myoglobin in their reactions with ligands. Amsterdam, The Netherlands: NorthHolland Publishing Co. Aranda IV, R., Cai, H., Worley, C. E., Levin, E. J., Li, R., Olson, J. S., et al. (2009). Structural analysis of fish versus mammalian hemoglobins: effect of the heme pocket environment on autooxidation and hemin loss. Proteins: Structure, Function, and Bioinformatics, 75, 217e230. Benesch, R. E., & Kwong, S. (1995). Coupled reactions in hemoglobin. Heme-globin and dimer-dimer association. Journal of Biological Chemistry, 270, 13785e13786.

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