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Squid sashimi is a popular food in Japan, where raw seafood is an intrinsic component of gastronomic culture. High-quality squid sashimi requires very fresh squid, but loss of freshness occurs more quickly with squid than with other fish and shellfish. High freshness sashimi is prepared from live squids to avoid use of the low freshness squid. Furthermore, short-term rearing and shipping of live squids performed to provide a stable supply of high freshness sashimi. It is a problem that squid is one of the most difficult animal to keep alive in tank or during shipping, and squid is visibly weaker during captive or shipping even with careful management of seawater quality and temperature. Yoshioka has reported that sashimi prepared from weakened squid had a lower free amino acid content, poorer taste and lower nutritional value. The report demonstrates that maintaining squid in a health condition enables the provision of high-quality squid as a sashimi material. In other words, a physical check-up way of squid will be good quality evaluation methods of squid.
Here, we suggest new quality evaluation method of a live squid as a sashimi material. We demonstrate hemolymph properties is useful for evaluating the physiological condition of squid. Furthermore, we focus on hemolymph hemocyanin as oxygen transporter, and discuss its molecule structure and the effect of protein folding on its oxygen-binding function.

Today, a lot of efforts are under way to reduce greenhouse gasses all over the world and fisheries cannot be an exception. The authors have been investigating how to reduce greenhouse gasses during the distribution of fishery products by utilizing LCA (life cycle assessment). It is a method for quantitatively and objectively evaluating environmental load throughout all stages from farm to table related to products.
For example, conservation of food in ultra low temperature such as -50 C is generally considered to consume more energy than chilled preservation.
It is true that making ultra low temperature requires more energy than chilled temperature but ultra low temperature can drastically extend the shelf life of food. The food of long shelf life can be transported by a ship that takes a long time but has less greenhouse gas emission. As a result, ultra low temperature possibly can reduce the environmental load throughout the entire food distribution system. Food conservation under ultra low temperature has been considered to have another defect, namely, food quality deterioration due to freezing. However, owing to currently developed technology, freezing damage have been much suppressed and there can be little difference between chilled food and frozen food.
In this presentation, I attempted to prove the above contents by using Japanese common squid (Todarodes Pacificus). I measured the quality of squids with the change of storage time under three conditions of frozen, refrigerated and active and evaluated the optimal transport form of squid with the environmental load of each condition obtained by LCA.

Introduction. Olive leaf (OL) is a good source of antioxidant properties. Antioxidant inclusion in farmed animal feed is a modern method used for improving the final product. We investigated the effect of OL powder supplemented to fish feed on biochemical properties of red sea bream (Pagrus major) muscle.
Methods. OL were dried, powdered and steamed before adding into feed. Red sea bream (63.10+8.74 g) were acclimated prior to feeding experimental diet. The sample fish group (n=3) was fed a commercial diet containing 5%(w/w) OL powder. The control fish group (n=3) was fed a commercial diet. Each group were fed 3 times for a day and raised for 25 days in a different aquarium under controlled condition. Dorsal muscle of fish was used in this study. Sample for antioxidant activity was prepared using 80% methanol. Acid-soluble collagen was extracted using 0.5 M acetic acid. Myofibril was isolated using 0.5 M NaCl-phosphate buffer (pH8.0) and molecular weight patterns were confirmed by SDS-PAGE. Sample for collagenase activity was prepared using 5 mM CaCl₂-Tris-HCl (pH8.0). Antioxidant activity was determined using DPPH assays. Acid-soluble collagen and myofibrils concentration were determined by Lowry method. Collagenase activity was evaluated by MOCAc-Pro-Leu-Gly-Leu-A₂pr(DNP)-Ala-Arg-NH₂ as a substrate.
Results. Antioxidant activity was not significantly different between sample and control group, but sample group tended to be a little bit higher. Acid-soluble collagen content of sample muscle was significantly higher than control while hydroxyproline was not difference between the two groups. There was no difference in SDS-PAGE pattern of myofibril but protein content was lower in sample than in control. Collagenase activity of sample looks lower than control.
Conclusion. Feeding olive leaf to red sea bream resulted in the increase in acid-soluble collagen. As collagen is responsible for meat texture, feeding OL may improve meat quality of the fish.

Fish is an important nutritive component of the human diet as it provides protein, energy and many other functional components and thus helps sustain health. Concerning fish quality, fish deteriorate more quickly post-mortem than meat derived from terrestrial animals, quick and simple methods are required to confirm fish freshness and quality. Concerning fish safety, about more than 50% of food intoxication caused by fish is occurred by histamine. Histamine is well known as the substance causing allergy-like food poisoning after ingestion of Scombroid fish that are not chilled adequately between harvest and consumption. The authors propose that fish should be confirmed the freshness and histamine contents to take in a good and safe fish, and to produce safety processing food.
Simultaneous determination freshness and histamine have done as followed. First step, freshness of fish flesh is measured by Freshness Checker (QS-SOLUTION), which is based on the paper electrophoresis method. After taking freshness data of fish on the electrophoresis paper by UV detection, histamine is developed by spraying histamine developing reagents on the same paper. By these quick and simple procedure, it can be avoided sea food intoxication and fish can be ingested and used for seafood processing in good and safely.

Sea cucumber Stichopus japonicus is one of the most important holothurian species in coastal fisheries. The farming of sea cucumber has rapidly developed in many Asian countries in recent decades. However, sea cucumber is susceptible to change of environmental factors and easily subjected to autolysis, which consequently leads to severe quality deterioration during handling and storage. Therefore, the objective of present study is to investigate the autolytic degradation of noncollagenous and muscular proteins of sea cucumber body wall. The effect of gelatinase from intestine on degradation of collagen from body wall was also determined. The results showed that the protein fractions removed from integument by extraction with SDS and urea were mainly noncollagenous proteins, which were identified as major yolk protein (MYP) and actin from holothurian species by LC-MS/MS with significant scores. Myosin heavy chains (MHCs), paramyosin and actin were the major proteins in S. japonicus muscle. The content of TCA-soluble oligopeptide was significantly increased in autolyzed sea cucumber integument and intestinal gelatinase-treated pepsin-soluble collagen (PSC).The autolytic degradation of MYP, MHCs and actin were partially inhibited by cysteine protease inhibitors, including Trans-epoxysuccinyl-L-leucyl-amido (4-guanidino) butane (E-64), iodoacetic acid and antipain. The activity of intestinal gelatinase was effectively inhibited by phenylmethanesulfonyl fluoride (PMSF), but not 1, 10-phenanthroline and E-64. PMSF showed partially inhibitory effects on intestinal gelatinase-induced degradation of PSC α chain. These results suggest that the cysteine and serine proteases play important roles in the autolysis of S. japonicus body wall.

Apoptosis induction is involved in UVA-induced autolysis in sea cucumber (Stichopus japonicus). Cellular Ca²⁺ signal has been implicated in the regulation of apoptosis. In this study, to further characterize the mechanism of apoptosis induction in UVA-induced autolysis of S. japonicas, the Ca²⁺ signals regulated in the progression of apoptosis was investigated. Fresh S. japonicus were exposed to UVA radiation for 30 min at the intensity of 15 W/m² and held at room temperature for the different autolysis development (0, 1, 2, 3 and 4 h). The coelomocytes from coelomic fluid of S. japonicus were subsequently prepared. Laser Scanning Confocal Microscopy (LSCM) and Flow Cytometry (FCM) were used to detect intracellular Ca²⁺ changes of coelomocytes during the development of S. japonicus autolysis. Apoptotic rates of coelomocytes and endoplasmic reticulum (ER) stress-related proteins (caspase-12 and CHOP) expression were also evaluated. The intracellular calcium concentration ([Ca²⁺]_i) in coelomocytes was significantly increased up to 2 h after UVA irradiation, and apoptotic rates of coelomocytes after treatment was also increased for 3 h. Pre-treatment with BAPTA sodium salt, a calcium chelator, significantly attenuated [Ca²⁺]_i elevation and apoptosis. Additionally, the expression levels of caspase-12 and CHOP markedly up-regulated after treatment. These results demonstrated that UVA-induced autolysis in S. japonicas at least partially involves the intracellular calcium overload-regulative apoptosis induction. The ER stress-associated pathway possibly operated in this apoptosis.

Frozen fish consumed as ‘Sushi’ and ‘Sashimi’ is required high freshness quality due to hygienic and good properties of texture, color, flavor, and taste. However, the traders can’t know the exact initial condition of frozen food instantly without thawing them. Moreover, the chemical methods for tracking the changes in the fish body are destructive and time-consuming. So, the present study was aimed to propose a noninvasive method using the fluorescence fingerprints (FFs) for monitoring the fish freshness frozen at the early stage after death. The study was especially focused on the changes of nicotinamide adenine dinucleotides (NAD and NADH) which are deeply involved in the progress of glycolysis and expected to be an indicator of the state of rigor mortis.
Fifty-six horse mackerel (Trachurus japonicus) were sacrificed and kept on ice for 14 different periods (0-48 hour) to prepare samples with different freshness, then filleted and frozen. FFs of the frozen samples were then acquired using fluorescence spectrophotometer (F-7000) aided with an external fiber optic probe at -30°C. Then, the NAD and NADH content of the same frozen samples were determined using relevant biochemical methods. The FFs were masked and partial least square (PLS) regression models were built by comparing with the chemical data.
By chemical analyses, it was confirmed that the NAD content decreased and NADH content increased gradually during the post-mortem changes. The PLS models of NAD and NADH content were observed with coefficient of determination (R²) of 0.71 and 0.88, respectively with 8 latent factors (LF). These fluorescent intrinsic compounds revealed a good correlation with the FF data. Thus, FFs coupled with the chemometrics could be a rapid approach for monitoring the post-mortem changes in frozen fish muscle nondestructively.

Recently, studies have investigated the production of frozen mackerel with high freshness for sashimi production in Japan. pH of mackerel meat with high freshness tends to decrease markedly after thawing due to the progression of glycolysis. As it is assumed that pH is correlated with the quality deterioration of thawed meat, the development of a suitable thawing method for frozen mackerel without pH decrease is required to improve the quality of thawed meat. Temperature control before thawing (TCBT), which is the method of storage at -20 to -2°C for several days before thawing, has been proposed as one of the effective methods to prevent a decrease in the pH of frozen fish meat. Although it is presumed that glycolysis is suppressed by using TCBT, the detailed mechanisms of TCBT have yet to be clarified. The objective of this study was accordingly to elucidate the biochemical changes in frozen spotted mackerel meat with high freshness during TCBT.
Frozen spotted mackerel caught in Kamaishi Bay and cultivated in a fish tank were used as samples. They were sacrificed instantly and blood was removed using the “Kubiore” procedure. The fish were subsequently frozen and maintained at -60°C until use. After TCBT at -10°C, the frozen meat was thawed at 5°C for 18 h. pH, ATP, NAD, and lactic acid in both frozen and thawed meats were measured.
In frozen meat without TCBT, pH decreased to a value of 6.0 after thawing. In contrast, in frozen meat subjected to TCBT at -10°C over 3 days, the pH was maintained at approximately 6.4 after thawing. Little lactic acid in the meat was produced during the thawing process and NAD in frozen meat was decreased during TCBT. It thus appears that glycolysis in the meat is suppressed during the thawing process as a consequence of TCBT.