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Mackerel water-soluble protein concentrate (SPC), which had been concentrated by an ultrafiltration system, were treated with several polymer coagulants to recover protein at high concentration. Over 90% of the protein in the SPC was aggregated by coagulating agents. The aggregation ratio depended on the pH of the reaction. The highest ratio of aggregation was obtained at about pH 5.0. In addition, the effects of alcohols and organic acid salts in aggregating the protein in the SPC were studied. The ratio of the aggregation increased slightly by the addition of alcohols, but not by the addition of organic acid salts.

Calmodulin (CaM) was purified from squid mantle muscle using hydrophobic chromatography. Relative molecular mass of the purified CaM deduced form the SDS-PAGE was exactly the same as that of bovine brain. Comparison of bovine brain and squid CaM indicates that each activates phosphodiesterase identically and that their activation curves are superimposable. Distribution of CaM in the squid tissues was also examined by measuring the activation level of PDE activity. The content of squid CaM in generative organ and fin is relatively higher than in pancreas, liver, and mantle muscle.

Mackerel water-soluble proteins' concentrates (SPC), which had been concentrated by the ultrafiltration system, were treated with three kinds of methods. The gel formation properties were also studied on the insolubilized SPC. Over 95% of protein in the SPC was insolubilized by two pH shifting methods: One was to adjust the primary pH of SPC lower than 3 then to 7, the other was to adjust the primary pH of SPC higher than 12 then to 7. The preferable gel was obtained by the pH shifting from 12 to 6. An elastic gel like Kamaboko was obtained from SPC by simple heating when SPC was highly concentrated (over 100mg/1). The alcohols caused insolubilization of SPC and the rate of the insolubilization increased with increasing of the carbon chain length of added alcohols. Insolubilized SPC with 50% ethanol formed a gel by heating, but its gelation property was poor. Fish water-soluble proteins were presumed not to cause gelation; we found, however, that the fish water-soluble proteins could form a gel if they were concentrated by an appropriate method like ultrafiltration.

This study was undertaken to indentify the key protein (s) which bring about the gel properties of kamaboko and other cooked gel products made from muscular proteins. Mixtures of rabbit actin and myosin of various ratios were prepared. To each mixture was added 2.5% NaCL and cooked in three differet ways: Method I, cooked at 85°C for 30min; Method II, held at 30°C for 30min and cookde as I; Method III, held at 4°C for 24h and cooded as I. Gels optained were submitted to the jelly strength measurement and the differential scanning calorimetry (DSC). The jelly strength values of the gels cooked by Method I increased with increasing myosin content. This indicated that myosin is the essential factor for high jelly strength. Effects of setting treatments of Method III in enhancing the jelly strength were more evidently manifested in the gels with higher myosin content. No effect was found with Method II. The R105 values of DSC which represent the water holding capacity of the gels paralleled the above jelly trength values, indicating that the highly hydrative property of myosin is an essential factor for the high jelly strength. Co-presence of actin and myosin was found essential to bestowing the precooking material with pastiness and consistency with pastiness and consistency which enable the precooking handling like molding or shape giving.

The object of this study is to estimate objectively the species difference and the changes in he physical properties of fish meat during the post-harvest storage and to search for the changes of the muscular matters underlying the property changes. Muscles of five fish species: skipjack, flyingfish, common horse mackerel, plaice, and channel rock fish were stored at 4°C for 14 days. Sample fish were taken out in turn and submitted to measurements of physical properties, namely penetration of a needle, firmness and cohesiveness by a General Foods type Texturometer, respectively. The measured values of each measurement item varied from species to species. During the storage, each of them shifted to the values reflecting the softening of the muscle usually observed. The rate of softening varied from species to species. The extractability of myofibrillar-, sarcoplasmic-, alkali-soluble-, and stroma-protein fractions of five fish muscles did not show any significant changes during storage. The SDS-polyacrylamide gel electrophoretic patterns of these extracted protein fractions and of the myofibrillar proteins extracted after M. H. STROMER showed no difference with respect to the storage time. The muscle samples were broken by rotating blades and sieved by 7 mesh, the percentage of the pieces remaining on the sieve decreased with the decrease of the freshness. The muscle samples were homogenized and the length of the myofibril fragments or the sarcomere number of each fragment was estimated under a microscope. The percentage of the small myofibrils varied by the fish species, and the degree of fragmentation increased with the storage period. These results denoted that changes in the physical properties, namely softening of meat, during the post harvest storage were affected more by the changes of the muscle tissue structures than by the changes of the component proteins.

Isolation of myosin from the obliquely striated muscle of squid mantle was studied by comparing the various conventional isolation methods used for the vertebrate and invertebrate muscles. A method suitable to the squid myosin is proposed. The purity of each obtained preparation was checked by SDS polyacrylamide gel electrophoresis. The myosin preparations obtained by the conventional methods did not give any band corresponding to the myosin heavy chain, but those less than 14×104 in molecular weight were not free from contaminating proteins such as paramyosin and actin. The myosin preparation obtained by the proposed method showed an electrophoretic profile with a heavy chain band (18-20×104) and two light chain bands (1.2 and 1.5×104) of myosin with a faint band of paramyosin (10×104) in 7.5% gel. The tryptic digestion of the squid myosin shows that it is less resistant than fish myosins, and much less than rabbit myosin. The difficulties in obtaining an intact heavy chain during the isolation procedures were attributed to the fragility of the squid myosin molecule and to the possibility of high catheptic activity in the squid muscle.

Tropomyosin preparations from obliquely striated mantle muscle of squid were obtained from low ionic strength extracts of natural actomyosin and their physico-chemical properties were studied. The sedimentation coefficient, 3.0 S, and the intrinsic viscosity, 0.31dl/g, of squid tropomyosin were very similar to those of skeletal tropomyosin. The relative viscosity decreased with increasing ionic strength, becoming constant over 0.1 M KCl. The molecular weight of the squid tropomyosin subunits, 3.5 × and 3.7 × 104, obtained by SDS disc electrophoresis was slightly higher than those of other species. There are some differences between squid and vertebrates in the contents of some amino acids, such as Pro, Trp and Arg. Moreover, the squid tropomyosin was poorer than the skeletal and the non-muscle tropomyosins in easily forming the paracrystals and did not form any crystals.

Denaturation of squid actomyosin during frozen storage was studied by measuring solubility, viscosity, and ATPase activity, and by ultracentrifugal analysis and SDS-electrophoresis. The additive effect of sodium glutamate, well-known for its cryoprotective effect on the freeze denaturation of carp actomyosin, was also checked. When solutions (0.6 M KCI) or suspensions (0.05 M KCI) of the isolated squid actomyosin were stored at -20 ??, the solubility, reduced viscosity and ATPase activity decreased with the length of frozen storage. The ultracentrifugal patterns showed that aggregation proceeded during frozen storage. Evidently, sodium glutamate prevented the freeze denaturation of squid actomyosin, as in the case of carp actomyosin. When the mantle muscle of squid was freeze-stored at -20 ?? and extracted with 0.6 M KCI, the amount of soluble actomyosin extractable from the frozen meat and the ATPase activity of the actomyosin were decreased only slightly even after a long freezing period. This differed from the results with isolated actomyosin.

G-Actin prepared from the obliquely striated mantle muscle of squid by a modified SPUDICH and WATT's method was ultracentrifugally monodisperse with a sedimentation coefficient, S020, w, of 3.3S, and its molecular weight was estimated to be 4.3×104 by SDS disc electrophoresis. The intrinsic viscosity of the squid G-actin was 0.12dl/g, and the viscosity increased by adding 0.1M KCl or 2mM MgCl2 revealing polymerization to F-actin. On ultracentrifugation, the squid F-actin showed 2 faster peaks with sedimentation coefficients corresponding to those of rabbit F-actin. Under the electron microscope, squid F-actin consisted of double stranded filaments, on which a periodicity of 35-36nm was recognized as was also the case with rabbit F-actin. There were no significant differences in amino acid composition among the actins of squid, fish, rabbit and plasmodium. Superprecipitation and viscosity response with added ATP were tested with positive results on the synthetic actomyosin from squid actin and carp myosin. The latter test was compared with one on the natural actomyosin of squid. Squid actin was found similar to the actins of rabbit, fish, and plasmodium actins in the ability to interact with vertebrate myosin. In contraction of the obliquely striated mantle muscle of squid, squid actin appears to be functionally similar to that of vertebrate striated muscle.

Contribution of the connective tissue content on the meat texture of fish species was studied. Firmness of texturometer tests and collagen content of the raw and cooked dorsal muscles of five fish species were determined. Five species selected were skipjack, flying fish, common horse mackerel, plaice and channel rock fish. The firmness value of raw meat varied in a decreasing order: plaice, channel rock fish, flying fish, skipjack, and common horse mackerel. That of cooked meat varied in a decreasing order: skipjack, flying fish, common horse mackerel, plaiceand channel rock fish. The order of the above series was in reverse between raw meat and cooked meat. The species with softer raw meat textures, on cooking gave a firmer texture. The amount of connective tissues as expressed by collagen content was determined on the muscle portion as the same with the texture measurements. Higher the collagen content, firmer the raw meat. There was a significant correlation (r: 0.697) between the collagen content and the firmness. The species with firmer raw meat texture contained higher collagen content than the species with softer texture. The amount of water soluble collagen at 20°C pH 7.0, assumed as an index of the exuding collagen during mustication, increased with the decrease of the firmness of raw meat (r: 0.650). The amount of water insoluble collagen gave higher correlation coe-fficient(r: 0.744) with the firmness. These results suggest that the properties and/or composition of collagen is varied by spies. However, between the cooked meat and the collagen content, such relationships was hardly recognized, and this was attributed to the collagen which is solubilized during cooking. There was no appreciable correlation between the amount of water soluble collagen at 70°C and the fumness of cooked meat, though cooked beef muscle has been reported to give such correlation. The connective tissue proteins must contribute to the raw meat texture, while the cooked meat texture does not depend upon the connective tissue proteins, but, probably on the other factors, like the muscle fiber characteristics as reported before.

Actomyosin solution isolated from carp muscle was frozen and stored with 20 amino acids. The cryoprotective effect of the additives was studied by following the changes in the solubility and the electron microscopic pictures of the protein. The results with the solubility change were classified into six groups with respect to the rate of the change; those with the electron microscopic pictures were classified into three groups depend-ing on the extent of the deformation of the filaments. Glu, Asp, and some others showed good cryoprotective effects in both the solubility and the electron microscopic pictures. Phe, Leu, and some others were less effective. Changes in solubility and in electron microscopic pictures were generally found to proceed in parallel to each other, but some exceptional cases were found for Pro, Gly, etc.

When dilute carp actomyosin solution was heated gradually the mixture became a jelly beyond 40°C in the presence of Na-glutamate or Na-EDTA, while the control mixture aggregated into fine particles. These additives were also found to be effective in raising the viscosity and in suppressing the change in ATPase activity and turbidity. Similar effects were found with other compounds which have difunctional ionic groups. In comparing the relative effects of the tested substances, a relationship similar to the HOFMEISTER series was found. This was compared with the known HOFMEISTER series which is observed in the setting (suwari) of fish meat mince and in the salting-out of egg albumin, with respect to the effect of additive ions. The series of ions found in the gelating effect was similar to but different from the so-called HOFMEISTER series found in studying the effects on acceleration of setting and on salting-out. A possible mechanism for accelerating the gelation is discussed.

Adenosinetriphosphatase (ATPase) activity of actomyosin of the obliquely striated mantle muscle of squid was studied to confirm and supplement the previous work. ATPase activity was assayed at ionic strength of 0.15 or 0.6, Tris-maleate buffer 30 mM (pH 7.0-7.5), protein 0.1mg/ml, ATP 3.2 mM, CaCl2, MgCl2 or EDTA 3.2 mM, and 25°C; for 10 min. Effects of Ca2+ and Mg2+ were tested in the function of temperature. Both Ca2+ and Mg2+ revealed marked activation at eithe ionic strength of 0.15 or 0.6, but EDTA none. At ionic strength 0.15, Ca2+-ATPase showed a steep peak at 30°C, while Mg2+-ATPase demonstrated a gradual peak with the top at 35°C. At ionic strenght of 0.6, the macimum of the former appeared at 25°C and that of the latter at 40°C. Mg2+ ion seemed to stabilize the ATPase. When the activety was assayed at various ratios of actomyosin and ATP, the full activity levels were attained below the ratio of 0.1-0.2 mg/ml protein per 3 mM ATP. Addition of Mg2+ to Ca2+-activated ATPase, reduced the activity to the level activated with Mg2+ alone, while addition of Ca2+ to Mg2+-activated ATPase, did not alter the activity. Effects of Mg2+ appeared to overcome that of Ca2+. During 60 min incubation of enzymic reaction mixture at 25°C prior to adding ATP, the activity was reduced appreciably. This seemed to satand with the so far reported lability of the squid actomyosin which was illustrated in changes of several physiconchemical properties.

The actomyosin ATPase of the obliquely striated squid mantle muscle, which has many characteristic properties in its solubility, electrophoretic behavior and lability as reported previously, was characterized. The ultracentrifugal patterns of squid actomyosin showed its peaks in aggregated states, while, on SDS-acrylamide gel electrophoresis, amount of actin and myosin accounted for 70% of total proteins. Paramyosin and tropomyosin were found as contaminants which are not readily removable at present. The ATPase activity measured in tris-maleate buffer at 0.05 or 0.6 ionic strength (I) and at 20°C was stimulated markedly with Ca2+ and moderately with Mg2+, but depressed with EDTA. [Stimulation by both Ca2+ and Mg2+ was most effective at 32 ram.] In the presence of Ca2+, the activity showed 2 maxima at pH 7.0-7.5 and pH 9.0, while, with Mg2+ present, it leveled off beyond pH 6.8-7.0. The activity was highest at I=0.25. The presence of membrane and mitochondrial ATPase was refuted since neither ouabain nor oligomycin depressed the activity. The stimulating effect of Mg2+ appears to he characteristic of the squid actomyosin ATPase.

This work was undertaken as an introductory step for isolating the biologically active components of the hydrolysate preparation of fur seal skeletal muscle proteins which contain some vasoactive factors as reported previously. This study was carried out with 2 preparations obtained at different fractionation stages, i.e., “unfractionated mixture” and “lyophilized powder”, the latter constituting the second fraction of gel filtration (Sephadex G-25) of the former. The 2 preparations differed in peptide composition, because their relative biuret, ninhydrin and ultraviolet absorption values differed respectively. By paperand thin layer-chromatography, peptide mapping as well as ion exchange column chromatography, both preparations were found to contain more than 22 peptide components, though the lyophilized powder had less than the other. The lyophilized powder showed an accelerating effect on the rates of both superprecipitaion (0.1 ionic strength, pH 7.0) and ATPase activity (0.15 ionic strength, pH 6.4) of fish actomyosins. The acceleration was also positive when Ca and Mg ions were removed by adding GEDTA or EDTA. These effects seem to be involved in the vasoactive and vasodilatory function of the fur seal peptide preparation.

We designed a new method to evaluate the gel properties of fish meat gel products, by a stretching test, and a film-shaped gel sample, ca. 1mm thick, is used as a test piece. This method had the following advantages: The method can be applied to a small amount of sample and less than 500mg of sample was needed for one measurement. It had high accuracy and reproducibility. It showed high correlations with the pushing-in method using cylindrical test pieces, and values could be converted to those from each method. The fracture test and measurement of Young's modulus (non-fracture test) can be performed at the same time, while for the pushing-in method using cylindrical gels, the fracture test and measurement of Young's modulus must be done sep-arately. Thus the present stretching method was effective particularly when a large amount of sample could not be obtained, and also possessed several other advantages as above. Therefore, the present method may contribute for evaluation of the gel properties of fish meat gel products.

In order to find suitable conditions for the fixation of fish muscles for electron microscopy, effect of the fixative concentration to the musculature of the freshwater and saltwater fish was examined. Carp and horse mackerel were used as the sample of the freshwate and saltwater fish, respectively. Muscle samples were prefixed in 3% glutaraldehyde-sodium cacodylate buffer (pH 7.2). Following the postfixation in 1% OSO4-sodium cacodylate buffer, muscles were dehydraed in ethanol and embedded in TAAB 812. The molarity of sodium cacodylate buffer was set to 0.05M, 0.1M or 0.2M for the freshwater fish, and to 0.1M, 0.2M or 0.3M for the saltwater fish, in both prefixation and postfixation Muscle samples were block-stained in 1% uranyl acette solution ater postfixation. Ultrathin sections were examined under electron microscope. The results show that the most suitable concentration of the fixatives is 0.1M and 0.2M for the freshwater and for the saltwater fish muscles, respectively. Use of the fixatives lower than the above strengths induced expansion of the mitochondria or the sarcoplasmic reticulum and broadening of the myofibrils in both fresh and salt water fish muscles. On the other hand, use of the fixatives stronger than the above induced deformation of the mitochondria, and obscuration of the filament structures.

Kasuzuke is a processing where fish meat and vegetables are cured in lees of sake (Japanese rice wine) for the purpose of preservation. In kasuzuke the fish meat cured in sake lees acquires a peculiar texture. Changes in muscle proteins were examined in relation with the texture changes. Spanish mackerel fillets were cured in sake lees after salting and were stored at 4°C for 1, 7, 14, 21 and 28 days. The fillets were submitted to the tests and measurements for firmness (after being cooked), extractability of proteins, viscosity and natural fluorescence of actomyosin solutions and the analysis of proteins by SDS-PAGE after various periods. For control, fillets were stored with the addition of 2% NaC1 or without any treatment. Firmness of the texturometer test increased after a day and 7 days, then decreased after 14 days and 21 days. Extractability of actomyosin and water soluble proteins did not change appreciably for 28 days. The changes in reduced viscosity and relative fluorescence intensity suggested that the conformations of actomyosin of muscle cured in sake lees have changed. SDS-PAGE patterns showed that the proteins of muscle cured in lees were decomposed during storage into fragments with smaller molecular weights. The decomposition of proteins were attributed to the effect of the acid proteinases in the sake lees. The texture changes during the curing in lees were suggested to come from the changes of proteins.

With an aim of studying the gel properties and its formation, differential scanning calorimetry (DSC) was applied to the fish meat gels (kamaboko) in parallel with the jelly strength measurements. DSC curves of the gels were characterized by a single deep hollow with a bottom slightly below 100°C which represents the evaporation of water. Each curve was divided into two fractions, below and above 105°C (Q1 and Q2), and a quotient, R105 (=(Q2/(Q1+Q2))×100), was adopted to represent the amount of captured water in the total water of the gels. Two series of gels were prepared from Alaska pollack frozen mince, one with various protein concentrations, and another containing various amounts of egg white. The gels were submitted to DSC and jelly strength measuremetns. In both cases, R105 values illustrated good correlationships with the jelly strength values. That the state of water in the gels affects the gel properties like the jelly strength has suggested.

The goal of this study is to obtain a basis for the research of recovering and reutilizing the water soluble proteins discarded into the waste wash waters of the frozen mince (surimi) plants.Effect of an ultrafiltration system (IHI Co., Ltd.) to recover the water soluble proteins was studied. Proteins from 5 fish species, cod, Alaska pollack, sardine, mackerel, and horse mackerel were studied. The water soluble proteins were extacted with 5-10 volumes of water and the extracts were applied to the ultrafiltration system giving rise to concentrates. The protein concentration of the extracts, 0.1-2%, was raised to 0.4-18% with yields of nearly 90%. The low molecular matters, inorganic and organic, were largely filtered off by a membrane. The molecular weight composition of the water extracts and the concentrates were analyzed by Sephadex G-100 or G-200 gel filtration. The water extracts consisted of 5-7 groups with molecular weight ranging 4, 000-150, 000 and the proteins higher than 10, 000 were recovered in the concentrates by ultrafiltration with sieve size 20, 000. Some packing effect on the sieve was suggested. These profiles were generally found for the five species tested. The applicability of the ultrafiltration system appeared optimistic.

The rheological properties of fish muscles of five species were characterized in terms of the creep compliance. A slight increase of the strain with time was observed in all species even without any load applied. The size of the linear region, where the stress increases in proportion to the strain, decreased when the loading time was prolonged. After 300s loading, the largest strain value of the linear region for each fish was, 9.5% in channel rock fish, 9.0% in plaice, 8.6% in flying fish, 8.5% in common horse mackerel, and lower than 2% in skipjack, respectively. On analysis of the creep compliance curves, a six-element model was found to represent each fish muscle, while three more elements, two ratchets and a spring, were needed to fit the creep recovery curves. The instantaneous modulus values of the creep compliance of flying fish and skipjack were larger than those of plaice and channel rock fish. Common horse mackerel gave an intermediate value. With the permanent viscosity modulus, plaice gave the largest value followed by flying fish, channel rock fish, common horse mackerel and then skipjack in a decreasing order. These results appeared to differ from the series obtained on our eating experience. It was suggested that our mouthfeel about “firmness” is not based on the difference in the elastic modulus of the muscles but on the ease of biting which is related to the collagen content as proved in our previous report.

To assess the mechanism of denaturation of fish muscle proteins during frozen storage, kinetic analysis of the rate of decrease in the solubility of the proteins was undertaken. Actomyosin solution isolated from carp muscle was frozen stored at various conditions of protein concentration, ionic strength, and pH, and the change of protein solubility was studied kinetically. Electron microscopic observations were also carried out to obtain information regarding changes in the shape of the actomyosin filaments. At pH 7.0, no single first order rate was found, but a two staged change consisting of two first order rate processes was illustrated regardless of the protein concentration and ionic strength. However, the mode of change varied depending on the pH. At a pH below 7.0, the change followed two staged process, while, at a pH above 7.5, it occurred in four successive stages, each of which was of a first order expression. From the results, three possible mechanisms have been proposed for the freeze denaturation of actomyosin.

In the previous paper, the ATPase accelerating peptides were found to be distinct from the vasoactive and vasodilatory peptides in the same hydrolysate. This work was undertaken to isolate the ATPase accelerating peptide from the fur seal muscle hydrolysate. Minced skeletal muscle of fur seal was digested with protease and the resulting hydrolysate was fractionated with 90%ethanol. The obtained precipitate and the effective agent was fund only in the precipitate. By continuous flow electrophoresis, the was divided into five major fractions, each of which was examind for accelerating effect on ATPase by two different methods, i.e., inorganic phosphate determination and pH-stat method. Among the five electrophoretic fractions, the second fraction E2 migrating to the anode showed the best accelerating effect, while the other fractions were less accelerative. Also the possible of Ca ions was refuted. E2 was also examined for physiological activity by kymographic recording of the contraction of rat intestine smooth muscle and showed an effect which is revealed by a contraction preceded by a slight relaxation. Thin layer chromatography revealed that there are more than two peptide components in fraction E2.

To obtain basic knowledge for processing of the meat products, such as, kamabokos, sausages, and hams, the myofibrillar proteins of rabbit, carp, and scallop were isolated and submitted to the differential scanning calorimetry (DSC). Myosin preparations gave broadly dispersed endothermic regions in each of which a peak was found at 62°C for rabbit(R), 50°C for carp(C), and 40°C for scallop(S). LMM's gave a single peak at 60°C(R)and 47°C(C). The peaks of myosins reflected the denaturation of the LMM portion of the myosin molecule. Actins showed a peak at 82°C(R), 75°C(C), and 77°C(S). In the crude actin, the actin's peak was accompanied by a peak at 58°C(R), 53°C(C), or 42°C(S), each of which was attributed to the regulatory proteins. The natural actomyosin preparations illustrated 2 peaks at 49°C and 62°C for rabbit and 41°C and 52°C for scallop, but that of carp gave 3 peaks at 43°C, 53°C, and 68°C. Each second peak was ascribed to myosin. None of the rest were found to be correlatable with any of the actin, myosin, or the regulatory proteins and thus these peaks were assigned to some conjugate derivatives of actin and myosin. The peak top temperatures, i.e., the heat denaturation temperatures, of the respective proteins were found to be different for the different animal species and an order of “rabbit, carp, and scallop” was commonly observed with a little exception in the case with scallop actin. The former descreasing order was related to the order of the habitat temperatures of the tested animals.

The preventive effect of carboxylic acids on freezing denaturation at -30°C for 4 to 7 weeks was examined by using an in vitro-model test system. The rate of denaturation was followed by estimating solubility, viscosity, ATPase activity as well as the degree of super-precipitation. The overall results can be summarized as follows: Additives exhibiting marked effect: malonic, maleic, lactic, malic, tartaric, gluconic, glycolic, methylmalonic, dimethyl malonic and glutaric acid. Additives displaying moderate effect: adipic, pimelic, formic, acetic and propionic acids. Additives with little or no effect: oxalic, succinic, fumaric, butyric and valeric acids. These results suggests that the preventive effect of an additive is influenced by the nature, position, number and configuration of the functional groups of the compounds, and that some physico-chemical properties such as water solubility are closely related to the preventive effect. The preventive effect of these additives during the thawing process was tested at various concentrations of KCl with results which support the view that additives function indeed during frozen storage.

For examing the factors involved in the elastic quality of kamabokos, the microstructures of the texture were examined by electron microscopy.The obtained microstructures were evaluated in correlation with the jelly strength values of the kamabokos. Some kamabokos were picked up at shops and others were prepared from frozen surimi of Alaska pollack, The electron micrographs demonstrated a variety of microstructure profiles and these were roghly classified into fivetypes: (1) a matrix with distinct network structures, similar to those of MIYAKE et al. (1971), (2) a matrix of randomly dispersed bundles of irregularly arranged filaments, but similar to (1), (3) a matrix with the Z-bands remaining, (4) a matrix including myofibrils remaining, and (5) a matrix including collagen-like objects. Matrix of the second type was found most frequently. In the same piece of kamaboko, some portions consisted of densely dispersed matter and other portions of loosely dispersed matter. In the latter portions, matrix of the first type was usually found. Some correlation was found between the microstructure profiles and jelly strength. Comparison of the kamabokos prepared from frozen surimi of various grades illustrated that the microstructure and the jelly strength of kamabokos vary depending on the myofibrillar structures and the state of the proteins in the surimi.

Dispersion profiles of proteins in “muen” (non-salt) and “kaen” (salt added) frozen surimi (fish mince) were examined under electron microscope. The cross sections of the muen surimis gave various profiles but no appreciable difference related with the jelly strength of the drived kamabokos was found. The dispersion profiles of the muen surimis were classfied roughly into following four types: (1) a profile demonstrating well reserved myofibrillar structures in which the M-lines and the Z-bands remain in nearly intact state, (2) a profile demonstrating moyfibrillar structures in which the M-lines are inappreciable and the vague Z-bands are shown, (3) a profile demonstrating parallel streams of fine filaments which are probably assingnable as actomyosin filaments, (4) a profile in which no uniform structure is appeciable. The dispersion profiles in the kaen surimis were similar to those found in kamabokos (cooked jellies) and showed dense or loose matrices of cross-linked filaments. Dilute homogenate suspensions of the surimis illustrated two types of filaments which were probably assignable as actomyosin filaments and as naked F-action filaments beside aggregates of myosin. The grade SA surimis and some grade C surimis demonstrated fine arrowheaded filaments of actomyosin and gave good jellies. Other grade C surimis demonstrated filaments of myosin-poor actomyosin filaments and gave poor jellies.

Shimesaba (acid-salt curing) is a traditional Japanese fish processing where mackerel fillets are cured in vinegar after salting. In shimesaba, acid-salt cured muscle acquires a peculiar palatable texture. Experiments were carried out to clarify the nature of the changes in texture. Changes in muscle proteins were examined for the possible major factor of the texture changes. Five hours after adding 10% NaCl, mackerel muscle were soaked in rice vinegar at 4°C for 15h and 50h and were submitted to sensory tests. Muscle cured in vinegar for 50h were harder and more fragile than those stored for 15h. As a model of shimesaba, the mackerel fillets (1.5cm thick) were stored in 4% acetic acid-2% NaCl solution at 4°C for 7 days, and were submitted to the tests for firmness, fracturability and fragility, and the analysis of the proteins by SDS-PAGE at various storage periods. For control, fillets were stored by adding 2% NaCl or without any treatment. Firmness by texturometer test and fragility by author's disintegration method increased by acid-salt curing. SDS-PAGE patterns showed that proteins of acid-salt cured muscle were decomposed into fragments with smaller molecular weights during storage. Actomyosin and water soluble proteins extracted from the muscle underwent similar decomposition after acid storage. Decompositions of the proteins were proved as attributable to the acid proteinases in muscle which had been activated at the acidic pH's. The texture changes of the acid-salt cured mackerel muscle were attributed to the decomposition of the proteins due to the function of the proteinases activated at the acidic pH of the curing.

A histological study was made on dark and white muscles of four freshwater fishes and foursaltwater fishes. In myofibrils, difference of cross sectional patterns was found between dark and white muscles of 8 fish species. Mitochondria in dazk muscles were observed not only around myofibrils but also in myofibrils, whereas they were located around myofibrils in white muscles. All the species studied here, with the exception of white muscle in blackthroat seaperch, were found to have I-bands which are narrower than in mammalian muscles. On the contrary, the I-bands of white muscles in blackthroat seaperch were almost similar to the skeletal muscles of rabbit. In dark muscle of Japanese char, rainbow trout, horse mackerel and mackerel, lipid droplets were found in the interspaces of myofibrils and there were many mitochondria surrounding the droplets. The dazk muscles of Japanese char, rainbow trout and blackthroat seaperch appeared struc-totally very similar to one another. These dark muscles were much smaller in size and less darker in color as compared with the dark muscles of other fishes studied here.

1) Some electrophoretic study was made on M-actomyosin, an actomyosin-like protein obtained from aqueous extract of the squid muscle. The actomyosin-like pre-paration showed an electrophoretic diagram including two or three peaks, which is similar to that of the actomyosin-like protein from salt extract of the squid muscle. Also in respect of the electrophoretic mobility each component corresponded with each other in both cases. So the two actomyosin-like protein, the one from aqueous extract and another from salt extract of the squid muscle, including the accompanying proteins, were found to be identical. There was found an indication that Component 2, the middle-speeded is rather scanty in the case with the protein from aqueous extract. 2) When a precipitate of the actomyosin-like protein from salt-extract was extracted repeatedly with water, a better part of the protein moved into solution. Moreover, marked streaming birefringence (SB) was found in the extracts as the case with the repeated aqueous extraction of the squid muscle. On the contrary, SB was never found in the case with carp actomyosin, whereas nearly half of the used actomyosin of the carp was brought in solution. 3) From these experimental bases, it was proposed that the actomyosin-like protein which occupies the dominant part of the squid muscle proteins should be referred to as “M-actomyosin” no matter whether it is isolated from salt extract or from aqueous extract.

A collaborative experiment was made on the grinding and mixing machine, wherein the authors took over a portion of the analysis of the mixing power. For the judgement of the proceeding of mixing, the variation of distribution of salt was pursued, which was added to the chopped meat of shark in amount of ca. 3% of the latter at the onset of mixing and grinding. The samples of meat were taken from 5 or 9 definite stations within the mill, 7 times at equal intervals during the whole procedure, being analysed for their salt contents, the data of which were subjected to the critical consideration, (Table 3). Upon the comparative studies with 2 representative types of this machine in Japanese market, type Y proved itself to be more effective than type A in its mixing power. On the other hand the latter was found to be more effective in that, of grinding, as the other colla borators reported on the basis of histological observations. It was shown, however, that both types can be used satisfactorily for the practical purpose in “Kamaboko” (a sort of fish paste product) industry.

Intending to know whether the quantitative relationship of water-extractable proteins to non-myosins in muscle is correlated with the development of SB in its aqueous extract, a comparative study was made with muscles of some Mollusca, Crustacea, fishes and bull-frog. (1) Muscles of used animals were found to be practically grouped into three types: Mollusca-, Crustacea-, and Fish-types (cf. Table 4). (2) In Mollusca-type muscles SB was detected in aqueous extracts and water soluble proteins far exceed non-myosins in amount (∑S>N). (3) In fish and Crustacea-types SB was not detectable in aqueous extracts, and ∑S ?? N in the former but ∑S>or ?? N in the latter. (4) Intensity of SB of salt extracts was apparently greater in Cephalopada and Crustacea than in fishes. (5) Muscles showed each characteristic pattern corresponding to the three types also in aspects of precipitates spun down in aqueous extraction mixture.

1) When squid muscle were repeatedly extracted with 2_??_20 times the amount oof water, streaming birefringence (SB) was observed in the second and the following extracts (Fig. 1).2) When squid muscle were extracted repeatedly the total amount of proteins passing into solution amounted to 55.5 per cent of total proteins. In carp and horse .mackerel muscle, however, the totalextractible proteins was not so much beyond the amount of non-myosins which was to be expected from the literature on the subject (Table 1)3) The amount of water soluble proteins of squid muscle increased with the in crease of the ratio of extracting water to muscle, while those of flat fish and horse mackerel muscle attained a constant value with the increasing ratio (Fig. 2).4) In squid, the amount of non-myosins was as small as one fifth to one third of the water extractible fraction, while in flat fish, horse mackerel, and carp, the .amount of ncn-myosins werefound to coincide roughly with that of the water extra-ctible fraction (Tables 1 and 2).5) If the aqueous extracts of squid muscle were dialysed against distilled water, some of the proteins precipitated on centrifugation. The precipitate from the seconct extract showed SB if it was mixed into an aqueous suspension, while the precipitate of the first extract did not.6) Some of the proteins precipitated out if KCI was added to aqueous extracts. of squid muscle so as to make up mixtures of varying ionic strengths ranging from 0.0005 to 1.0. The relationship of solubility to ionic strength seems to suggest the presence of both myosin proper and actomyosin in the aqueous extracts (Figs. 3 and 4).7) In view of these facts it was suggested that in the case of squid muscle, myosin might be dispersed into the aqueous suspension and give rise to the characteristic SB.The authors wish to express their gratitude to Miss Tomiko ARAI and Mr. Yoshio. Isxiro for their devoted assistance in the experimental work.The authors thank Mrs. Dorothy GILNER who has kindly consented to read overthe manuscript.

It was found in preliminary experiments that “myosins” of squid muscle are apt to become soluble at lower ionic strengths (I) and often escape from estimation (Table 1). The present work was planned to comfirm this and to find factors involved in this change. The muscles of different freshness were extracted with KCl-phosphate buffer of I=0.6 and pH ?? 7.5, myosins in the extract being determined by dilution method in three ways, immediately after the extraction, after keeping it at 30°C. for 2 hr., and after 2 day cold storage. In every case, myosins were precipitated at pH 6.5, 7 and also 9 (Fig. 1). Myosins of squid was found to become less precipitable, unless the extract was diluted immediately after extraction. This change was more marked in media of pH higher than 7, and when the used muscle was not fresh (Figs. 3 and 4). Consequently in determination of myosins in squid muscle, myosins should be precipitated immediately after extraction and at pH near and not excceeding 7. Observation of streaming birefringence of the extract suggested that asymmetry of dissolved proteins might hardly change even under the above condition which causes decrease of precipitability of myosins in the extract. With carp muscle, on the other hand, no such marked change in amount of precipitating myosins was observed (Figs. 6 and 7). ?? A protein fraction which precipitates at lower ionic strenghts (I=0.05-0.2) from saltextracts was called here “myosins”.

1) As far as the salting-in curve is concerned, it appeared that 2 or 3 components are contained in M-actomyosin preparation obtained by adding KCl to the streaming birefringent aqueous extract of squid muscle. Among these components, Component I which is least soluble in lower ionic strengths (I) was found to be responsible for the characteristic responses to ATP and thus was inferred to be M-actomyosin. 2) Observations were made and discussed on the purification of Component I by reprecipitation. Thus it was inferred that reprecipitation is useful for purification, but that it is questionable if the purification was complete or not. It was also found that Component I is considerably unstable and is apt to change its solubility behaviors which is not the case with rabbit and carp actomyosins. 3) The salting-in range of M-actomyosin of squid was found to cover I = 0.2 ?? 0.5. In this respect, it appeared not very different from rabbit or carp actomyosins.

1) The distribution pattern of squid muscle proteins was studied by the method of DYER et al. as well as by a similar method which was modified in some points so as to fit the properties of squid muscle proteins. 2) Squid muscle proteins were found to consist of 12 ?? 20% of non-myosins, 77 ?? 85% of myosins and 2 ?? 3% of stroma. Subdivision of non-myosins into myogen, myoalbumin and globulin X did not give a uniform pattern from analysis to analysis. As far as the gross distribution pattern of muscle proteins is concerned, squid seems to resemble fishes, although properties of its myosins are distinct from those of fishes as shown in the previous reports.