THE PROTEINS OF WASHED, MINCED FISH MUSCLE HAVE SIGNIFICANT SOLUBILITY IN WATER

After washing the minced muscle tissue of 5 species of white-fleshed fish twice with water and a third time with 0.15% (26 mM) NaCl solution, a considerable, portion of the remaining protein was soluble when extracted with water at a 1:20 ratio. The amount of protein extracted was 20–36% of the protein extracted by buffered 1 M Lid solution, a good protein extractant. This amount of protein indicates that the extracted proteins were primarily myofibrillar. When soluble protein was plotted versus the logarithm of the NaCl concentration in the extracting solution, an inverse linear relationship was observed. The moisture content of the fish paste was related linearly to the log of the water-soluble protein with a correlation coefficient of 0.945. The solubility of the proteins of washed, minced Atlantic mackerel was much less than that of the five white-fleshed species tested.     

ROLE OF pH IN SOLUBILITY AND CONFORMATIONAL CHANGES OF PACIFIC WHITING MUSCLE PROTEINS

This study was conducted to better understand biochemical changes of fish muscle proteins as affected by novel surimi process, acid- or alkali-aided solubilization. At 10 mM NaCl, between pH 5 and 10, the solubility of Pacific whiting muscle proteins was low but increased dramatically as the pH was shifted to either acidic or alkaline pH. At 600 mM NaCl, the isoelectric point was shifted to the acidic direction by about 2 pH units, resulting in aggregation of proteins at low pH, but improving the solubility of MHC (myosin heavy chain) between pH 6 and 10. ANS surface hydrophobicity (ANS-S_O) showed much greater values than PRODAN surface hydrophobicity (PRODAN-S_O) for samples treated at pH 2 - 4 perhaps due to an enhancement of the electrostatic interactions between the ANS probe and proteins. At very high pH, according to hydrophobicity results, proteins were partially refolded when the ionic strength increased. Under acidic conditions, SDS-PAGE demonstrated the degradation of MHC at 10 mM NaCl. The formation of MHC polymers was observed under alkaline treatment with a concomitant decrease of SH content.     

SOLUBILITY OF THE PROTEINS OF MACKEREL LIGHT MUSCLE AT LOW IONIC STRENGTH

Over 90% of the proteins of mackerel light muscle were soluble in solutions of physiological ionic strength or less. To accomplish this solublization, it was necessary to extract certain proteins at moderate ionic strength and neutral pH before extracting the rest of the myofibrillar and cytoskeletal proteins in water. Six proteins were favorably solubilized by sodium chloride solutions of moderate ionic strength at neutral pH under conditions that allowed later dissolution of myofibrillar and cytoskeletal proteins in water. The possibility is suggested that three of these proteins were involved in preventing the solubilization in water of other myofibrillar and cytoskeletal proteins of mackerel light muscle. Based on molecular masses and relative abundance, these proteins could possibly be M-protein (166 kDa), α-actinin (95 kDa) and desmin (56 kDa).     

POSSIBLE ROLE OF MUSCLE PROTEINS IN FLAVOR AND TENDERNESS OF MEAT

Evidence suggests that both the myofibrillar proteins and collagen play important roles in meat flavor and tenderness. The probable contributions of the purified proteins to flavor are reviewed in terms of their amino acid composition, especially the sulfur containing and certain other amino acids that have been implicated in meat flavor development. Myofibrils solubilized in sodium dodecylsulfate (SDS) undergoproteolysis on warming to room temperature overnight or on storing for several days at 0–4°C as demonstrated by extra protein bands. The extra proteins appear to be due to the presence of indigenous muscle proteases. The implications of some indigenous muscle proteases are reviewed in terms of their probable role in tenderization of postmortem meat.     

HYDROXYL RADICAL MODIFICATION OF FISH MUSCLE PROTEINS

A xanthine oxidase-based system was used to generate hydroxyl free radicals in washed, minced cod muscle. Oxidation of protein was measured by increase in protein carbonyl content; the system used produced approximately 0.1 mol of carbonyl groups per 10⁵ g of protein. This degree of oxidation had only minor effects on the SDS-PAGE patterns of the muscle proteins. The solubility of the proteins was not affected by this amount of oxidation unless they were also subjected to a freeze/thaw cycle. With a freeze/thaw cycle, a highly significant decrease in protein solubility occurred compared to that which took place in a sample not exposed to the free radical system. Lowering the pH from 6.8 or 6.5 to 6.0 or 5.5 had a strong negative impact on protein solubility. Protein oxidation appeared in two phases in washed cod mince, an initial rapid increase followed by a second phase that may have been linked to oxidation of the small amount of lipid in the sample. Comparison of protein carbonyl formation in stored mackerel fillets or mince indicated that the range of oxidation studied in the cod model system was similar to what occurs in stored mackerel muscle postmortem.     

EFFECT OF IN SITU FORMALDEHYDE PRODUCTION ON SOLUBILITY AND CROSS-LINKING OF PROTEINS OF MINCED RED HAKE MUSCLE DURING FROZEN STORAGE

Three forms of minced red hake muscle representing whole-mince, mince with the low molecular weight fraction removed (reconstituted-minced) and mince with low and high molecular weight soluble fractions removed (washed-minced) were stored frozen with added Fe⁺² and ascorbate (trimethylamine oxide (TMAO) was added when necessary). The production of DMA and free formaldehyde was measured as were the decreases in water-soluble and salt-soluble proteins and TMAO as a function of increasing concentrations of ascorbate. Dimethylamine (DMA) production and loss of overall protein extractability were greatest in minced muscle, followed by reconstituted-minced muscle, and least in washed-minced muscle. The minced muscle lost water-soluble proteins, however, less rapidly than the reconstituted-minced muscle. The percentage of formaldehyde that was bound was highest in the minced, next in the reconstituted-minced and least in the washed-minced muscle. This supports earlier data and indicates that formaldehyde reacts with both the small molecular weight fraction and the water-soluble proteins as well as the contractile proteins. Loss of protein extractability in all samples appeared to be heavily dependent on hydrophobic interactions. Disulfide interactions appeared to occur to some extent in the reconstituted-minced and washed-minced muscle but were a minor factor with the minced muscle samples. Surface hydrophobicity of the proteins was inversely related to their extractability. In the sample of minced muscle with the highest concentration of added ascorbate where approximately 79% of the proteins became inextractable, some 2% of the muscle proteins were covalently linked in polymers with molecular weights greater than that of the myosin heavy chains. The data indicate that cross-linking of protein components occurs as well as hydrolysis of a considerable amount of the protein.     

EFFECTS OF ANTI-LMM ANTIBODIES ON THE SOLUBILITY OF CHICKEN SKELETAL MUSCLE MYOSIN

Monoclonal antibody Fab fragments which bind epitopes in the rod domain of skeletal muscle myosin had specific effects on the solubility properties of chicken muscle myosin in vitro. Two antibodies (NA4 and 5C3), which bind at the C-terminal portion of the rod domain caused myosin to remain soluble and monomeric in 0.1 M KCl, pH 7.2, conditions in which myosin normally aggregates into filamentous structures. Other antibodies (EB165 and AB8), which bind in the middle of the rod did not alter either myosin solubility or the morphology of the myosin assemblies formed. These results demonstrate the importance of the C-terminus of the myosin rod as a domain involved in regulating myosin interactions and solubility.     

CONTINUATION OF SYMPOSIUM: FUNDAMENTAL PROPERTIES OF MUSCLE PROTEINS IMPORTANT IN MEAT SCIENCE: ROLE OF NEW CYTOSKELETAL ELEMENTS IN MAINTENANCE OF MUSCLE INTEGRITY

Evidence suggests that desmin, titin and nebulin, three recently discovered proteins, have cytoskeletal roles in muscle cells. The three proteins have been purified from mature skeletal muscle and partially characterized. Properties of the three proteins are described, with special regard to their probable roles and importance in maintaining muscle cell integrity. Results will be shown that demonstrate ability of purified desmin to self-assemble into synthetic 10-nm (intermediate) diameter filaments. Taken together with immunoelectron microscope results (Richardson et al. 1981), it is evident that desmin is the major component of 10-nm filaments of mature skeletal muscle cells and that the desmin filaments link adjacent myofibrils at their Z-line levels and seemingly tie the myofibrils into the cell cyto-skeleton. Desmin is degraded at about the same rate as is the highly susceptible troponin-T in bovine semitendinosus muscle postmortem. Alterations in desmin and other recently discovered cytoskeletal proteins would be expected to disrupt muscle cell integrity and to have marked effects on properties of muscle important to its use as food.     

ROLE OF pH IN GEL FORMATION OF WASHED CHICKEN MUSCLE AT LOW IONIC STRENGTH

This work was designed to test the hypothesis that it is not solubilization of the myofibrillar proteins per se that is required to form good gels at low salt concentrations, but the protein‐containing structures must be disorganized. Gels were made from washed minced chicken breast muscle at 0.15, 0.88, and 2.5% sodium chloride. The gels made with varying salt concentrations were evaluated either at pH 6.0–6.5 or pH 7.0–7.4. Strain values, an indicator of protein quality, were high only at neutral pH in the gels containing 0.15 or 0.88% salt. At 2.5% salt, strain values of gels made at acid pH were superior to those at the low salt concentrations at acid pH, but inferior to gels with 2.5% salt at neutral pH. Poor gels were obtained at 0.15% salt and low pH whether or not there was an intermittent adjustment to neutral pH. A neutral salt wash markedly increased the water content of the mince, suggesting that solubility‐inhibiting proteins were removed. Good quality gels were obtained in the absence of any detectable solubilization of myosin and only minimal solubilization of actin.     

THE EFFECT OF pH, POLYPHOSPHATES AND DIFFERENT SALTS ON WATER RETENTION PROPERTIES OF GROUND TROUT MUSCLE

The water binding potential (WBP) and expressible moisture (EM) curves of ground rainbow trout white muscle versus pH are different, particularly in the region from pH 5.0 to 7.0. The effect of different salts such as CaCl₂, Mg Cl₂, NaCl, Nal and Na₂SO₄ on WBP suggests that for the same salts, cations cause a smaller change than anions. On the other hand, the EM measurements suggest that cations have a greater effect on this measurement than anions. In the case of WBP, the effect of tripolyphosphate and pyrophosphate can be attributed solely to their effect on pH. These results indicate the differences between the two methods and suggest that they measure different components of the water retention properties of a complex system, and thus water holding capacity measurements must be interpreted with care.     

STABILITY OF FISH MUSCLE PARVALBUMINS AT DIFFERENT pH VALUES

Parvalbumins of herring (Clupea harengus) and carp (Cyprinus carpio) are essential components of the pattern ofsarcoplasmic proteins used for fish species identification by isolelectric focusing. In fishery products oflowpH, like marinated herring (pH ～ 4), parvalbumins were not detectable. It was demonstrated for herring and carp that parvalbumins were not precipitated at pH 4, but were degraded by fish muscle proteases.     

ROLE OF MUSCLE PROTEINASES IN MAINTENANCE OF MUSCLE INTEGRITY AND MASS

Current evidence indicates that, of the thirteen known lysosomal peptide hydrolases, only seven, cathepsins A, B, C, D, H, L, and lysosomal carboxypeptidase B are located inside skeletal muscle cells. Only one of the reported neutral and alkaline proteases is located inside skeletal muscle cells', this neutral protease is the Ca²⁺-dependent proteinase, CAF. With the possible exception of cathepsin N, which can degrade collagen, it seems probable that any protease that contributes to postmortem tenderization needs to be located inside muscle cells. Because very little degradation of myosin or actin occurs in postmortem muscle, most of the small amount of proteolytic degradation of the myofibrillar proteins that occurs during postmortem storage must be due to CAF, which is unique in being unable to degrade myosin and actin. It is not certain that postmortem proteolysis by CAF causes increased tenderness; some recently discovered actin-fragmenting proteins could be involved.