Interactions between hemoglobin and cod muscle constituents following treatment at extreme pH values.

UNLABELLED: Acid and/or alkaline solubilization is a recent method developed to separate proteins from muscle foods with good functional properties. However, exposure of the muscle and its components to low pH values has been shown to promote lipid oxidation, limiting therefore the applications of this novel method. This research aimed primarily to study the physicochemical changes of the fish membranes brought about during acid or alkali solubilization processes. The effect on lipid oxidation and the possible role of the water soluble fraction of the muscle (press juice) as a potent antioxidant were also investigated. Model systems comprising minced cod muscle or cod microsomal suspensions were used. Results showed that acid or alkaline treatment (pH < 3.5 or pH > 10.5) of cod membranes significantly delayed lipid oxidation. Added triacylglycerols to washed cod system treated at low pH did not enhance hemoglobin-mediated lipid oxidation. Decreased precipitation of hemoglobin was observed with the alkali-treated membranes at all protein concentrations compared to the acid-treated and the untreated membranes. Finally, the addition of press juice to washed cod muscle tissue or to the membrane model system, significantly delayed hemoglobin lipid oxidation. PRACTICAL APPLICATION: The results of this study can be used to improve pH-shifting technologies to avoid or decrease lipid oxidation problems. Also, the use of press-juice from cod muscle as means of protecting the muscle against lipid oxidation is suggested.

Impact of citric acid on the tenderness, microstructure and oxidative stability of beef muscle.

Acidification of meat can improve texture however it also increases susceptibility to lipid oxidation. The effect of injection and marination of citric acid to acidify and sodium carbonate or sodium tri-polyphosphate to increase pH of beef on tenderness, microstructure and oxidative stability was determined. Water-holding capacity and tenderness of beef semitendinosus muscle increased significantly at pH 3.52 upon addition of citric acid and returned to the level of untreated sample after pH was increased (pH ∼5.26) by sodium tri-polyphosphate. The microstructure of the muscle was lost upon acidification but reformed upon increasing muscle pH. Lipid oxidation was inhibited in cooked beef blocks and ground muscle acidified with citric acid. Lipid oxidation was also inhibited in citric acid acidified beef that was readjustment to pH values equal to or greater than the raw beef muscle with sodium tri-polyphosphate or sodium carbonate. In addition, citric acid that was adjusted to the pH of the raw beef so that it did not alter the pH of the beef also inhibited lipid oxidation. These results indicate that citric acid and not sodium tri-polyphosphate or pH adjustment was responsible for inhibiting lipid oxidation in beef. These results suggest that the best acid marination technique for beef would be citric acid since it is effective at both improving texture and inhibiting lipid oxidation.

Effect of low pH on the susceptibility of isolated cod (Gadus morhua) microsomes to lipid oxidation.

During the extraction of muscle to produce protein isolates by acid or alkali solubilization, membranes are exposed to abnormally low or high pH. Low but not high pH treatment induces rapid oxidation of membrane phospholipids in the presence of hemoglobin. The goal of this research work was to study the oxidative stability of microsomes under the conditions met during acid solubilization. Isolated microsomes from cod muscle were used as a model system. At pH 5.3 or lower, 99% of isolated cod membranes sedimented at low centrifugation speeds. Isolated membranes that were exposed to pH 3.0 were less susceptible to hemoglobin-mediated lipid oxidation. Cod hemoglobin exposed to pH 3 was rendered less pro-oxidative than the untreated cod hemoglobin. However, when microsomes and hemoglobin were together exposed to low pH, oxidation was promoted. Citric acid and calcium chloride, as well as press juice isolated from cod muscle, were able to inhibit lipid oxidation of microsomal suspensions.

Effect of chitosan and chitin on the separation of membranes from proteins solubilized by pH shifts using cod (Gadus morhua).

Studies with isolated membranes and isolated membranes suspended in muscle proteins solubilized at pH 3 showed that mixing chitosan and membranes at this low pH followed by a pH adjustment to 10.5 could sediment membranes effectively at 4000 g. In the solubilized muscle homogenate, the effectiveness of membrane removal by chitosan at 4000 g for 15 min was molecular weight dependent. About 80% of the phospholipids and 28% of proteins were sedimented from solubilized muscle homogenate by mixing muscle homogenate (10 g of muscle tissue homogenized with 90 mL of distilled water) with 10 mL of MW 310-375 k chitosan (10 mg/mL in 0.1 N HCl) before solubilizing it at pH 10.5, whereas 55% of the phospholipids and 12% of proteins were sedimented by mixing muscle homogenate with the MW 310-375 k chitosan before solubilizing the homogenate at pH 3. Low molecular weight chitosans (at MW 1k or 33k) showed little effect on membrane sedimentation under the same conditions. Chitin was not useful for removing membranes at either pH 3 or 10.5, whether added before or after pH adjustment.

Separation of muscle membrane from alkali-solubilized fish muscle proteins.

Treatment with Ca2+ and citric acid improved membrane removal from muscle homogenates solubilized at pH 10.5 by centrifugation at 4000 g for 15 min. The percentage of phospholipid removed from muscle homogenates increased with increasing Ca2+ concentrations at 1 mM citric acid. More than 85% phospholipid and 45% protein in the muscle homogenates were removed at Ca2+ concentrations of >20 mM in the presence of 1 mM citric acid. At 8 mM Ca2+, addition of citric acid at 5 mM improved phospholipid removal to approximately 78% from 58% in its absence. Because treatment with 8 mM Ca2+ alone can remove significant amounts of phospholipid, it is likely that Ca2+ played the major role in membrane removal in muscle homogenates solubilized at pH 10.5.

Model system for testing the efficacy of antioxidants in muscle foods.

The objective of this research was to study the effect of the antioxidants, delta-tocopherol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary butylhydroquinone (TBHQ), and propyl gallate in a model system of lean muscle and canola oil and to compare the effects with those in minced herring. Two carrier solvents with different dielectric constants (epsilon), ethanol (epsilon = 24) and oil (epsilon= 2), were used. Oxidation was measured using thiobarbituric acid reactive substances (TBARS) and sensory analysis. In both the lean muscle-canola oil model system and in herring muscle, the hydrophilic antioxidants, propyl gallate and TBHQ, were more effective in providing oxidative stability than the lipophilic antioxidants, delta-tocopherol and BHT. The oxidative stability of a cod muscle-canola oil system in the presence of propyl gallate, and delta-tocopherol was not affected by the dielectric constant of the carrier solvent, while BHA was more effective as an antioxidant when added in the polar solvent ethanol.

Separation of membranes from acid-solubilized fish muscle proteins with the aid of calcium ions and organic acids.

Calcium chloride, and to a lesser extent MgCl2, aided in the separation of membranes by centrifugation from cod (Gadus morhua) muscle homogenates solubilized at pH 3 in the presence of citric acid or malic acid but not lactic acid. Adding citric acid and Ca2+ before solubilizing the cod muscle homogenates was needed for the effect. At 1 mM citric acid, 70-80% of the phospholipid and 25-30% of the protein were removed at 10 mM Ca2+. At 8 mM Ca2+, citric acid showed an optimal effect on phospholipid removal at 5 mM with 90% of the phospholipid and 35% of the protein removed. The treatment with citric acid and Ca2+ was also effective in separating the membrane from solubilized herring (Clupea harengus) muscle homogenate. Ca2+ and citric acid might exert their influence by disconnecting linkages between membranes and cytoskeletal proteins.

Effect of pH on hemoglobin-catalyzed lipid oxidation in cod muscle membranes in vitro and in situ.

The effect of pH and hemoglobin on oxidation of the microsomal lipids of cod was determined in isolated microsomes and in washed cod muscle. An increase of hemoglobin concentration from 0.5 to 15 microM accelerated lipid oxidation in both systems. In cod microsomes the rate of lipid oxidation increased in the order pH 6.8 >> pH 7.6 > pH 8.4 > pH 6.0 > pH 3.5. However, in washed cod muscle a decrease of pH from 7.8 to 6.8 greatly increased the lag phase and decreased the rate of lipid oxidation. A further decrease in pH to 3.5 decreased the lag phase and increased the rate of lipid oxidation further. A decrease of pH from 7.6 to 6.4 greatly reduced the affinity of hemoglobin for oxygen. Formation of methemoglobin due to autoxidation occurred more rapidly at pH 6.0 than at pH 7.5. Structural changes of the isolated microsomal membranes could be the reason for the unexpected slow lipid oxidation in microsomes at pH 6.0 and below.

Distribution of exogenous delta-tocopherol between the membrane lipids and triacylglycerols of a cod muscle-triacylglycerol model system.

Membranes of muscle foods are more susceptible to oxidation than triacylglycerols. Hence, directing a lipid-soluble antioxidant into the membranes may reduce the oxidative deterioration of muscle tissue. The objective of this research was to use a model system of cod muscle and triacylglycerol to study the distribution of exogenous delta-tocopherol between the membranes and triacylglycerol fractions of muscle. When ethanol was the carrier solvent, more tocopherol was incorporated into the membranes than when oil was the carrier. Addition of tocopherol to the muscle before the triacylglycerol was added allowed more antioxidant to be incorporated into the membranes than for the case when the oil was added before the antioxidant. When the triacylglycerol was solid, the amount of tocopherol incorporated into the membranes was higher than if the triacylglycerol was liquid and the amount of tocopherol incorporated into the membranes was less dependent on the order of tocopherol and triacylglycerol addition. There was a competition between the membrane lipids and triacylglycerol for uptake of the delta-tocopherol. In some circumstances, some of the tocopherol did not enter either the membrane lipid or triacylglycerol phase.

The effect of acid and alkali unfolding and subsequent refolding on the pro-oxidative activity of trout hemoglobin.

The pro-oxidative activity of trout hemoglobin was significantly increased at low pH (2.5-3.5) in a washed fish muscle (WFM) system. It was found that the more unfolded the hemoglobin was the more exposed its heme group was, which increased its pro-oxidative activity. The amount of oxidation products produced (TBARS) were, however, lower at low pH vs neutral pH. At pH 10.5-11, the pro-oxidative activity of hemoglobin was greatly suppressed. The conformation of hemoglobin was significantly more stable at high pH as compared to pH 7 as judged by its visible absorption spectrum. Hemoglobin readjusted from low pH to pH 7 had a higher pro-oxidative activity (i.e., more rapid oxidation) in WFM than native hemoglobin at pH 7, even though TBARS values were lower than in the untreated sample at pH 7. The results suggest that the WFM becomes slightly more susceptible to oxidation after low pH treatment but also produces less TBARS. The increased pro-oxidative activity after pH readjustment correlated well with an incomplete recovery in the native structure on pH readjustment. A longer unfolding time and a lower pH led to a less refolded hemoglobin with increased pro-oxidative activity. Hemoglobin was less pro-oxidative at low pH in the presence of 500 mM NaCl. The presence of salt did, however, increase the pro-oxidative properties of hemoglobin after readjustment to pH 7. The treatment of washed fish muscle at alkaline pH followed by adjustment to pH 7 led to a slight delay in hemoglobin-mediated lipid oxidation in WFM as compared to native hemoglobin at pH 7. The results suggest that WFM becomes less susceptible toward oxidation after pH readjustment from alkaline pH. These results clearly show that for muscle protein extraction/isolation processes requiring highly alkaline or acidic conditions, alkaline conditions are preferred if the lipid oxidation originating from hemoglobin is to be minimized.

Hemoglobin-mediated oxidation of washed minced cod muscle phospholipids: effect of pH and hemoglobin source.

Lipid pro-oxidative properties and deoxygenation/autoxidation patterns of hemoglobins from nonmigratory white-fleshed fish (winter flounder and Atlantic pollock) and migratory dark-fleshed fish (Atlantic mackerel and menhaden) were compared during ice storage at pH 7.2 and 6. A washed cod mince model system and a buffer model system were used for studying lipid changes and hemoglobin changes, respectively. TBARS and painty odor were followed as markers for lipid oxidation. At pH 6, all four hemoglobins were highly and equally active as pro-oxidants. At pH 7.2, pro-oxidation by all hemoglobins except that from pollock was slowed down, and activity ranked as pollock > mackerel > menhaden > flounder. The higher catalytic activities of the hemoglobins at pH 6 than at pH 7.2 corresponded with higher formation of deoxyhemoglobin and methemoglobin. Pollock had the most extensive formation of deoxy- and methemoglobin at both pH values, which could explain its high catalytic activity. The pro-oxidative differences among the other hemoglobins at pH 7.2 did not correlate with deoxygenation and autoxidation reactions. This indicates involvement of other structural differences between the hemoglobins such as differences in the heme-crevice volume. It is suggested that a biological reason for the species differences was their adaptations to different depths/water temperatures.

Changes in trout hemoglobin conformations and solubility after exposure to acid and alkali pH.

The effect of different acid and alkali treatments followed by pH readjustment on solubility and conformation of trout hemoglobins was investigated. At low pH (1.5-3.5) hemoglobin was unfolded at faster rates as the pH was lowered. Inclusion of 500 mM NaCl at low pH significantly increased the rate of unfolding. At alkaline pH (10-12) the conformation of hemoglobin was much less affected than at acid pH, and the presence of salt had little additional effect. When hemoglobin solutions were adjusted to neutrality at different stages of unfolding, the recovery of native structure on refolding was proportional to the extent of unfolding prior to pH readjustment: the more unfolded the protein, the less was the recovery of native structure. The presence of salt led to a smaller recovery of native structure. The more improperly unfolded the hemoglobin was (and hydrophobic), the lower was its solubility. Results suggest that the presence of NaCl (25-500 mM) may not only interfere with the refolding process but also enhance the hydrophobic interactions of improperly refolded hemoglobin, possibly due to charge screening. These results show that proper control of unfolding and refolding time and ionic strength in processes using highly acidic or alkaline conditions can minimize loss of hemoglobin solubility.

Changes in conformation and subunit assembly of cod myosin at low and high pH and after subsequent refolding.

Conformational and structural changes of cod myosin at pH 2.5 and 11 and after subsequent pH readjustment to pH 7.5 were studied. Results suggest that on acid unfolding, the myosin rod may fully dissociate due to electrostatic repulsion within the coiled coil, while it does not dissociate at alkaline pH. Both pHs led to significant conformational changes in the globular head fraction of the myosin heavy chains, suggesting that it takes on a molten globular configuration. A large part of the myosin light chains are lost on both pH treatments. On pH readjustment to neutrality, the heavy chains take on a structural form similar to the native state with the coiled-coil rod reassociating from acid pH while leaving the globular head less packed, more hydrophobic and structurally less stable. The irreversible change brought about in the globular head region leads to the failure of light chains to reassemble onto it, a drastic loss in ATPase activity, and more exposure of reactive thiol groups. The acid and alkali processes therefore lead to substantial changes in the globular part of the myosin molecule and perhaps more importantly to different molecular changes in myosin, depending on which pH treatment is employed.

Effect of low and high pH treatment on the functional properties of cod muscle proteins.

The functional properties of cod myosin and washed cod mince (myofibrillar protein fraction) treated at high (11) and low (2.5) pH were investigated after pH readjustment to 7.5. The solubility of refolded myosin was essentially the same as the native myosin. The pH-treated myofibrillar proteins had increased solubility over the whole ionic strength range studied. Acid and alkali treatment gave myosin and myofibrillar proteins improved emulsification properties, which were correlated with an increase in surface hydrophobicity and surface/interfacial activity. Enhanced gel strength was observed with acid- and alkali-treated myosin compared to native myosin, while the same treatment did not significantly improve the gel strength of acid- and alkali-treated myofibrillar proteins. The acid- and alkali-treated protein samples unfolded and gelled at a lower temperature than did the native proteins, suggesting a less conformationally stable structure of the refolded proteins. Functional studies show that acid and alkali treatment, which leads to partial unfolding of myosin may improve functional properties of cod myosin and myofibrillar proteins, with the greatest improvement being from the alkali treatment. The results also show that improvements in functionality were directly linked to the extent of partial unfolding of myosin on acid and alkali unfolding and refolding.

Consistency and solubility changes in herring (Clupea harengus) light muscle homogenates as a function of pH.

Fish muscle proteins can be isolated from a variety of low-value raw materials by solubilization in either acid or base. If the consistency of the resulting solution is sufficiently low, it is possible to recover most of the solubilized proteins and remove most of the lipids by centrifugation. Lipid removal should greatly stabilize the isolated proteins. In a previous investigation into the use of herring for production of these protein isolates, it was observed that this species had particularly high consistency values when the proteins were solubilized. This study was undertaken to determine the consistencies obtained with herring light muscle tissue over the pH range covered by the two processes, from about pH 2.7 to 10.8. Protein solubility was compared to consistency of the resultant solutions. Maximum consistencies of the homogenates, approximately 220 and approximately 175 mPa.s, were obtained at pH values of approximately 3.5 and 10.5, respectively. Consistency began to increase approximately when solubilization began. Storage of homogenates at pH 2.7 decreased the consistency over a 10 min time period. The magnitude of the consistency peaks at both acid and alkaline pH values increased when using ice-stored as well as frozen-stored herring, especially in the acid range. Protein solubility at pH <4 and pH >/=10.8 slightly decreased after post-mortem storage of the herring muscle. It is suggested that the observed changes in consistency result from the expansion and solvation of protein aggregates which eventually dissociate into smaller units, perhaps even monomers.

Aqueous extracts from some muscles inhibit hemoglobin-mediated oxidation of cod muscle membrane lipids.

It was evaluated whether trout hemoglobin (Hb)-mediated oxidation of minced washed cod muscle lipids could be prevented by an aqueous isolate from cod and some other muscle sources. Lipid hydroperoxides and painty odor developed approximately 4 days faster in washed than unwashed cod mince. When adding back an aqueous fraction (press juice) isolated from unwashed mince to washed mince at 2-6-fold dilutions, development of hydroperoxides and painty odor was either delayed or completely prevented. The inhibitory substances were heat stable, and their effect was slightly reduced at reduced pH. The <1 kDa fractions of whole and heated press juices were as inhibitory as the unfractionated press juices. Inhibition by the unheated, heated, and ultrafiltered (30 kDa) press juices was lost after dialysis. These findings implied the presence of one or more highly effective aqueous low molecular weight antioxidants in cod muscle press juice. The same antioxidative properties were found in heated haddock, dab, and winter flounder muscle press juices but not in heated herring and chicken muscle press juices. Unheated chicken press juice was however highly inhibitory.

Recovery of functional proteins from herring (Clupea harengus) light muscle by an acid or alkaline solubilization process.

Proteins from herring (Clupea harengus) light muscle were extracted using acidic or alkaline solubilization; 92 and 89% of the initial muscle proteins were solubilized at pH 2.7 and 10.8, respectively, of which 96 and 94% were recovered during precipitation at pH 5.5. Consistency of the pH-adjusted muscle homogenates increased with increased raw material age and homogenization intensity; it declined following holding on ice. Some hydrolytic myofibrillar protein degradation occurred during cold storage of the acidified (pH 2.7) homogenates. With alkalized homogenates, hydrolysis was negligible. The total lipid content changed from 0.13 g/g of protein in the muscle to 0.04 g/g of protein in both the acid- and alkali-produced protein isolates. Corresponding values for the phospholipid content were from 0.037 to 0.02 g/g of proteins. Acid- and alkali-produced proteins made gels with equal strain and color. Stress values were equal or lower in acid- versus alkali-produced protein gels. When ice-stored raw material was used, strain and stress values of gels were reduced.

Partitioning of exogenous delta-tocopherol between the triacylglycerol and membrane lipid fractions of chicken muscle.

The partitioning of exogenous delta-tocopherol, added dissolved in ethanol, between the neutral triacylglycerols and membranes of chicken leg muscles was investigated. The two lipid fractions were separated using differential ultracentrifugation techniques. Triacylglycerols were obtained after high-speed centrifugation of the minced muscle at 130000 g for 30 min. Membranes were collected from a muscle-buffer homogenate (pH 7.5) between 10000 g for 20 min and 130000 g for 30 min. The triacylglycerols collected represented from 15 to 80% of the total triacylglycerols of the minced muscle, the yields increasing with increasing muscle triacylglycerol content. The phospholipids in the isolated membrane fraction represented from 20 to 35% of the total phospholipids of the muscle. At low muscle total lipid contents (3-5%), the added delta-tocopherol was present in approximately the same concentration in both muscle lipid fractions. At higher total lipid contents, achieved by adding exogenous triacylglycerols, the delta-tocopherol concentration in the membranes increased relative to that in the triacylglycerols.

Added triacylglycerols do not hasten hemoglobin-mediated lipid oxidation in washed minced cod muscle.

Hemoglobin-mediated lipid oxidation in washed, minced cod muscle was related to the triacylglycerol to membrane lipid ratio. The same rapid development of thiobarbituric acid reactive substances (TBARS) and painty odor occurred with and without the presence of up to 15% menhaden oil. Without hemoglobin, development of TBARS and painty odor was slow, despite a high amount of hydroperoxides in samples with oil added (1135 micromol/kg muscle). This suggested that hemoglobin reacted by cleaving preformed hydroperoxides into secondary oxidation products. Nearly doubling the hemoglobin concentration approximately doubled the extent of lipid oxidation with and without added oil. This indicated that hemoglobin was limiting for the oxidation reaction. The noneffect of added oil suggests that membrane lipids and/or preformed membrane lipid hydroperoxides provided sufficient substrate in hemoglobin-catalyzed oxidation of washed minced cod muscle. Fe(2+-)ADP did not induce any oxidation of washed minced cod with/without added oil. Results suggest that lipid oxidation in fatty fish may be more related to the quantity and type of the aqueous pro-oxidant and the membrane lipids than to variations in total fat contents.

Contributions of blood and blood components to lipid oxidation in fish muscle.

There was a wide variation in the amounts of hemoglobin extracted from the muscle tissue of bled and unbled fish. Averaged values suggested that the residual blood level in the muscle of bled fish was substantial. Myoglobin content was minimal as compared to hemoglobin content in mackerel light muscle and trout whole muscle. Hemoglobin made up 65 and 56% of the total heme protein by weight in dark muscle from unbled and bled mackerel, respectively. Bleeding significantly reduced rancidity in minced trout whole muscle, minced mackerel light muscle, and intact mackerel dark muscle but not minced mackerel dark muscle stored at 2 degrees C. The reduction was in the number of fish that had a longer shelf life; muscle from certain bled fish had rancidity that was comparable to the rancidity in unbled controls. The soluble contents of erythrocytes accounted for all of the lipid oxidation capacity of whole blood added to washed cod muscle. Limiting lysis of erythrocytes delayed lipid oxidation, which was likely due to keeping hemoglobin inside the erythrocyte. Apparent breakdown of lipid hydroperoxides occurred only when a critical level of hemoglobin was present. Blood plasma was slightly inhibitory to oxidation of washed cod lipids. These studies suggest that blood-mediated lipid oxidation in fish muscle depends on various factors that include hemoglobin concentration, types of hemoglobin, plasma volume, and erythrocyte integrity.