Various aquatic invertebrates including crustaceans and some bivalve mollusks contain a copious amount of free d-alanine in their tissues. In these invertebrates, d-alanine is largely accumulated with the l-form under a high-salinity environment for maintaining cell volume. d-Alanine is a major osmolyte in these invertebrate tissues, together with glycine, l-alanine, l-glutamine, and l-proline, and is responsible for intracellular isosmotic regulation. Alanine racemase, catalyzing the interconversion of d- and l-amino acids, has been isolated to homogeneity from the muscle of black tiger prawn, and its cDNA has been cloned from the muscle and hepatopancreas of kuruma prawn Marsupenaeus japonicus. This is the first time cloning was achieved in eukaryotes other than yeast. Common carp Cyprinus carpio is an omnivorous fish that often feeds on crustaceans and mollusks containing free d-alanine, but that contains only a trace amount of d-alanine in their tissues. A cDNA of d-amino acid oxidase has been cloned from carp hepatopancreas. Carp d-amino acid oxidase is an inducible enzyme. The activity and mRNA levels of d-amino acid oxidase increase in the intestine and are followed by the hepatopancreas and the kidney. Carp d-amino acid oxidase is structurally similar to the porcine kidney enzyme but is enzymatically similar to the yeast enzyme. d-Amino acid oxidase is thought to be an important enzyme responsible for the efficient utilization of the carbon skeleton of food-derived d-alanine.

ABSTRACTIn order to make effective reuse ofkamabokoprocessing wastes and to minimize the amount of waste disposal, the production of two kinds of fish sauces was tried on a small industrial scale from the wastes with or without the addition of the meat of deepsea smelt (Glossanodon semifasciatus). They were fermented for 6 months at room temperature using salt andkojimold. As a control, a fish sauce was also produced only from the deepsea smelt meat. The recovery of fish sauce from the initial mashed mixture ranged between 75 and 79%, depending on the ingredients in the three fish sauce products. The total nitrogen content of the waste sauce and the mixed sauce was lower than that of the control. The levels of the original additives tokamabokoproducts,β‐carotene and sorbic acid, were very low in these fish sauces. The taste‐active components of the waste sauce and mixed sauces were lower than those of the control. Sensory evaluation revealed that the former two waste sauces were less bitter and higher in saltiness than the control. However, no difference was found in umami taste between these products. These findings suggest that the wastes fromkamabokoprocessing factories could be reused as fish sauce for food condiments.PRACTICAL APPLICATIONSRecent development in the food industry in Japan has enabled the surimi‐based products to be mass produced and standardized even in the fish gel,kamabokotrade. However, a serious problem has arisen in the development ofkamabokoprocessing through the discharge of the wastes, i.e., nonstandard products or fragments, fromkamabokoprocessing factories. It is clear that the waste fromkamabokoprocessing factories is transformed effectively into fish sauce by using soy saucekojimold. As a result, the amount of the discharged wastes fromkamabokofactories are able to be minimized because the liquefaction ratio of the fish sauce mushes (moromi) from the wastes after fermentation was high and the products have a high umami taste and agreeable soy sauce‐like flavor.

Fourspine sculpin (Cottus kazika), a euryhaline and catadromous teleost, was artificially hatched and reared in seawater (SW). Some one-year-old fish had been reared in fresh water (FW) were transferred to SW, and further reared in FW or SW for four months. Free amino acids in the skeletal muscle of wild fish and those reared in FW or SW were compared at the stages of downstream migration (December) and upstream migration (August). The results showed that the skeletal muscle of the sculpin contains large amounts of glycine followed by alanine and lysine, and the lysine content was lower but alanine content was higher in SW-reared fish than in FW-reared fish in both seasons. It seems that season of migration and the actual changes in environmental salinity affect amino acid contents of the skeletal muscle. In addition, total free amino acids in the skeletal muscle of wild fish and those reared in FW or SW showed no significant difference but the muscle taurine content was higher in wild fish compared with cultured ones. Taurine content, however, largely decreased in SW-acclimated fish at the stages of upstream migration, Therefore taurine in the skeletal muscle may be complementary to other amino acids to maintain total amino acids in the skeletal muscle of this species.

The extractive constituents, which are known as taste active components of fisheries products in many research works have been reviewed..This review found glutamate, glycine, alanine, arginine, proline, valine, methionine, phenylalanine, tyrosine, inosine 5’-monophosphate (IMP), adenosine 5’monophosphate (AMP), guanosine 5’-monophosphate (GMP), trimethylamine, trimethylamine oxide (TMAO), glycine betaine, lactate, succinate as important contributors to the taste of raw and processed fisheries products. Sweet, salty, bitter, sour, and umami are the basic tastes, defined by these taste active components. Sweet taste is imparted by glycine, alanine, TMAO while bitter taste by arginine and other hydrophobic amino acids. Glutamate has a role in sour taste, and contributes to umami taste through synergetic effects in co-existence of IMP, GMP and AMP. The large amount of alanine or glutamate suppresses the sweetness effect of glycine through antagonistic effect. However, these taste-producing components vary with species, environments, various processing methods and relative quantities among them.

The distribution of D-amino acids was examined on several tissues of kuruma prawn Marsupenaeus japonicus. D-Alanine was found in all tissues, and the ratio of D-alanine to total alanine ranged from 18.7 to 43.7% depending on the tissues. Of these tissues, muscle, heart, and gill contained a relatively large amount of D-alanine. Nervous tissue and eye, on the other hand, contained a large amount of D-aspartate. D-Glutamate was specifically detected in testis. The percentage of D-glutamate to total glutamate was over 50% in testis, suggesting the existence of the biosynthetic enzyme in this tissue. The changes of alanine racemase activity were determined in the muscle and hepatopancreas of M. japonicus before and after molting. The activity after molting increased twice in the muscle. On the other hand, it was not changed in the hepatopancreas. These data suggest that D-alanine plays an important role in the muscle during ecdysis. However, the free D-alanine level in the muscle was not changed significantly before and after ecdysis. From these data, several D-amino acids are considered to be utilized in some essential physiological phenomena in the different tissues of the prawn.

This study investigated the effect of antioxidants, i.e., carnosine and its Trolox- (water-soluble analog of alpha-tocopherol) acylated derivatives (S,S)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl-beta-alanyl-L-histidine (S,S-Trolox-carnosine, STC) and (R,S)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonyl-beta-alanyl-L-histidine (R,S-Trolox-carnosine, RTC) on the life span of the fruit fly Drosophila melanogaster. Adding carnosine to foodstuff was accompanied and followed by a 20% increase in the average life span of males, but it did not influence the average life span of females. At the same time, adding STC to foodstuff prolonged average longevity both in males (by 16%) and females (by 36%), but the addition of RTC to foodstuff had no influence upon the average life span of insects of either gender. The compounds studied have previously been shown to protect neurons of the rat brain from oxidative stress in the descending order of efficiency: RTC > STC > carnosine. The finding obtained in the present study suggests another order of efficacy regarding the effect on life span in male insects: STC > carnosine > RTC (inefficient). No correlation between antioxidant protection of rat neurons and the effect on life span of the fruit fly makes it possible to suppose the presence of additional cellular targets to be acted upon by exposure of D. melanogaster to these compounds.

The three-dimensional structure of the flavoprotein D-amino acid oxidase (DAO, EC 1.4.3.3) from carp hepatopancreas (chDAO) and its active site cavity was modelled using ProModII. The structural features relevant for the overall conformation and for the catalytic activity are described. Secondary structure topology consists of 11 -helices and 17 -strands, which differs slightly from pig kidney, and Rhodotorula gracilis DAOs. chDAO showed a theoretical 'head-to-head' mode of dimerization. The presence of a short 'lid' in chDAO covering the active site, commonly found in mammalian DAO but absent in R. gracilis DAO, is interpreted as the origin of the differences in kinetic mechanism among these enzymes. This lid has been proposed to control the access of the substrate to the active site and to regulate dissociation of products. The conformational change in the large size active site loop determines the overall rate of turnover of DAOs. The shorter active site loop found in chDAO might be responsible for the higher turnover rate in chDAO compared to that of pkDAO.

Aquatic crustaceans and some bivalve mollusks are known to contain copious amounts of free d-alanine in their tissues. For the first time in the animal kingdom, we have isolated a cDNA clone encoding alanine racemase from the muscle and hepatopancreas of the kuruma prawn Marsupenaeus japonicus. The recombinant enzyme expressed in Escherichia coli exhibited alanine recemase activity. The deduced amino-acid sequence showed only 2331 identity to bacterial alanine racemases. However, the active site residues and some residues that interact with pyridoxal 5-phosphate were also conserved in M. japonicus enzyme. There was higher alanine racemase mRNA expression in hepatopancreas than in muscle. In contrast, the d-alanine content in hepatopancreas was lower than that in muscle, suggesting that the physiological functions of free d-alanine may differ among tissues. These data suggest that the alanine racemase gene has been conserved from bacteria to invertebrates throughout a long evolutionary time scale.

Using a series of omission and addition tests and sensory evaluation of synthetic extracts, the taste-active components of the mantle muscle of the oval squid (Sepioteuthis lessoniana) were determined to be glycine, alanine, proline, glutamate, arginine, adenosine 5′-monophosphate, trimethylamine oxide, glycine betaine, as well as potassium, sodium, and chloride ions. A simplified synthetic extract prepared from these 11 taste-active components was found to almost reproduce the taste of the complete synthetic extract containing 45 components of S. lessoniana muscle. This was also the case for three other squid species; Loligo bleekeri, Loligo edulis, and Todarodes pacificus. When certain components were added to the simplified synthetic extracts of these three squid species – sodium ions and glycine for L. edulis, sodium ions and arginine for L. bleekeri, chloride ions, glycine, and arginine for T. pacificus – the taste of the simplified synthetic extracts approximated the extract from S. lessoniana, which had the most preferred taste.

Extractive components were determined for the mantle muscle and liver of three species of Loliginidae squid, Sepioteuthis lessoniana, Loligo bleekeri, and L. edulis, and one species of Ommastrephidae, Todarodes pacificus, as a control. Total free amino acid levels and the major free amino acids in muscle, taurine, proline, glycine, alanine, and arginine, were significantly higher in Loliginidae squids than in T. pacificus. The main nucleotide was adenosine 5′-monophosphate, which did not differ significantly between species. Malate was the organic acid found in muscle in highest concentrations. The muscle of these species contained a large amount of trimethylamine oxide. A large amount of glycine betaine was also detected in the muscle, but showed no large species difference. From these results, the muscle of Loliginidae species is considered to have a much sweeter taste than that of T. pacificus. Compared with muscle, squid liver was characteristic with high contents of taurine, glutamate, bitter amino acids, succinate, propionate, trimethylamine, and glycine betaine, and with low contents of sweet amino acids, arginine, nucleotides, malate, and trimethylamine oxide. These results suggest that squid liver is characterized by a complicated taste containing umami, bitterness, sourness, fishy flavor, and less sweetness.

In response to hypoxia at PO2 1.3-1.7 mg/L for 6 h, the kuruma prawn Marsupenaeus (Penaeus)japonicus showed a dramatic decrease in phosphoarginine storage in muscle, with normal levels restored during 4-h post-hypoxic recovery. Large stores of muscle glycogen only decreased between 4 and 6 h during hypoxia, but greatly diminished during recovery. Muscle ATP levels and energy charge decreased only slightly under hypoxia. Lactate levels increased slightly during hypoxia and promptly returned to control levels during recovery. These data indicate that phosphoarginine works in muscle as an ATP buffer during hypoxia and glycogen is utilized as an energy source during recovery. Under hypoxia, up- and down-regulated proteins were identified after 2D electrophoresis and partial sequences were obtained after protease digestion. Fructose bisphosphate aldolase was down-regulated during hypoxia, suggesting the suppression of glycolysis under hypoxia. Several partial sequences from three protein spots up-regulated under hypoxia were all assigned to arginine kinase, suggesting the existence of several isoforms of arginine kinase in the muscle of M. japonicus. This arginine kinase up-regulation under hypoxia may indicate a provision for oxygen re-supply after anaerobiosis. This is consistent with the prompt replenishment of phosphoarginine stores during recovery from hypoxia. (c) 2006 Elsevier Inc. All rights reserved.

To make use of underutilized fish species and produce high-quality condiments substitutable for those of mammalian origin, fish sauces were produced on a small industrial scale from three fish species, the flyingfish Cypselurus agoo agoo, the small dolphinfish Coryphaena hippurus, and the deepsea smelt Glossanodon semifasciatus, using salt and koji mold. During 180 days of fermentation, the pH decreased to approximately 4.5 and total nitrogen and free amino acids increased to approximately 2 g/100 mL and 6000 mg/100 mL, respectively. Halophilic and extreme halophilic bacterial counts dramatically increased from 106 to 108 colony forming units/mL between days 14 and 30 and decreased the reafter. In the final products, the pH and salt concentration decreased to between soy sauce and a Vietnamese fish sauce, Nuoc mam. The total amino acid and organic acid contents were almost the same as those of Nuoc mam and soy sauce. Sensory evaluation found these products to have lower smell, saltiness, and bitterness, and higher sweetness and umami taste than Nuoc mam. During fermentation, the histamine content increased in one or two of three fermentation tanks for each fish species. As a result, the histamine content of the final products was higher than that in Nuoc mam.

The quality of a new surimi-based product (healthy kamaboko) prepared from fish sauce, katstiobushi-kelp extract and yeast extract was compared with that of a commercial control product with chemical condiments, which is commonly eaten in Toyama district. Sensory evaluation revealed that the toughness, elasticity and agreeable fish flavor of the new product were superior to those of the control product, although the external appearance of the new product was judged inferior to that of the control. No difference was found in umami taste strength. Results indicated the following : 1. The values of breaking strength and breaking strain of the new product were slightly higher than those of the control. 2. The free amino acid content, particularly for glutamic acid and glycine, was lower in the new product than in the control. 3. The amount of inosine 5 '-monophosphate was lower in the new product than in the control, but adenosine 5 '-monophosphate and guanosine 5 '-monophosphate were higher in the new product than in the control. These nuclectides were thought to provide sufficient umami taste by synergistic taste effects with glutamic acid. Additionally, the new product contained volatile components found in fish sauce and did not contain the sorbic acid and 2-methylthiophene that was found in the control product. The new product could be stored for 40 days at 5 degrees C without any bacterial proliferation while the control product contained sorbic acid as a preservative.

Live spear squid and Japanese common squid were sedated in seawater containing 20 MM MgSO4 (Mg-SW) For sedation, seawater temperature was slowly reduced from 15 to 7 degrees C with ice bags. After sedation, live spear squid and Japanese common squid were individually accommodated in a highly gas impermeable plastic bag with 0.6 and 1.0 L of Mg-SW, respectively, and the bags were filled with oxygen and sealed tightly. They were transported at 5 degrees C taking about 27 h by truck. For comparison, samples quickly frozen in liquid nitrogen and instantly killed samples were also transported at -20 degrees C and 5 degrees C, respectively. Changes in various components were measured. Arginine phosphate and ATP contents decreased greatly by transportation in sedated Japanese common squid. There was no clear difference in sensory scores between sedated and chilled Japanese common squid. However, arginine phosphate and ATP contents did not decrease by transportation for spear squid. The mantle muscles of sedated spear squid were more transparent and had a more favorable texture than chilled or frozen samples.

We have purified a novel enzyme from eel white muscle which catalyzes the syntheses of imidazole dipeptides, such as carnosine (β-alanyl-l-histidine), anserine (β-alanyl-π-methyl-l-histidine), and balenine (ophidine; β-alanyl-τ-methyl-l-histidine), directly from their precursors. The enzyme was purified 1130-fold from eel muscle by a series of column chromatographies. Although eel muscle contains a large amount of carnosine and only trace amounts of anserine and balenine, the anserine synthesizing activity was by far the highest. From gel permeation chromatography, the molecular mass of the enzyme was calculated to be 275kDa. SDS-PAGE of the purified enzyme represented a band around 43kDa, suggesting that the native enzyme is a hexamer or heptamer. The optimal pH and temperature were around 9.5 and 60°C, respectively. Km values for β-alanine and π-methyl-l-histidine were 44 and 89mM, respectively. The enzyme was greatly activated by Zn2+ and inhibited by EDTA. The N-terminal amino acid sequence of 25 residues of the purified enzyme showed 52% amino acid identity to 38–62 residues of zebrafish haptoglobin precursor. The purified enzyme also exhibited hydrolytic activity against these imidazole dipeptides.

Aquatic crustaceans and some bivalve mollusks contain a large amount of free D-alanine (up to 100 mumol/g wet wt.) in their tissues. Under high salinity stress, crustaceans and bivalve mollusks largely accumulate D- and L-alanine irrespective of species examined, together with L-glutamine, L-proline, and glycine of which increases are species dependent. These data indicate that D-alanine is one of the major compatible osmolytes responsible for the intracellular isosmotic regulation in the tissues of crustaceans and bivalves. Alanine racemase has been proven to catalyze the interconversion of D- and L-alanine in these invertebrates. The enzyme has been isolated to homogeneity from the muscle of black tiger prawn Penaeus monodon and its cDNA has been cloned from the muscle and hepatopancreas of kuruma prawn Penaeus japonicus for the first time in eukaryotes other than yeast. Several fish species fed on crustaceans and mollusks contain D-amino acid and D-aspartate oxidases that catalyze the decomposition of D-amino acids. A cDNA of D-amino acid oxidase has been cloned from the hepatopancreas of omnivorous common carp Cyprinus carpio. During oral administration of free D-alanine to carp, the activity and mRNA of D-amino acid oxidase increased rapidly in hepatopancreas and the increases were highest in intestine followed by hepatopancreas and kidney. These data suggest that D-amino acid oxidase is inducible in carp and an important enzyme responsible for the efficient utilization of carbon skeleton of D-alanine in their feeds.