DISTRIBUTION AND SOME CHARACTERISTICS OF TRIMETHYLAMINE N-OXIDE (TMAO) DEMETHYLASE ACTIVITY OF RED HAKE MUSCLE

It has been suggested by some workers that decomposition of trimethyl N-oxide (TMAO) to dimethylamine (DMA) and formaldehyde in gadoid fishes occurs via enzymic processes while others have suggested a nonenzymic pathway. DMA production in frozen red hake muscle is shown in this study to be enzymic by the necessity of the high molecular weight soluble or insoluble fraction from red hake to convert the low molecular weight components of flounder muscle to DMA. In red hake muscle the TMAO demethylase activity is approximately evenly divided between the high molecular weight soluble and the insoluble fractions: the amount of potential activity in either fraction is 60–100 times that required for the production of DMA that normally occurs during frozen storage of the muscle tissue. The K_m for TMAO of the soluble enzyme was approximately 3 mM; the concentrations of TMAO in red hake muscle range from 60 to 140 mM (calculation based on water content of 80%). Thus, it seems unlikely that TMAO or TMAO demethylase limit the rate of the reaction. On the other hand, the K_m values for flavin mononucleotide and NADH are higher than the concentrations of these components found in the tissue suggesting that the cofactors limit the rate of TMAO breakdown to DMA and formaldehyde in the stored muscle. This supports other studies (Landolt and Hultin 1982; Banda and Hultin 1983) in which the same conclusion is reached based on other considerations.     

ROLE OF COFACTORS IN BREAKDOWN OF TMAO IN FROZEN RED HAKE MUSCLE

Previously indicated results that cofactor concentrations were the rate-limiting factor in the production of DMA in frozen red hake muscle were confirmed. Both the Fe-reducing agent (ascorbate, cysteine) and the flavin-NADH systems which had been previously shown to be effective in vitro, were demonstrated to also function in minced muscle and in reconstituted muscle, i.e., muscle from which the low molecular weight fraction had been removed. The evidence implies that an oxidation-reduction cycle of Fe is involved in the breakdown of TMAO to DMA. Activity with the flavin system is greatly increased in the presence of glucose oxidase and glucose which would remove O₂ from the system. Fe was effective in increasing DMA production under all conditions, whereas Fe⁺³ was effective only in the presence of reducing agents and/or anaerobic conditions. Enzymes capable of destroying cofactors when added to minced muscle tissue before frozen storage inhibited the rate of formation of DMA. The percentage of formaldehyde produced which was bound (unreactive to the Nash reagent) was much higher in minced muscle than in reconstituted muscle, indicating that a large fraction of the formaldehyde produced reacts with the low molecular weight fraction.     

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.     

MASS TRANSFER IN PACIFIC HAKE (Merluccius australis) PACKED IN REFRIGERATED MODIFIED ATMOSPHERE

The objective of this research was to develop, and experimentally validate a fully mathematical model, to predict mass transfer phenomena in Pacific Hake (Merluccius australis) packed in refrigerated modified atmosphere. A mathematical model to predict mass transfer of CO₂, O₂, N₂ and water vapor was developed and validated. The diffusion model was developed utilizing Fick's second law, considering fish fillet as an infinite slab and applied to Pacific Hake (lean fish species) under refrigeration conditions. CO₂ diffusivity of Pacific Hake was determined by an inverse procedure at OC and resulted in a value of 5.19 × 10^?10 [m²/s] that is in accordance with values reported in the literature. However, postmortem variations of pH could affect solubility and diffusivity of CO₂ in fish muscle.     

SOME CHARACTERISTICS OF THE NAD(P)H-DEPENDENT LIPID PEROXIDATION SYSTEM IN THE MICROSOMAL FRACTION OF CHICKEN BREAST MUSCLE

Incubation of the microsomal fraction isolated from chicken breast muscle in the presence of NADPH, ADP and Fe⁺⁺⁺ ions was shown to result in lipid peroxidation. NADH was able to replace NADPH as the source of reducing equivalents but was less efficient. The pH optimum for malonaldehyde (MDA) production was found to be around pH 6. 7. Oxygen uptake by the system was shown to have a pH optimum of 5.5. Oxygen uptake was approximately linearly dependent on temperature but malonaldehyde production exhibited a sharp increase between 25°C and 37°C. Inhibitor studies and the virtual absence of cytochromes indicate that this system is not similar to that isolated from rat liver microsomes. The activity of the lipid peroxidation system was maintained after storage for 7 days at pH 7.25 and 4°C. At pH 5.6 a reduction in activity was noted after 7 days.     

LIPID CHANGES IN FROZEN STORED FILLETS FROM PRE- AND POSTSPAWNED HAKE (MERLUCCIUS HUBBSI MARINI)

The lipid composition of frozen stored fillets from pre‐ and postspawned hake was studied. The total lipid (TL) content in the chloroform/methanol extract from unfrozen postspawned hake was four times higher than that of prespawned fish. After freezing, the TL content of postspawning hake muscle remained unchanged whereas the TL extracted from prespawning fish muscle increased about 90%. The TL extractability of muscle from fish in both different gonadal conditions was not affected by frozen storage. Lipolysis in frozen stored fillets from prespawned hake occurs principally by hydrolytic action on phospholipids (PL), and phosphatidylcholine was the main PL hydrolyzed. Triacylglycerols were the main substrates hydrolyzed in frozen stored fillets from postspawned hake. Freezing and frozen storage affected polyenoics and n‐3 fatty acids (FA). The decrease in the contents of n‐3 FA in fillets from postspawned hake was lower than that observed in fillets from prespawned fish.     

CHARACTERISTICS OF TMAO DEGRADING SYSTEMS IN ATLANTIC SHORT FINNED SQUID (ILLEX ILLECEBROSUS)

The decomposition of trimethylamine oxide (TMAO) to dimethylamine (DMA) was studied in the mantle of squid, Illex illecebrosus. The production of DMA in either whole or ground mantle stored at – 20°C was low or nonexistent. However, significant DMA was produced from TMAO during heating of squid or squid extracts. Conversion of TMAO to DMA was shown to reside in the soluble fraction of the squid mantle which catalyzed the breakdown of TMAO in an assay system consisting of FeCl₂ and ascorbate at room temperature. On the average, some 10–15% of the activity was accounted for by a high molecular weight, thermolabile substance, presumably an enzyme. The rest of the activity was in a small molecular weight, thermostable fraction. The low molecular weight fraction produced significant quantities of TMA as well as DMA, had low activity with flavin-NADH, and was not activated by methylene blue.     

CHARACTERIZATION OF THE PROTEASES INVOLVED IN HYDROLYZING PADDLEFISH (POLYODON SPATHULA) MYOSIN

An extract from paddlefish surimi possessed activities of B, L, and H‐like cathepsins. The optimal pH was around 5.0 for cathepsins B and L, and was between 6.0–6.5 for the H‐like cathepsin. The enzyme activities were not impaired by heating at 40Cfor 20 min. However, the protease extract lost about 20% of its cathepsin B, 50% B+L, and 90% H‐like cathepsin activities after heating at 50C for 20 min. The activity of H‐like cathepsin was not inhibited by E‐64, suggesting that it did not belong to ike known cysteme protease group. The protease extract was capable ofhydrolyzing myosin heavy chain, producing a major fragments) around 140 kDa. Degradation of myosin by the protease extract was substantially reduced by protease inhibitors including E‐64, a protease inhibitor mixture, and bovine plasma powder.     

ABSENCE OF PROTEOLYSIS IN PURIFIED MYOFIBRILS OF POSTSPAWNED HAKE (MERLUCCIUS HUBBSI MARINI) DURING IN VITRO STORAGE AT 37C

The biochemical behavior of myofibrils from postspawned hake during in vitro storage at 37C was investigated. SDS‐PAGE, densitometric analysis of the band areas corresponding to the major myofibrillar proteins, and TCA soluble peptides determination showed no evidence of proteolysis in myofibrils after 44 h of incubation either in presence or in absence of a cocktail of protease inhibitors (1 mMPMSF+1mMiodoacetic acid + 1mM of EDTA). The absence of proteolysis in stored postspawned hake myofibrils contrasts with the autolysis in those from prespawned fish previously reported, indicating an influence of the reproductive cycle on the proteolytic activity closely associated to myofibrils.     

RELATION BETWEEN THE FREE AMINO ACIDS, ANSERINE AND THE TOTAL VOLATILE BASIC NITROGEN PRODUCED IN MUSCLE OF HAKE (MERLUCCIUS MERLUCCIUS, L.) DURING ICED STORAGE

This work studied the development of free amino acids (FAAs) and dipeptide anserine as quality indices for gutted hake stored in ice for 25 days. The correlation of these compounds was determined with total volatile basic nitrogen (TVBN) which has been used as a quality index, for fish stored in ice. The most abundant free amino acids in hake muscle were found to be threonine, glycine, alanine, glutamic acid, β-alanine methylhistidine. lysine and the dipeptide, anserine. The only hydrophobic free ammo adds which exhibit significant differences (P<0.05) throughout storage was tryptophan. moreover, this amino acid exhibited a very high correlation (r=0.951) with TVBN. A significant decrease in anserine (P<0.05) correlated with the increases in 1-methylhistidine and β-alanine throughout storage. These changes also exhibited a very high correlation with TVBN. Therefore, 1-methylhistidine, β-alanine anserine and tryptophan could be used as quality parameters for hake stored in ice.