AKIRA SHINAGAWA

We analyzed the bacterial flora and chemical properties of the Japanese traditional anchovy product called saltedEtari, which is distributed in Nagasaki Prefecture in the western part of Japan. The pH and NaCl concentrations ranged from 6.4 to 6.6 and from 9.6% to 13.9%, respectively. The lactic acid and total free amino acid concentrations were determined to be between 122 and 344 mg/100 g of sample and between 2850 and 4783 mg/100 g of sample. The bacterial cell counts were determined to be 10(3)-10(8)and 10(4)-10(8)cells/g under aerobic and anaerobic conditions using a plate medium containing 10% NaCl, respectively. The isolates were classified into six groups on the basis of the results of PCR-restriction fragment length polymorphism (RFLP) analysis targeting the 16S rRNA gene. According to the results of 16S rRNA gene sequencing, the predominant PCR-RFLP group was identified as the halophilic lactic acid bacteriumTetragenococcus halophilus, and the remaining groups were identified as bacteria belonging to the generaChromohalobacter,Halomonas, andStaphylococcus.The results of culture-independent analysis, namely, terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the 16S rRNA gene, indicated thatT. halophilusis the bacterium commonly found in saltedEtari.

【目的】牛乳は良質のたんぱく質やカルシウムの給源として学校給食にも導入され、家庭科における食品群の学習にも明記されてきた。一方、近年は乳アレルギーなどの問題も指摘されている。そこで、本研究では小学校～中学校の家庭科の教科書において「牛乳・乳製品」がいつごろから、どのように取り扱われてきたのかを明らかにすることを目的とした。<br>【方法】公益財団法人教科書研究センター附属教科書図書館所蔵の学習指導要領に準拠した平成27年までに発刊・使用されてきた小学校家庭科、中学校技術・家庭科の教科書を分析対象とした。<br>【結果・考察】「牛乳・乳製品」は、小・中学校を通じて「三色食品群」、「6つの基礎食品」などに一貫して必ず記載されてきた。中学校家庭科においては、我が国の食生活における「牛乳・乳製品」の受容の変化に伴い、「食品の保存方法」や「食品添加物の使用例」、「特定保健用食品」、「アレルギー原因物質」、「食糧自給率の例」など多彩な記載となり、調理実習における使用例も増加した。しかしながら、牛乳の種類による栄養成分の比較や殺菌方法などの記載はなく、今後は、調理科学的な視点や消費者として適切な食品選択、消費生活と環境の視点なども必要であろう。

We analyzed the chemical constituents of Pacific oysters (Crassostrea gigas) collected in the Konagai district in NagasakiPrefecture and further evaluated the taste characteristics using a taste-sensing system. We compared the results with those obtainedfor oysters collected in Hiroshima Prefecture, which are most widely distributed oysters in Japan. We analyzed the taste components,including free amino acids, adenosine triphosphate (ATP) and related compounds, and glycogen contents, in the oysters by meansof spectroscopy and high-performance liquid chromatography (HPLC). The levels of serine, alanine, and β-alanine, which arefree amino acids that confer sweetness, were significantly higher in oysters cultured in Konagai than those cultured in Hiroshima.Glutamic acid levels were not significantly different between oysters from the two districts. Oysters in Konagai contained significantlylower levels of aspartic acid, which is associated with a slight umami taste, than those in Hiroshima. The levels of valine, methionine,isoleucine, leucine, and phenylalanine, which are associated with bitterness, were significantly lower in oysters from Konagai than inthose from Hiroshima, although the overall proportion of these amino acids among the total free amino acids was low. No significantdifference in total free amino acid levels and glycogen levels was observed between the oysters cultured in the two districts. Withregard to ATP and its related compounds, adenosine mono-phosphate (AMP) levels were shown to be significantly higher in oystersfrom Konagai than in those from Hiroshima. The results of the taste sensor analysis showed significantly higher values of umami andastringency, with significantly lower values of aftertaste of bitterness, in Konagai samples than in Hiroshima samples. The umamitaste, which is primarily derived from glutamic acid, is known to be enhanced by the synergic effect of inosine mono-phosphate (IMP)and AMP, which is structurally similar to IMP. Thus, the taste sensor used in this study might reflect the umami taste enhanced bythe synergic effects on the human tongue. These findings indicate that the oysters collected in Konagai might have a stronger sweet,astringent, and umami taste and a weaker aftertaste of bitterness than those from Hiroshima. The results of the taste sensor analysisdid not contradict with those of the chemical analysis. The use of a taste-sensing system to evaluate the taste of raw oysters may serveas a potential alternative to the conventional evaluation of heated oysters that are deemed more hygienic.

We compared the taste components of Pacific oysters (Crassostrea gigas) cultured using the single seed method (single seed oysters) off the coast of Konagai (Nagasaki Prefecture, Japan) that were cultured for different periods. Single seed oysters that had been farmed up to the first harvest year (first-year oysters), the procedure typical in this area, and those that had been kept for an additional summer and into the second harvest year (second-year oysters) were compared. We analyzed taste components including free amino acids, ATP and related compounds, and glycogen levels in the oysters that were obtained in the different harvest seasons. The experiments were repeated at 4 different times between February 2013 and February 2014 because of chemical constituent fluctuations under various environmental conditions. First, we used 2-way ANOVA to analyze all groups of the data collected in the experimental replicates and then we independently evaluated differences between the 2 groups, first- and second-year oysters, using t-tests. The second-year single seed oysters showed significantly higher values of whole body weights and soft-tissue weights than those of first-year oysters. However, the soft tissue ratios, meaning soft tissue per gram of the whole body weight including the shell, were significantly lower in second-year oysters than first-year oysters, which suggested that second-year oysters had less edible contents. Significantly higher glycine, alanine, and serine levels were observed in first-year oysters than in second-year oysters, indicating that first-year oysters might be more sweet-tasting. In contrast, no significant differences in AMP, IMP, and glycogen levels were observed between the 2 groups of single seed oysters. Moreover, in a taste evaluation test of the 2 oyster groups performed in January 2014, different culture periods had little or no effect on taste, except for their better shape and larger size.

The Pacific oyster was cultured off the coast of Konagai along Isahaya Bay, Nagasaki Prefecture using the conventional suspension method (CSM) and the single seed method (SSM). By using the SSM, oysters were said to have good appearance and taste better than those by CSM, although no comparison report was found on free amino acid constituents affecting the taste between the two different culture methods of CSM and SSM. We investigated the taste-associated free amino acids of the oysters obtained from the same district in December 2012 and March 2013 and cultured using either of the two methods. Besides, we made another comparison of the free amino acid constituents between the two collection periods of winter and spring. Consequently, we observed that the mean total levels of amino acids in oysters from both collection periods was higher in oysters cultured using the SSM than in those using the CSM. Especially in December 2012, we found that the mean total level of free amino acids was significantly higher in the SSM-cultured oysters by ca. 36% than in the CSM-cultured oysters. The level of glutamic acid, related to umami, was significantly higher in oysters cultured using the SSM than the CSM by ca. 70% and 81% in December 2012 and March 2013, respectively. At both collection periods, serine, glycine, alanine, and β-alanine, which were related to sweetness, showed higher levels in the oysters cultured using the SSM than the CSM. Especially in December 2012, the levels of threonine, serine, alanine, and β-alanine were significantly higher in the SSM-cultured oysters than in the CSM-cultured oysters by ca. 139%, 167%, 78% and 81%, respectively. In March 2013, the levels of glycine and β-alanine were significantly higher by ca. 26% and 32%, respectively. These findings exhibited the SSM-cultured oyster had stronger tastes in umami and sweetness than the CSM-cultured oyster. On the other hand, oysters obtained in March showed higher level of total free amino acid and contained more free amino acids related to umami and sweet tastes than those in December during the same harvest season in Konagai. Accordingly, the results correspond to the feedback of consumers that the oyster cultured using SSM tastes better than that using CSM, and further, that the spring oyster tastes better and richer than the winter oyster, which might be due to the abundance of amino acids that impart the sweet and umami tastes.

An experiment for preventing mass mortality of cultured short-neck clams due to hypoxic and anoxic conditions was conducted at the Kama tidal flat in northern Isahaya Bay, Nagasaki, in the middle of August 2008. Before the experiment, the starting date (11 August 2008) of hypoxia formation in the experimental site was predicted by reviewing historical data including dissolved oxygen (DO) in the bottom water, and other environmental conditions such as tidal range, wind speed and red tide incidence. A hypoxia prevention system that was composed of barriers against the hypoxic water intrusion and a micro-bubble generator for aeration was set in the experimental area (20m×20m), and time changes in DO concentration in the bottom water were monitored and compared with the time changes in the control area. The differences in survival rates of short-neck clams and contents of acid volatile sulfides in the sediment after the experiment were also compared between the experimental and control areas. Despite some troubles during the experiment it was revealed that the system mentioned above was effective for preventing hypoxia and for avoiding mass mortality of cultured short-neck clams at the Kama tidal flat.

The present study aimed at examining a possible role of low salinity in causing mass mortalities of the Manila clam Ruditapes philippinarum in culture areas. Laboratory experiments were conducted on the effect of low salinity on survival, hemolymph osmolality and tissue water content at 25 &deg;C. Also, seawater salinity was monitored from June 2003 through October 2005 in a culture area in Isahaya Bay, Kyushu, Japan.<BR>No clam died during 96 h exposure to 15 psu, while substantial mortalities occurred below 10 psu. When clams were exposed to salinities below 20 psu, hemolymph osmolality declined gradually and became iso-osmotic with the external media after 48 h. In contrast, forced opening of valves resulted in a rapid reduction of hemolymph osmolality. Thus, clams appear to maintain hemolymph osmolality by closing the valves when exposed to low ambient salinities. Tissue water content increased from 78.3-80.6% to 81.4-88.3% during 72-96 h exposure to 22.5-10 psu. Salinity remained over 25 psu when mass mortalities occurred in 2003 and 2004 in the culture area. These results led us to conclude that low salinity was not a major cause of the mass mortality in the two years.

A series of laboratory experiments was conducted on four bivalves, Corbicula japonica, from Lake Shinji, and Anadara subcrenata, Ruditapes philippinarum, and Musculista senhousia from Lake Nakaumi, to evaluate their tolerance to water temperature, salinity, anoxia, and hydrogen sulfide. All species survived well at 30&deg;C, whereas only C. japonica tolerated at 34&deg;C. At the range of salinity from Opsu to 32 psu, C.japonica survived at salinities&le;20 psu, which is the common salinity range in Lake Shinji, while the three species from Lake Nakaumi survived at>10 psu. The order of tolerance to anoxia was C. japonica>A.subcrenata>R, philippinarum&ge;M.senhousia, with C. japonica's tolerance being remarkably strong. Tolerance to hydrogen sulfide showed a similar trend as to anoxia.

Sulfide tolerance in the bivalve Corbicula japonica was determined based on laboratory experiments using individuals collected from Lake Shinji, an estuarine lagoon in Japan. The median tolerance time (LT₅₀) of individuals immersed in a 50 mg/l sulfide solution at 18&deg;C was 23 days, while the LT₅₀in 1.0mg/l sulfide solution at 28&deg;C was 21 days. At any given water temperature, the tolerance time decreased as sulfide concentration increased. The sulfide tolerance seemed similar between juvenile and adult C. japonica. C. japonica has a much higher sulfide tolerance than other macrobenthos that was reported at sulfide tolerance, which may reflect the adaptation of C. japonicato brackish environments where salinity stratification commonly causes anoxia and sulfide formation at the bottom.

The tolerance ofCorbicula japonicato low concentrations of dissolved oxygen (DO) was examined in laboratory experiments using individuals collected from Lake Shinji, Japan. Tolerance varied with water temperature. In anoxic conditions, C. japonicasurvived for more than 17 days at 10&deg;C and 20&deg;C, while all individuals at 30&deg;C died with in 10 days. The tolerance to anoxia seemed no difference between juveniles and adults. Oxygen consumption of C. japonicawas higher at 30&deg;C than 20&deg;C. Even after conditions became anoxic due to respiration, all individuals maintained at 20&deg;C survived more than 50 days. Individuals maintained at 30&deg;C, however, died with in 8 days. The minimum oxygen requirement at 28&deg;C with in a 30-day period was between 1.0 mg/l and 1.5 mg/l.

The brackish water bivalve, Corbicula japonica was found to tolerate short-term exposure (24h) to waters temperatures ranging from 0 to 35&deg;C. During long-term exposure (30 days) to high temperatures, the upper lethal limit of C. japonica was about 32&deg;C.<BR>At 30&deg;C, individuals first acclimated to 25&deg;C survived longer than those acclimated to 10&deg;C or 20&deg;C.<BR>The temperature tolerance of adults was similar to that of juveniles.

The brackish water bivalve, Corbicula japonica, was found to tolerate exposure to salinities ranging from 0 to 35 psu at 15&deg;C. Gradual exposure to different salinities resulted in equilibration of the body fluid with the external medium, indicating that this species is a euryhaline osmoconformer.<BR>Salinities ranging from 1.5 to 22 psu were not lethal for C. japonica acclimated to either 10&deg;C or 25&deg;C. At a high salinity of 32 psu, bivalves survived a longer period of time after acclimation to low temperature as compared to high temperature. In addition, adults were more tolerant of concentrated media than juveniles.

Water extracts from the brackishwater clam (Corbicula japonica) were found to be lethal to mice upon i.v. injection. Muscular tissues (foot muscle, adductor muscle, mantle muscle, mantle and siphon) were all toxic while gill and viscera (mid-gut gland, testis and ovary) were nontoxic; the highest toxicity was observed with foot muscle. The toxin was judged to be a thermolabile, basic protein with a mol. wt of about 13,000.

The Japanese spiny lobster Panulirus japonicus tolerated exposure for 24 h to osmolarities ranging from 760 to 1240 mOsm/l at 20 degrees C. Acute exposure to hypo- and hyperosmotic stress resulted in hyper- and hypoosmotic regulation of hemolymph, respectively, indicating that this species is a stenohaline osmoregulator. Free amino acids and betaines in the muscle, midgut gland, ovary, and gills were measured after the lobsters were exposed to hypo- and hyperosmotic stress for 24 h. Results obtained showed that glycine, glycine betaine and homarine are important osmoeffectors. The decrease in glycine under hypoosmotic condition and the increase in glycine, glycine betaine, and homarine under hyperosmotic conditions were noticeable in the tissues studied: suggesting that these nitrogenous components play a significant role in intracellular osmoregulation. Changes in free amino acids and betaines in the hemolymph during the exposure of lobsters to different osmolarities were opposite to those in the above-mentioned tissues.

Six species of marine sponges belonging to the class Demospongiae were analyzed for free amino acids and betaines, and changes in their concentrations in two species of intertidal sponges exposed to hypo-and hyperosmotic stresses were investigated.<br> The concentration profile of individual free amino acids and betaines in those sponges was characterized by a high level of glycine. Especially, two intertidal species, Halichondria okadai and H. japonica, were very rich in this amino acid. The amount of cyclic betaines such as homarine and trigonelline more or less exceeded that of &omega;-betaines such as glycinebetaine, &beta;-alaninebetaine, and &upsih;-butyrobetaine. The trigonelline level in H. japonica was particularly high.<br> Although most of free amino acids and betaines in the two intertidal sponges fluctuated in response to salinity changes, the major portion of the total change in those components was at-tributable to glycine. Under high salinity conditions, trigonelline in H. japonica also played a significant role in intracellular osmoregulation.

A method is described for the HPLC analysis of glycinebetaine, &beta;-alaninebetaine. and &gamma;-butyrobetaine in aquatic animals. The betaines in marine animals were extracted with 80% methanol and converted to p-bromophenacyl esters by treatment with p-bromophenacyl bromide in the presence of 18-crown-6 and KH₂PO₄. The esters of betaines having UV absorption maximum at 262nm were separated on Shimpack FLC-CN using 10mM KH₂PO₄ containing 2% acetonitrile, pH 2.10 as a mobile phase, The recoveries of betaines added to the muscle of marine animals were in the range from 93 to 104%.

Arsenic contents and its chemical forms in shellfishes were examined in connection with their feeding habits. The shellfishes used were 5 species of plankton-feeding bivalves, 3 species of herbivorous gastropods, and 7 species of carnivorous gastropods.<br> The arsenic content (wet-weight basis) in soft tissue or muscle of bivalves and herbivorous gastropods was below 10 ppm in most specimens. In contrast, the arsenic content in muscle of carnivorous gastropods was significantly high; the mean values ranged from 16.8 ppm (Neptunea arthritica) to 67.9 ppm (Reishia bronni). The arsenic content in mid-gut gland was higher than that in the soft tissue or muscle in the cases of bivalves and herbivorous gastropods but lower in the case of carnivorous gastropods. Regardless of the species and tissues, the ratio of water-soluble organic arsenic to the total arsenic in soft tissue or muscle was somewhat lower in carnivorous gastropods than in the other two groups. These results suggested that carnivorous gastropods can be distinguished from bivalves and herbivorous gastropods with respects to arsenic contents and its chemical forms.

Selective determination of inorganic arsenic (III), (V) and organic arsenic by the solvent extraction method was done with 20 species of marine organisms, including fish, ascidian, holo-thurian, sea urchin, crustacean, shellfish, cephalopod, polychaete, and seweed. In all species, inorganic arsenic, which is more highly toxic than organic arsenic, comprised only a small part (0-7%) of the total arsenic; the one exception was in a seaweed Hizikia fusiforme, in which inorganic arsenic accounted for 60% of the total arsenic. Furthermore, when arsenic in water-soluble and fat-soluble fractions was determined, the ratio of water-soluble arsenic reached 58-97% of the total arsenic. Especially in the cephalopod (3 species) and fish (6 species), more than 84% of the total arsenic was found in the water-soluble fraction. These results strongly suggested that most arsenicals in marine organisms are present in water-soluble organic forms.

The major arsenic compound in the muscle of an octopus Paroctopus dofleini was purified by column chromatographies on ion-exchangers and Bio-Gel P-2. The arsenic content of the purified compound was estimated to be 44%; this is close to that (41%) of arsenobetaine. In thin layer chromatography and electrophoresis, the purified arsenic compound gave a single spot positive to the Dragendorff reagent at the same Rf and mobility as arsenobetaine. No characteristic UV absorption was observed with the purified compound. The ¹H-NMR spectrum of the purified compound showed two significant signals at &delta; 1.88 (s, 9H) and 3.31 (s, 2H), which corresponded to those at &delta; 1.87 (s, (CH₃)₃ AS) and 3.28 (s, CH₂) reported for arsenobetanine. No difference was found between the IR spectra of the purified compound and arsenobetaine. Judging from these results, the major arsenic compound in the muscle of P. dofleini was concluded to be arsenobetaine (CH₃)₃AS⁺CH₂COO-. Moreover, arsenobetaine accounted for more than 90% of the total arsenic in P. dofleini.