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The study was conducted to evaluate the meat texture, muscle proximate composition, lipid class composition, and collagen content of cultured amberjack and to compare these parameters with those of the yellowtail. Our results showed that the meat texture of cultured amberjack was tougher and had a lower degree of seasonality than that of cultured yellowtail. Muscle lipid and collagen content also varied in the two fish species over the study period. Meat breaking strength was not correlated with any of the muscle constituents, indicating that variations in the meat texture of cultured amberjack was not directly influenced by the changes in the muscle biochemical constituents.

In order to elucidate the mechanism of the changes in gel forming characteristics of fish meat by pH-lowering, the gelation-temperature curve and the gelation-moisture content curve were examined using the acidified walleye pollack surimi or neutralized one after acidification. In the gelation-temperature curve, the gel strength was highest at 30°C and lowest at approximately 50–60°C, irrespective of pH shifting. The gel strength at 30°C and 80°C decreased with the decrease in pH value. The neutralization of acidified surimi improved the gel strength, but it was considerably lower than the original gel strength. The gel strength at 50–60°C was not affected by pH lowering. The gel strength at 80°C could not be revived to the original by pH readjustment, either in the presence or in the absence of EDTA. These results suggest that irreversible changes of meat protein take place under the low pH, and the oxidation ability of sulfhydryl (SH) groups of protein molecule is not affected by pH-shifting.

The post-mortem changes of type I and V collagens in Japanese flounder muscle during chilled storage were examined. The muscle softened significantly within 12 h under chilled storage. Both type I and V collagens isolated by ion-exchange chromatographies showed no remarkable changes in band patterns even after 24 h of storage, suggesting that degraded collagen molecules may be removed from the collagen fraction by a conventional preparation method. In contrast, type I and V collagen molecules were detected by Western blotting with each specific antibody in a saline extract and gradually increased during chilled storage. These results suggest that both type I and V collagens may participate in the post-mortem softening of fish muscle.

The present study was undertaken to assess the lipid composition and deposition in muscle at three anatomical locations in cultured yellowtail and to investigate the effect of lipid composition and deposition on meat texture. Lipid deposition in muscle was studied by histochemical staining of lipid with Sudan dye. Lipid class composition analysis showed that neutral lipids were the main constituents of lipid in cultured yellowtail and accounted primarily for the variation in muscle lipid content with the anatomical location of meat, as well as with season, whereas the polar lipid content remained almost constant. Furthermore, muscle neutral lipid content was correlated negatively with meat breaking strength; however, no correlation was observed between muscle polar lipid content and meat breaking strength. The histochemical study revealed that, in yellowtail muscle, lipid is preferentially deposited in the myosepta and, with increases in muscle lipid content, additional fat is deposited along sparsely distributed thin connective tissue. It was also observed that the greater the lipid deposition in collageneous connective tissue, the lower the meat breaking strength; presumably, higher lipid deposition in the connective tissue resulted in weakening of the muscle structure.

トカゲエソ肉塩すり身の40°Cでのミオシン重鎖(HC)分解は肉を晒処理しても抑制されなかった。NaClはHC分解を促進し糖類やみりんは影響しなかった。弱アルカリ性でHC分解が最も大きかった。塩すり身の40°C付近での加熱時間が長いほどHC分解が進み,同時にかまぼこのゲル強度が低下した。従って,かまぼこ製造工程においてトカゲエソ肉塩すり身はHC分解によるゲルの劣化を生じやすいことが示された。

In order to examine the effect of meat oxidation on the gel forming ability before grinding the meat with salt, fish meat was washed with CuCl2 solution, and the gel strength as well as total sulfhydryl (SH) groups and sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) patterns were analysed. Washing with CuCl2 solution resulted in a decrease in the total SH content of fish meat and the formation of myosin heavy chain (MHC) dimer through disulfide bonding. The plot of logarithmic gel strength versus protein concentration after heating the washed meat at 80°C in the presence of 3% NaCl to form a gel illustrated that the gel forming ability of meats washed with CuCl2 solution was weaker than the control meat. The gel of meat washed with CuCl2 showed the polymerization of MHC and MHC dimers through disulfide bonding much more than the control meat gel, although a small decrease in the SH group content after heating. Further washing with ethylene diamine tetra-acetic acid (EDTA) solution to remove CuCl2 from the CuCl2-washed meat also resulted in similar behavior for MHC polymerization and SH content as the CuCl2-washed meat, and the gel was still weaker than the control gel. It was found that the oxidation of SH groups during washing with CuCl2 solution accompanied by MHC dimer formation in the meat results in the weakening of its gel forming ability.

To clarify the contribution of polymerization of myosin heavy chain (MHC) by disulfide bonding to increased gel strength of cooked gel via preheating, the pastes of walleye pollack surimi (SS and C grades) were preheated at 25°C and 40°C for a variety of hours prior to heating at 80°C for 20 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) patterns of cooked gels were analyzed with and without reducing the samples, which were solubilized in 8 M urea–2% SDS solution. The formation of polymers by disulfide bonding in cooked gels was almost constant in each of the SS and C grade surimi gels despite the period of preheating. Therefore, it was suggested that polymerization by disulfide bonding occurred during cooking at 80°C and not during preheating.

To confirm the contribution of polymerization and degradation of myosin heavy chain (MHC) during preheating to the gel-forming ability of fish meat paste, walleye pollack surimi paste was preheated at 30∞C and 50∞C prior to heating at 80∞C in the presence of various inhibitors. At 30∞C, ethyleneglycol bis(2-aminoethyl ether)-N,N,N¢,N¢-tetraacetic acid (EGTA) and ethylenedi- aminetetraacitic acid (EDTA) inhibited gel formation as well as the polymerization of MHC, whereas dithiothreitol (DTT) and leupeptin promoted gel formation, which was accompanied by the enhance- ment of MHC polymerization and decreased MHC degradation, respectively. At 50∞C, leupeptin inhib- ited MHC degradation and improved gel strength, whereas EGTA, EDTA and DTT had no effect on MHC polymerization and degradation and did not affect gel formation. The results demonstrate that the gel strength of cooked gel (80∞C) is not affected by preheating at 30∞C and 50∞C and does not inhibit polymerization and degradation. Results suggest that the gel strength of cooked gel is depen- dent on the polymerization and degradation of MHC during preheating.

本研究で用いた魚肉水溶性画分中のミオシン重鎖(HC)分解抑制因子(MDI)は市販トリプシンおよびキモトリプシン活性を阻害したことから, セリンプロテアーゼインヒビターと考えられた。一方, 水晒しによって戻りが誘発される魚種の晒肉塩ずり身の40℃付近でのHCの分解は市販セリンプロテアーゼインヒビターで阻害された。従って, MDIは戻りを誘発する晒肉中のセリンプロテアーゼのインヒビターとして作用するものと推察した。単離したMDIは分子量80Kの単量体であった。

The separation of a factor, which inhibits the degradation of myosin heavy chain in washed meat paste around 40°C, from the sarcoplasmic protein of fish meats was examined. The factor was extracted easily with water from meat. After the extract was mixed with DEAE-ion exchange resin, the factor was eluted with 0.1 M NaCl solution. On the other hand, when the extract was fractionated by ammonium sulfate, the factor was contained in a 50-70% saturated fraction. Then ammonium sulfate fractionation, after ion exchange treatment, gave an almost single peak chromatogram in gel filtration. The peak was 80, 000-90, 000 in molecular weight and showed no transglutaminase activity. Furthermore, this factor was separated from various kinds of fish meats, that is even from fish meat of which modori at 40°C is not induced by meat washing, which varies in modori behavior.

Proximate, fatty acid, and free amino acid compositions in the muscle of cultured red sea bream fed moist pellet (MP-fish) were compared with those of the fish fed fresh bait (FB-fish). Lipid accumulation in the abdominal cavity of both fish was also examined.The lipid content in the muscle of MP-fish was almost the same as that of FB-fish. On the other hand, the content of lipid in the abdominal cavity of MP-fish was higher than that in FB-fish. In the fatty acid composition of muscle non-polar lipid, the percentages of C18:1 n-9 and C18:2 n-6 in MP-fish were higher than in FB-fish, while the percentage of C22:6 n-3 was lower in the former than in the latter. Fatty acid composition of muscle non-polar lipid was affected by the bait.Free amino acid composition was also affected by the bait. The content of Anserine in MP-fish was higher than that in FB-fish, while the contents of Lys, His, and Arg were much lower.

When carp myosin was heated at 80°C with cysteine, the polymerization of protein through SS bonding (SS polymerization) was promoted. The SS polymerization was promoted even in the presence of both EDTA and cysteine, though it was not promoted in the presence of only EDTA. Cysteine was oxidized into cystine. Cystine also accelerated the SS polymerization of myosin heavy chain during the heating, and it was reduced into cysteine.From these results, it was suggested that acceleration of SS polymerization of myosin heavy chain by adding cysteine is due to the promotion of intermolecular SS bonding by the oxidative effect of cystine which was produced from cysteine during the heating.

The quantitative methionine requirement of juvenile yellowtail was determined by feeding diets containing six levels of L-methionine ranging from 0.57 to 1.58% of dry diet in about 0.2% increments. Duplicate tanks, each containing 20 fish, were assigned to each diet. The fish were fed to satiation four times a day for 30 days and weight gain, feed utilisation parameters, hepatosomatic index (HSI), body and liver compositions, and serum components were measured. Weight gain, feed efficiency, protein efficiency ratio, nitrogen retention and HSI increased with increasing levels of methionine up to 1.20% and remained nearly constant thereafter. Protein and fat contents of body and liver were significantly different between treatments. Fish fed the diet with the lowest methionine content gave poor haematological characteristics and serum components. Based on broken line analysis of percent weight gain and feed efficiency, the methionine requirement of juvenile yellowtail was estimated to be 1.11% of the diet or 2.56% of dietary protein in the presence of 0.31% cystine.

To decide the subfragment in carp myosin responsible for the polymerization of myosin heavy chain (HC) through SS bonding during the heating at 80°C, carp myosin was digested with α-chymotrypsin into S-1 and rod, and was heated. From the SDS-PAGE patterns (3% polyacrylamide gel) of the heated samples solubilized in 8M urea-2% SDS, two components were found to be produced. The one was observed on the top of the polyacrylamide gel, and the other was just over HC and was 260 kDa in molecular mass. By treating each component with 10% 2-mercaptoethanol, it was found that the former component was made only from S-1 (80 kDa) and the latter was only from rod (130 kDa).Therefore, it was concluded that in the polymerization of HC through SS bonding during the heating of myosin, SH groups on rod is responsible for the formation of HC dimer and those on S-1 is responsible for the HC polymer formation.

Myosin from carp muscle was heated at 80°C in 0.6M NaCl solutions containing various kinds of buffer (pH 6.8), and the polymerization behavior of proteins through SS bonding (SS polymerization) was analyzed by SDS-polyacrylamide gel electrophoresis. In the phosphate buffer and Tris-HCl buffer, the oxidation of SH groups in myosin and SS polymerization of myosin heavy chain were promoted, which suggested that metal ions from the reagents used might be related to them. In Tris-maleate buffer and Tris-maleic anhydride buffer, the content of SH groups decreased more than in the phosphate buffer and in Tris-HCl buffer, but the decrease was not inhibited by a metal chelating reagent (EDTA) Moreover, in Tris-maleate buffer, SS polymerization was inhibited, and in Tris-maleic anhydride buffer, SS polymerization through covalent bonding other than SS bonding occurred. Therefore, it was concluded that these two buffers are not suitable for the research of SS polymerization.

The polymer of protein through SS bonding on the top of 5% SDS-PAGE gel was found to be made up of only a myosin heavy chain from the analysis of two-step SDS-PAGE, and from the results it was a dimer, trimer, tetramer, and above-decamer with 1.8% SDS-PAGE gel. The polymerization was suppressed in the presence of N-ethylmaleimide, dithiothreitol, or EDTA. The polymerization of myosin light chains as well as myosin heavy chain was promoted in the presence of transition ion (Cu+, Cu2+), while Ca2+ and Mg2+ did not affect the polymerization. From the above results, it was made clear that the polymerization of protein through SS bonding during the heating of myosin is the reaction that the SH groups of myosin heavy chain are oxidized in the presence of catalysts such as copper ion, and contribute to the smooth and swift formation of the polymer larger than decamer.

For the sake of utilizing the washed meat from Type IV (the fish which exhibits a modori at around 40°C in the washed meat, not in the unwashed meat) fish, this meat was mixed with the unwashed meat and the washed meat from the fish Types I and II which have excellent gel-forming ability. Type I is the fish which does not exhibit a modori at all in the unwashed meat and the washed meat, and Type II is the fish which exhibits the modori at 60°C only in the unwashed meat. The mixed meat pastes with the unwashed meat of these two types did not exhibit the modori at 40°C, while that with the washed meats exhibited it. However, the modori at 60°C was occurred in the mixture with the unwashed meat of Type II. The gel-forming ability at 80°C was improved in the mixed meat pastes. Sarcoplasmic protein fraction of the meat of not only Type IV but also Type I inhibited the modori at 40°C as well as the myosin heavy chain (HC) degradation of the washed meat pastes from Type IV. The floating lipid on the wash water did not have the inhibitory effect. Therefore it was suggested that an inhibiting factor against the modori at 40°C of the washed meat may be contained in the sarcoplasmic protein fraction of fish meats.

Two-step heating (2S heating) is one of the processes used for strengthening kamaboko gel. The usefulness of 2S heating on meat pastes from various modori-inducing types of fish (Type I-IV) was examined. 2S heating was carried out by heating at 40°C for various periods, and subsequently at 80°C for 20 min. The gel strength of 2S heated gels was compared with that of gel heated only at 80°C for 20 min to evaluate the effectiveness of 2S heating. 2S heating was not effective for the washed meat gels from Type IV, whose washed meat indicates modori-phenomenon at 40°C. The gel strength of the 2S heated gels decreased with heating time at 40°C, accompanied by degradation of myosin heavy chain. These results imply that the degradation of myosin heavy chain is responsible for gel disintegration and that the degraded product of myosin heavy chain does not contribute to the gel forming ability of the meat. The results also indicate that 2S heating (40→80°C) to strengthen the gel fails when the washed meat of fish Type IV is used.

To clarify the effect of microbes on the formation of amino acids during the ripening of squid shiokara, changes in the viable cell counts in 2.5% and 10% NaCl-BPG medium, 10% NaCl-PDA medium, and 10% NaCl-LBS medium, and amino acid contents etc. during ripening at 20°C were compared to shiokara containing 10% NaCl with and without antibiotics. Though the viable cell counts in the shiokara without antibiotics markedly increased during the ripening in contrast to those with antibiotics, neither the total nor the individual amounts of amino acids significantly differed between the samples, suggesting that microbes have a slight effect on the formation of amino acids. The initial bacterial flora of the shiokara without antibiotics were dominated by Staphylococcus, irrespective of the medium used for counting. The dominant flora in the later stage of ripening, however, differed depending on the media: Staphylococcus dominated on the 2.5% NaCl-BPG medium, while Micrococcus dominated and Streptococcus appeared in the deteriorating stage on the 10% NaCl-BPG medium.

The gel-forming characteristics of unwashed and washed meat from three lizardfish species (maeso Saurida undosquamis, wanieso Saurida wanieso, and tokageeso Saurida elongata) were investigated at various heating temperatures from 30 to 80°C for 20 minutes and 2hours. Judging from evaluation of gel strength by tensiometer and a folding test accompanied by degradation of myosin heavy chain (MHC) by SDS-PAGE analysis, the gel degradation of unwashed meat gels occurred at 40, 60, and 70°C in tokageeso, while it occurred at 60 and 70°C in maeso and at 60°C in wanieso. Washing of meat did not improve the gel strength or MHC degradation of meat gel at 40°C in tokageeso or at 60°C in maeso. Moreover, washing of meat showed an enhancing effect on MHC degradation of maeso and wanieso meat gels at 40°C. These findings indicate that the gel-degrading factors (GDFs) which existed in these species were not only sarcoplasmic (Sp) and myofibrillar (Mf) type active at 60°C but also Mf type active at 40°C and Sp type active at 70°C.

Gel-forming ability at 30-90°C of unwashed or washed meat from 19 fish species, which were caught off Kochi Prefecture and are usually used for Age-mono (a fried product of fish meat paste), was investigated to utilize their meat as raw materials for Kamaboko.The fish species used could be classified into the following 4 groups. Type I (3 species): neither unwashed nor washed meat gels disintegrated at any temperature examined. Type II (3 species): unwashed meat gels disintegrated at about 60°C, but washed ones improved. Type III (6 species): both unwashed and washed meat gels disintegrated at about 60°C. Type IV (7 species): unwashed meat gels disintegrated at about 60°C, but washed ones newly disintegrated at about 40°C. The gel forming ability at 80°C of Type I (unwashed or washed meat) and Type II (washed meat) was enough for Kamaboko and that of Type III was also improved to some extent by washing.It was suggested that washing the meat is not necessary for Type I and is useful for Types II and III, but that it is not good for Type IV. It should be emphasized that the fish of Type IV are a novel group in view of the fact that the Modori-phenomenon was newly induced at around 40°C by washing the meat, accompanied by the degradation of myosin heavy chain.

The inhibitory effects of several selected proteinase inhibitors, soybean trypsin inhibitor, leupeptin, antipain, E-64, NEM and EDTA, on myosin heavy chain (MHC) degradation of unwashed and washed meat gels from three lizardfish species, maeso or true lizardfish Saurida undosquamis, wanieso Saurida wanieso, tokageeso or shortfin lizardfish Saurida elongata, were investigated in order to identify the proteolytic activities of gel-degrading factors (GDFs) existing in mat. It was apparent that the proteolytic activities of GDFs responsible for MHC degradation of meat gels from all species at 40°C resulted from two myofibril-associated (Mf) type proteinases, mainly of serine proteinase and slightly of cysteine proteinase. In addition, as indicated by the amino acid patterns obtained from TCA-soluble fraction of meat gels with the presence of various inhibitors, these proposed proteinases were not distinct in these three species. With regard to the MHC degradation at 60°C, the proteolytic activity present in GDFs of maeso, proposed as myofibril-associated gel degrading factors (Mf-GDFs), belonged to cysteine proteinase while that of wanieso and tokageeso were sarcoplasmic-type serine proteinases. The Mf-GDF corresponding to the MHC degradation at 60°C, which occasionally occurs in wanieso meat gel, showed the proteolytic activities of serine proteinase.

°No carboxypeptidase A activity was detected in the extract of pancreas of the cafish Parasilurus asotus. However, when the extract was left at 4C, carboxypeptidase A activity increased with the time and reached to a maximum level after 4 days. These results indicate that carboxypeptidase A is stored as zymogen in the pancreas.Carboxypeptidase A was isolated from the autoactivated extract of the catfish pancreas by CM-cellulose column chromatography and affinity chromatography. The homogeneity of the enzyme was demonstrated by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated as 34, 000 by SDS-polyacrylamide gel electrophoresis. The amino acid composition of the catfish enzyme was found to be similar to those of the carboxypeptidases A from other species.

Crude stromata were prepared as the residual fraction of salt-extraction of muscles from carp Cyprinus carpio and Japanese mackerel Scomber japonicas. They were divided into alkali-soluble (AL-S, 0.1N NaOH-or 0.01N NaOH-soluble), acid-soluble (AC-S, 0.5M acetic acid-soluble), autocleave-extractable (AU-E, extraction at 120°C for 2h), and residual (RES) fractions.Constituent proteins in each fraction were examined by the analyses of amino acid and SDS-polyacrylamide gel electrophoresis. It was found in both species that AL-S consisted mainly of denatured myofibril proteins, and AC-S and AU-E mainly of collagen. In both species, the amino acid composition of RES was also found to be similar to connection.

ELLMAN method was modified as follows. To 3ml of sample, 2ml of 0.2M phosphate buffer (pH 6.5) and 0.5ml of 0.1% DTNB solution (pH 6.5) were added. The mixture was incubated at 40°C for 10 min, the absorbance was determined at 412nm. HABEEB method was modified as follows. To 3ml of sample 1ml of 10% sodium borohydride was added. The mixture was incubated at 40°C for 30 min and adjusted to pH 6.5 with 1ml of 0.5M phosphate buffer and stood for 5 min at room temperature. Sulfhydryl groups in the solution were determined. The sulfhydryl groups and disulfide bonds in the fish meat which was heated at 80°C upon addition of KBrO3 to the meat were determined as follows. To 1.5ml of samle, 0.25ml of 1.2% L-ascorbic acid solution, 0.25ml of 1% potassium bromide solution and 0.5ml of 10% sulfuric acid were added in order to decompose potassium bromate. After the mixuture was incubated at 40°C for 15 min, and neutralized with 0.5ml of 15% sodium hydroxide. The solution was used for the determination. It was suggested that sulfhydryl groups in fish meat turn not only to disulfide bonds but also to sulfonic acid groups by potassium bromate. In other words, the decrease in sulfhydryl groups does not permit the idea of the formation of disulfide bonds.