Carotenoids are yellow to red isoprenoid polyene pigments; approximately 650 carotenoids are known from bacteria, fungi, plants and animals. They occur not only as free forms but also as esters, glycosides, sulfates and carotenoproteins. A recent discovery of antitumor activity in carotenoids, such as vitamin A and β-carotene, has brought attention to marine carotenoids. This review describes the structures of 117 carotenoids isolated from sponges, coelenterates, mollusks, crustaceans, echinoderms, tunicates and fishes. The comparative biochemistry and metabolic pathways of these carotenoids are also discussed.

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The dolphin fish, Corphaena hippurus Linnaeus, is one of the most important migratory fishes on the east coast of Taiwan. Present results are based on an investigation of 1439 specimens caught using long-line and driftnet, and set net collected monthly between September 1996 and September 1997. The sex ratio of female to male and female combined was 65%, demonstrating a female predominance. Oocytes became mature and transparent when they attained 1.0 mm in diameter. The total number of oocytes per ovary was estimated to range from 2.78×10⁵ to 23.48×10⁵, but batch fecundity was noticeably lower than the total number of ovarian eggs, ranging 5.3-32.7% (average 30.1%), and the relative fecundity ranging 10-344/g bodyweight (mean 111/g bodyweight). Minimum body size at sexual maturity was estimated to be 51 cm for both sexes. Dolphin fish spawned throughout the year with reproductive activity peaking in February to March. It is suggested that the dolphin fish has an extended spawning season, during which it lays eggs almost continuously.

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Relationships between the growths of five different clonal strains of Heterocapsa circularisquama and intracellular bacteria were investigated using culture experiments. Although each H. circularisquama strain culture was established from one cell by repeated and careful washings with micropipettes, intracellular and extracellular bacteria in each strain culture were still observed under a epifluorescence microscope using the DAPI (4', 6-diamidino-2-phenylindole) staining technique. The extracellular bacteria are derived, presumably, from the inside of algal cells after the death and collapse of algal cells in culture. Using an electron microscope, bacteria were constantly observed in the cytoplasm and food vacuoles of H. circularisquama cells. The growth of five algal strains containing bacteria was compared with that of a bacteria-free strain using culture experiments under combined conditions of five different light intensities and five different strengths of culture medium. Bacteria showed no significant effect on the growth or survival of the algal cells. During the algal exponential growth phase, the intracellular bacterial cell numbers per algal cell decreased, whereas the total bacterial cell density in each algal culture increased. Final cell yield (total number) of the intracellular and extracellular bacteria varied considerably according to the algal strains. These results suggest that the intracellular bacteria of H. circularisquama grow or survive depending on the host alga, and that the alga can grow independently.

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Morphology of the photoreceptor cells and tapetum of the Japanese anchovy Engraulis japonicus was studied by histologically and by chemical analysis. The Japanese anchovy has a duplex retina. The cones form parallel rows consisting of alternately placed long cones and bifid cones. Both types of cones are intimately associated and form triple units that are regularly spaced along the row of cones. The rods are grouped and stacked. This fish has a retinal tapetum lucidum composed of guanine and hypoxanthine. Three structures of the tapetum lucidum were recognized: platelet, diamond and rod types. Photomechanical changes include movements of the photoreceptor cells and the retinal tapetum. The retina of the Japanese anchovy is thought to be highly sensitive and well adapted to a dim light environment.

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The surface-migratory myctophid fish, Myctophum asperum, of the western North Pacific was found to have daily growth increments of its sagittal otolith, which also exhibited lunar periodicity in the deposition of increments. Daily deposition of the otolith increments was verified because the width of the marginal increment increased during the night and early morning between 20.00 h and 08.00 h and its growth stopped during the day. An autocorrelation analysis of the increment widths, which were measured consecutively in 11 specimens covering 33 synodic months, also showed a lunar periodicity in increment deposition. The mean increment widths during five days around the time of a full moon were significantly narrower than those around a new moon in 18 of the 33 full moon cases (P<0.01: Student's t-test) and, on average, tended to be narrow in 29 cases. The cause of this tendency is thought to be slower growth caused by staying in deeper and colder habitat due to the suppression of diel vertical migration and/or lower food availability resulting from the possible dispersion of zooplankton during the full moon period.

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The effect of an n-3 essential fatty acid (EFA) -deficient diet on spawning and on the lipid and fatty acid contents of eggs, semen and livers of rainbow trout Oncorhynchus mykiss was investigated. Fish were split into two groups and fed either of two diets for a period of 4 months prior to the start of the spawning season. The control group was fed a commercial diet, containing n-3 EFA, whereas the experimental group was fed an n-3 EFA-deficient diet. Fish were 3 years old at the time of spawning. Eggs and semen were stripped off five females and five males from each diet group and cross-fertilized. Two of the five males fed the deficient diet showed a lower sperm motility, resulting in slightly lower mean hatching rates when crossed with eggs of either group. Higher lipid contents in the EFA-deficient diet were reflected in the egg and semen lipid contents, whereas the lipid contents of male livers of both diet groups were higher than those of female livers. In livers and eggs, the main polar lipid was phosphatidylcholine accompanied by phosphatidylethanolamine in sperm polar lipids. The non-polar lipids of eggs were mainly triacylglycerols but in livers and semen, free fatty acids and free sterols were more abundant. Essential fatty acids, namely n-3 highly unsaturated fatty acids and linolenic acid, were generally lower in eggs, semen and livers sampled from the EFA-deficient diet group. These results indicate that the fertilization, eyed and hatching rates obtained from crossings with males fed the EFA-deficient diet were slightly lower because of the dietary effect on sperm motility. Moreover, lipids of eggs, semen and livers of male and female rainbow trout were influenced greatly by their dietary availability.

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The complete nucleotide sequence of the mitochondrial genome for the Japanese anchovy Engraulis japonicus (Teleostei: Clupeiformes) was determined. The entire genome was purified by gene amplification using the long polymerase chain reaction (PCR) technique, and products were subsequently used as templates for PCR with 56 fish-versatile primers that amplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR products demonstrated that the genome (16 675 base pairs [bp]) contained the same 37 mitochondrial genes (two ribosomal RNA, 22 transfer RNA and 13 protein-coding genes) as those found in other vertebrates, with the gene order being identical to that in typical vertebrates. A major non-coding region between the tRNA^Pro and tRNA^Phe genes (1024 bp) was considered to be the control (D-loop) region, as it has several conservative blocks characteristic to this region.

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In order to ascertain whether the use of drugs as feed additive for fish may cause drug resistance of bacteria in the intestinal tract, viable counts and bacterial flora in the intestine of yellowtail cultivated with the feeds in the presence or absence of various drugs, drug resistance of the flora were determined. Viable counts in the intestine were lowered remarkably by the administration of each drug, suggesting that bacterial growth in the intestinal tract was inhibited by various drugs. Bacterial flora in the intestine consisted mainly of coryneform bacteria, Staphylococcus and Bacillus regardless of whether drugs were administered or not. In these floras, significant differences could not be observed for their resistance to respective drugs among the isolates from fish cultivated with feeds containing or not containing each drug. In addition, these floras mainly showed resistance to various combinations comprising from one to seven drugs; moreover, the majority of resistant strains were multidrug-resistant.

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Genetic polymorphism in kingfish, collected from coastal waters of Japan, Australia and New Zealand, were examined using microsatellite (MS) DNA and mitochondrial DNA (mtDNA) control region markers. Sixteen to 25.7 alleles per locus were observed in three MS markers, while the average observed (and expected) heterozygosities were 0.782 (0.918), 0.750 (0.809) and 0.650 (0.888) for Australian, Japanese and New Zealand kingfish, respectively. Twelve mtDNA haplotypes were detected by the digestion of control region sequences with five endonucleases: Haelll, Hinfl Mbol, Rsal and Taql. Significant genetic divergence was observed between the kingfish population from Japan and those from Australia-New Zealand. There was no significant differentiation among the Australian and New Zealand population samples.

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To analyze the effect of thin twine on gill net size selectivity, a series of fishing experiments using gill nets of two twine thicknesses (no. 0.8 of 0.16 mm diameter and no. 3 of 0.28 mm diameter) and three nominal mesh sizes (41 mm, 46 mm and 51 mm) was conducted in outdoor water tanks containing a rainbow trout (Oncorhynchus mykiss) population of known size frequency. For nets of the same nominal mesh size, mesh openings of the no. 0.8 twine nets were approximately 2 mm larger than those of the no. 3 twine nets. The effects of both mesh opening and twine thickness are combined in the conventional analysis with the selectivity curve against fish length. Selectivity against the ratio of fish girth to mesh perimeter was calculated to avoid the direct effect of changes in mesh openings due to twine diameter at the same nominal mesh size. Results demonstrated that the thinner twine gill net was more likely to capture rainbow trout of smaller girth than the mesh perimeter. Fish with girths smaller than the mesh perimeter were often retained by the net with its body tangled by the thinner twine, possibly due to the increased flexibility of the thinner twine.

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Random amplified polymorphic DNA (RAPD) was studied to detect genetic markers for three economically important Japanese laminarian species, Laminaria japonica Areschoug, L. religiosa Miyabe and L. ochotensis Miyabe, which were sampled from their representative localities on the coasts from Tsugaru Straits to the Sea of Japan. DNA templates for RAPD were extracted from lamina using a DNA extraction kit and were purified with glassmilk. Reproducible RAPD markers for these three species were detected using three random primers from a total of 15 tested. In L. japonica and L. religiosa, these RAPD markers were confirmed to be useful for populations from other localities. The three species studied showed high intraspecific and interspecific band sharing index (BSI) values. Like annual typical L. religiosa from other localities, biennial individuals identified as putative L. religiosa from Atsuta could be discriminated from L. japonica but not from L. ochotensis by any of the RAPD genetic markers studied so far.

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To clarify the quantitative changes in the transport of orally intubated protein into the blood circulation as macromolecules in development, immunoglobulin Y (IgY) extracted from chicken eggs was administered orally to juvenile Japanese eel, Anguilla japonica. For the first experiment, which was performed before the commencement of artificial feeding, the oral delivery of 2.0 μg/0.1 g bodyweight of IgY resulted in a rapid increase in plasma IgY to a maximum of 2.30 μg/mL. However, the transport of IgY into the blood decreased significantly in the experiments that followed, which were performed after 12, 25 and 42 days. During this period, bodyweight increased approximately by a factor of eight, and rapid growth of the stomach was observed histologically. Possible contributions for the development of the alimentary canal to the diminishment of intestinal protein assimilation are discussed.

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Morphological changes in the oocytes of the Japanese eel, Anguilla japonica, induced to undergo ovarian development by repeated injections of salmon pituitary homogenate, were examined using electron microscopy. Oil droplets were closely associated with organelles, especially mitochondria, and increased in number as oocyte growth proceeded. They fused at the migratory nucleus stage. During vitellogenesis, two types of cortical alveoli were distinguished, one having filamentous contents, the other having latticated contents. As oocytes reached maturity, the structure of the cortical alveoli was exclusively filamentous. Yolk globules were homogeneous and highly electrondense, but electrondensity decreased during hydration. The structure of the zona radiata of previtellogenic oocytes consisted of two layers, and an additional reticular network structure was formed on the inside of the zona radiata during the vitellogenic stage. The zona radiata lost the reticular network structure and assumed a layered structure of uniform electrondensity at the migratory nucleus stage. These structural changes during oocyte development were mostly comparable to those in other teleosts. Results of the present study should assist in developing improved methods for full control of artificial maturation in the Japanese eel.

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The present study was designed to quantify changes in serum vitellogenin (VTG) levels and to localize VTG in hepatic cells immunohistochemically during artificial maturation of the Japanese eel Anguilla japonica. Hormone treatment resulted in a gradual rise in serum VTG levels, in which the highest values were attained at the migratory nucleus stage. Hepatocytes responded to hormone treatment by increased immunoreactivity; both staining area and intensity were enhanced with ongoing gonadal development. Moreover, cytoplasmic vacuoles increased in number during artificial maturation, occupying large areas inside hepatocytes. The unusual appearance of hepatocytes may reflect the effect of artificial maturation.

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This study clarifies the annual reproductive cycle and the lunar-synchronized spawning of the spiny rabbitfish (Siganus spinus) that inhabit the Okinawan waters. Annual and weekly changes in the gonadosomatic index (GSI) and the histological features of the ovaries were checked. Gonadosomatic index was high during the months of May to July, and yolk-laden oocytes were observed in the ovaries from March to July. Some of the ovaries collected during June and July contained oocytes at maturation stage or ovulatory follicles. These results suggest that the spiny rabbitfish undergo active vitellogenesis and spawning from May to July. During the reproductive season (May to July), collection of fish according to the lunar phase revealed that a high GSI occurred around the time of the new moon. Cyclic oocyte development with peaks around the time of the same moon phase was also observed, suggesting that, in Okinawan waters, this species is a lunar-synchronized spawner and spawns three times.

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The rate of oxygen consumption and energetic cost of locomotion of captive harbor porpoises Phocoena phocoena were studied in Hokkaido, Japan, using respirometry together with externally attached devices (a data logger) that recorded swim speed and dive depth. Harbor porpoises swam freely in a tank at a swim speed ranging 0.5-4.2 m/s. Swimming during most dives was relatively slow with mean swim speed being 0.76-0.91 m/s. Oxygen consumption rate of the swimming harbor porpoise increased with swim speed according to a cubed function. The minimum cost of transport during underwater swimming in the harbor porpoise was 2.39-2.43 J/kg per m at an average swim speed of 1.3-1.5 m/s. However, porpoises usually swam slower to conserve energy and dived aerobically. This explains why harbor porpoises can dive repeatedly and continuously without extended rest at the sea surface.

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Four new bromophenols and a known bromophenol derivative have been isolated from the Palauan sponge Phyllospongia dendyi. The structures of these new compounds were established using nuclear magnetic resonance and mass spectral data. They showed antimacroalgal activity with IC₅₀ values of 0.02-0.05 p.p.m. Moreover, all compounds were antimicroalgal against various classes of algae at concentrations of 0.5-5.0 p.p.m.

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The responses of enzyme activities and metabolic intermediate concentrations to a long burst of exercise and following resting were examined in muscle and the hepatopancreas of carp Cyprinus carpio. A 15 min burst of exercise made the fish so exhausted that they could not swim any more. In muscle, the exercise decreased glycogen content significantly and increased lactate content significantly, resulting in a lowered pH. Furthermore, the burst of exercise decreased phosphofructokinase (PFK) activity significantly, although it changed adenosine-5'-monophosphate, adenosine-5'-diphosphate, adenosine-5'-triphosphate, fructose-6-phosphate and citrate concentrations within ranges that could activate PFK. In the hepatopancreas, the exercise increased glucose-6-phosphatase and fructose-1, 6-bis-phosphatase activities, and glucose and lactate concentrations in the serum, and it decreased glycogen content. Even at 3 h resting after the burst of exercise, the fish had not completely restored many parameters. These results suggest that although the fish tried to enhance in vivo muscular glycolysis through the activation of PFK by changes in metabolic intermediate concentrations, glycolysis seemed to decrease markedly through the inhibition of PFK as a result of the lowered pH. However, the hepatopancreas made a contribution to muscular exercise through glucose supplementation by enhanced gluconeogenesis and glycogenolysis, indicating the presence of an interdependence of carbohydrate metabolism between muscle and the hepatopancreas in the fish.

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The present study used squid gill as a source of transglutaminase (TGase) because it has extremely high TGase activity compared with other tissues. The enzyme was purified using successive chromatographies of Sephacryl S-300 and hydroxyapatite columns. The yield and purification-fold of the enzymatic activity was 12.6% and 14.1-fold, respectively. The molecular mass of the purified enzyme was estimated to be 94 kDa by using sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. Enzyme activity was enhanced 15-fold with an increase in NaCl concentration. Although the activity was dependent on Ca²⁺ concentration, it was not sufficiently activated even by 50 mM CaCl₂ in the absence of NaCl, but could be fully activated with 10 mM CaCl₂ in 0.7 M NaCl. However, in the absence of substrates, the enzyme was rapidly inactivated. The pH and temperature optima of the enzyme were approximately pH 8.0 and 20°C, respectively. It was stable in the absence of Ca²⁺ at pH 7.5-9.0 and had a rate constant (K_D) of 1.6×10^-5 s^-1 for thermal inactivation at 50°C. These results in which squid gill TGase could be activated at higher concentrations of Ca²⁺ and NaCl than at a physiological concentration, suggest that contact with seawater or body fluid seems to activate the enzyme if the tissue is disrupted.

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The effect of pH on thermal gelation and transglutaminase (TGase; EC2.3.2.13)-induced suwari (setting) of surimi and actomyosin pastes was investigated. A strong and elastic gel was produced from walleye pollack surimi paste at pH 7.0 in the presence of Ca²⁺ using a two-step heating method. In contrast, walleye pollack actomyosin paste formed a weak gel under the same conditions as a result of the low concentration of endogenous TGase. In the presence of EGTA [ethyleneglycol bis(2-aminoethylether) tetraacetic acid], weak gels were formed at pH values of 7.0 and 6.0. Non-proteolytic modori (gel weakening) occurred extensively in the course of actomyosin gelation, but not in surimi gelation. Maximum TGase-induced myosin heavy chain cross-linking was observed at a slightly higher pH of 7.5 than at the optimal pH of endogenous TGase activity; the difference being derived from different substrates. Gelation of carp actomyosin paste at pH values of 5.5, 6.0, 6.5 and 7.0 was monitored by measuring storage modulus (G') and loss modulus (G''). A weak gel was formed at all pH values, but a slightly rigid and less elastic gel was obtained at lower pH values. The addition of microbial TGase (MTGase) formed strong elastic gels at pH 7.0 and 6.5. MTGase cross-linked myosin heavy chains even at pH 5.5, but contributed neither to suwari response nor strong gel formation. Overall, results suggest that the optimal pH for the gelation of surimi paste from easy-setting fish species is a compromise between the pH-optima of TGase activity and of preferable actomyosin conformation for myosin cross-linking.

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Grammistins are hemolytic and ichthyotoxic peptides in the skin secretion of soapfishes and are structurally characterized by their abundance in amphiphilic α-helicity. In the present study, their interaction with lipids and lipid vesicles as well as antibacterial activity were examined using four grammistins (Gs 1 and Gs 2 from Grammistes sexlineatus and Pp 1 and Pp 3 from Pogonoperca punctata). The hemolytic activity of grammistins was inhibited by phospholipids but not by cholesterol. Moreover, grammistins released carboxyfluorescein entrapped within liposomes made of phosphatidylcholine. In contrast, grammistins were found to have antibacterial activity with a broad spectrum against nine species of bacteria, including both Gram-negative and Gram-positive groups. The potency of their antibacterial activity was not related to that of hemolytic activity, suggesting that grammistins bind to membrane phospholipids but lyse erythrocyte and bacterial membranes via different mechanisms. Conclusively, grammistins are new members of the family of cell non-selective membrane-lytic peptides with amphiphilic α-helices, being similar to pardaxins, which are secreted from the skin of soles, and to melittin, which is derived from bee venom.

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The major allergen (named Oct v 1) in the muscle of the octopus Octopus vulgaris was purified by gel filtration on Sephacryl S-300, anion-exchange fast protein liquid chromatography on Mono Q and reverse-phase high-pressure liquid chromatography on TSKgel Octadecyl-4PW. In addition to the molecular mass, amino acid composition and cross-reactivity with Tur c 1 (turban shell Turbo cornutus allergen), the determined partial amino acid sequence clearly demonstrated that Oct v 1 is tropomyosin, similar to the known molluscan and crustacean allergens. Using peptide fragments isolated from the lysylendopeptidase digest of Oct v 1, competitive enzyme-linked immunosorbant assay inhibition experiments showed that IgE-binding epitopes of Oct v 1 are contained in two peptides (77-112 and 148-160) in the central region and one peptide (269-281) in the C-terminal region. In the peptide 77-112, the same sequence as the IgE-binding epitope proposed for Cra g 1 (oyster Crassostrea gigas allergen) is recognized at 92-105. Moreover, the peptide 148-160 partly overlaps with the IgE-binding epitopes suggested for Pen i 1 (shrimp Penaeus indicus allergen) and Pen a 1 (shrimp Penaeus aztecus allergen), and the peptide 269-281 with those for Tur c 1 and Pen a 1.

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The effects of trimethylamine-N-oxide (TMAO) on the urea-resistibility of requiem shark myofibrils were investigated, using Ca²⁺- and Mg²⁺-ATPase activities as a parameter. Both activities were hardly changed or activated up to 0.6 M urea. In contrast, the two activities both decreased to less than 50% in the presence of TMAO up to 0.5 M. When measured at a 2 : 1 molar ratio of urea and TMAO, Ca²⁺- and Mg²⁺-ATPase activities were similar to those in the presence of TMAO alone, indicating that TMAO reduced the urea-resistibility of myofibrils. Myosin, the most abundant protein in myofibrils, from requiem shark exhibited the effects of urea and TMAO on its Ca²⁺-ATPase activity, which was primarily similar to those of myofibrils. However, Ca²⁺-ATPase activities in the coexistence of urea and TMAO for actomyosin reconstituted from requiem shark myosin and chicken F-actin were approximately average of those measured independently in the presence of either urea or TMAO alone. Carp myofibrils, reconstituted actomyosin and myosin, which were used as teleost references, all showed a tendency in the effects of urea and TMAO on Ca²⁺-ATPase activities that was similar to those of requiem shark counterparts.

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To clarify the cause of differences in the temporal change of K-value among fish species living in the same habitat water temperature, the influence of the interposition of pink muscle fibers into dorsal ordinary muscle on 5'-inosine monophosphate (5'-IMP) degrading activity was examined. Fourteen fish species from the Sakishima Islands (habitat water temperature 28°C) and from Nagasaki (habitat water temperature 17°C) were used for the sample fishes. Each of 5'-IMP and ρ-nitrophenol phosphate (ρ-NPP) degrading activities showed a peak at near pH 8.0 and near pH 5.0, respectively. These activities were somewhat higher in fish from Nagasaki than in fish from the Sakishima Islands. The interposition percentage of pink muscle fibers into dorsal ordinary muscle correlated significantly (P<0.05) with the 5'-IMP degrading activity at pH 7.0. Furthermore, the activity at pH 7.0 correlated significantly (P<0.001) with the increasing rate of K-value at 32°C (ΔK₃₂). These results suggest that the difference in ΔK₃₂ among fish species in the same habitat water temperature might be caused by differences in 5'-IMP degrading activity because of the differences in the interposition rates of pink muscle fibers into dorsal ordinary muscle.

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Poly (α-L-guluronate) lyase, as one of alginate lyases, was purified from the culture medium of a marine bacterium, Pseudomonas sp. strain F6, to an electrophoretically homogeneous state. The enzyme was shown to have a molecular mass of 36 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and was most active at around pH 7.5 and was stable between pH 6.5 and pH 8.5. In the thermal stability experiments, the enzyme's activity diminished through an intermediate state with increasing incubation temperatures and was finally lost when heated at 100°C for 15 min. The addition of hen egg-white lysozyme to the enzyme decreased thermal stability dramatically. The apparent retention of enzyme activity (approximately 50%) was observed after the addition of 6 M guanidine hydrochloride and 8 M urea. Enzyme activity was lost completely with 10 mMSDS, while the ordered structure, which is considered likely to be β-structure, was markedly created. The similar conformational feature has also been created in marine bacterial and mollusc enzymes and the β-structure is commonly observed in polyuronate lyases. The divalent cation (Ca²⁺) promoted the activity of the calcium chelator-treated enzyme significantly, suggesting that Ca²⁺ is involved in the formation of the active intermediate between the acidic uronate (s) and amino acid side-chain (s) of the enzyme.

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It is suspected that the proteolytic breakdown of extracellular matrix proteins is responsible for the postmortem tenderization of fish muscle during chilled storage. In order to identify the type (s) of proteinases involved in this phenomenon, the effect of proteinase inhibitors, EDTA (ethylenediamine tetraacetic acid), 1, 10-phenanthroline, ρ-APMSF [(ρ-amidinophenyl) methanesulfonyl fluoride hydrochloride] and E-64 [L-trans-epoxysuccinyl-leucylamido-(4-guanidinobutane)] on tenderization was investigated by using Japanese flounder. Proteinase inhibitor solution was injected into a blood vessel in a caudal portion of live flounder and the firmness of muscle was then evaluated as a shear force value at 0 h and 6 h after death. Metalloproteinase inhibitors, EDTA and 1, 10-phenanthroline, significantly suppressed postmortem tenderization. These findings suggest that metalloproteinases are candidates for proteinases involved in the postmortem tenderization of fish muscles. Although not significantly, ρ-APMSF, a serine proteinase inhibitor, partially suppressed muscle tenderization, which suggests that serine proteinases are also implicated in postmortem tenderization. A cysteine proteinase inhibitor, E-64, showed no effect, suggesting that cysteine proteinases are not involved.

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A rapid method to enumerate bacteria adhered on a surimi-based product (kamaboko) by flow cytometry (FCM) is described. To remove Escherichia coli cells from the surface of kamaboko, ultrasonic energy was used. Almost all cells can be removed from kamaboko in 3 min with ultrasonic treatment. Because the sample might contain various non-bacterial particles such as food additives and debris of products, propidium iodide was used to discriminate bacterial cells from non-bacterial particles. Fluorescence scattergrams could distinguish bacteria from the particles, and the FCM method could be used to enumerate bacteria adhered on the surface of kamaboko during storage. Cell numbers determined by FCM paralleled well with those measured using a traditional colony counting method in the range of 10⁴-10⁸ cells/g. The FCM assay could enumerate cells within 1 min and the total assay time, including sample preparation, was less than 30 min.

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Hydroxy fatty acid isomers derived from phosphatidylcholine hydroperoxides (PC-OOH) in the livers of three fish species, including sweet smelt, yellowtail and rainbow trout, were determined by selected ion monitoring of gas chromatography/mass spectrometry. Total molar amounts of all hydroxy fatty acids determined in the present study coincided well with those of PC-OOH reported previously, suggesting that hydroxy fatty acid composition reflects hydroperoxide composition. The total amount of hydroperoxide isomers accumulated in the livers of sweet smelt was much higher than those of yellowtail and rainbow trout. The amounts of certain isomers, including 10-hydroperoxy octadecanoic acid, 12-hydroperoxy eicosanoic acid and 14-hydroperoxy docosanoic acid, were significantly higher than those of rainbow trout and yellowtail. These results suggest that differences in the contents and compositions of certain hydroperoxide isomers, which are possible precursors of a watermelon-like or cucumber-like aroma, result in differences of fresh fish aroma between aromatic fish and non-aromatic fish.

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