Observation of the feeding behaviour of reared Japanese eel Anguilla japonica leptocephali fed picocyanobacteria Synechococcus spp.

The authors observed the feeding behaviour of artificially reared Japanese eel Anguilla japonica leptocephali, 7.5-19 mm total length (10-61 days post-hatch), fed Synechococcus sp., which is considered a potential food source of anguilliform larvae. Three strains of Synechococcus sp. (NIES-972, 976 and 979) were tested as the food material. Larvae across the entire length range could effectively ingest a suspension of pico-sized cyanobacteria (1-3 µm in diameter). Video observations of the mid-hindgut of larvae under an epifluorescence microscope confirmed that the movement of microvilli of the intestinal epithelium allowed the cell particles to circulate in the mid-hindgut, before becoming solidified in the anal region. Significant differences in food intake were observed between larvae fed two strains of Synechococcus (NIES-972 and 976), and among different cell densities, which suggests feeding selectivity and density dependence. Comparisons of feeding behaviour under the light group (9L:15D) and the dark group (24D) showed significantly higher food intake (measured as an index of intestinal fullness) in the light group, although substantial and continuous ingestion was observed in the dark group, indicating continuous feeding by swallowing sea water. The authors hypothesise that the feeding ecology of anguilliform leptocephali is based on a survival strategy whereby the larvae do not compete with various higher-trophic-level fishes for food in an oligotrophic environment.

Alterations of stool metabolome, phenome, and microbiome of the marine fish, red sea bream, Pagrus major, following exposure to phenanthrene: A non-invasive approach for exposure assessment.

The present study aimed to assess the impact of phenanthrene (Phe) on fish health by addressing the alteration of fecal characteristics, in lieu of collecting biomarkers that often involves injurious or even fatal sampling of organisms. The marine fish red sea bream, Pagrus major, was exposed to Phe at a concentration of 18 µg/L for 16 days followed by depuration for 13 days. We collected feces from Phe-exposed or control (Phe-free) fish and then analyzed the fecal metabolite profile (metabolome), carbon utilization of microbiota (phenome), and bacterial 16s rRNA gene sequence (microbiome). Along with the increase in physiological stress markers (SOD and EROD) in serum and liver, we noted the possible role of intestine as a Phe reservoir. Furthermore, abnormal fecal appearance (green coloration) and remarkable changes in fecal characteristics were observed. These changes include alterations of cholesterol and putrescine metabolism and the enhanced utilization of putrescine as a carbon source. Phe also altered the microbial community, with an increase in Phe-degrading bacteria such as Pseudomonas. Interestingly, these enteric impairments were ameliorated by depuration. Taken together, our findings suggest that these alterations in feces were associated with adaptive responses to environmentally relevant Phe exposure scenarios, and that stool samples are potential candidates for exposure assessment in fish.

Development of koji by culturing Aspergillus oryzae on nori (Pyropia yezoensis).

Koji is a traditional fermentation culture medium, based on Aspergillus oryzae, which is commonly used in the manufacture process of Japanese fermented products such as soy sauce, miso, and sake, and promote enzymatic degradation. Koji is usually prepared by culturing a mold on cereals such as wheat flour, soybean, or rice, but that cultured on seaweeds has not been developed yet. This study prepared the koji by culturing A. oryzae on seaweed nori (dried piece of Pyropia yezoensis), and, then, characterized on this nori koji. The nori koji contained 0.85 µg N-acetylglucosamine, estimated as 6.1 µg mold cells, per gram dry matter and showed various kind of enzymatic activities in glycosidase, protease, and phosphatase as well as traditional soy sauce koji and rice koji. The suitability of these characteristics for degradation of nori was tested on nori sauce culture with and without the addition of the nori koji. After 167 days of culture, the fermentation tank with the nori koji showed over 74% recovery of supernatant while that without the nori koji had less than 57% recovery. The supernatant of culture mashes contained more than two times larger quantity of total nitrogen compounds in nori koji test group against control group. The present study prepared koji on seaweed nori for the first time and demonstrated its advantages to shorten the culture period and increase taste value in nori sauce manufacture. Development of seaweed koji enables a method to prepare cereal allergen free fermented sauces from seaweeds.

Sample treatment optimization for fish stool metabolomics.

Gut microbiota play an essential role in an organism's health. The fecal metabolite profiling content reflects these microbiota-mediated physiological changes in various organisms, including fish. Therefore, metabolomics analysis of fish feces should provide insight into the dynamics linking physiology and gut microbiota. However, metabolites are often unstable in aquatic environments, making fecal metabolites difficult to examine in fish. In this study, a novel method using gas chromatography-mass spectrometry (GC-MS) was developed and optimized for the preparation of metabolomics samples from the feces of the marine fish, red sea bream (Pagrus major). The preparation methodology was optimized, focusing on rinsing frequency and rinsing solvent. Feces (collected within 4 h of excretion) were rinsed three times with sterilized 2.5% NaCl solution or 3.0% artificial seawater (ASW). Among the 86 metabolites identified in the NaCl-rinsed samples, 57 showed superior recovery to that in ASW-rinsed samples, indicating that NaCl is a better rinsing solvent, particularly for amino acids, organic acids, and fatty acids. To evaluate rinsing frequency, fecal samples were rinsed with NaCl solution 0, 1, 3, or 5 times. The results indicate that three or more rinses enabled robust and stable detection of metabolites encapsulated within the solid fecal residue. Furthermore, these data suggest that rinsing is unnecessary when studying sugars, amino acids, and sterols, again highlighting the need for appropriate rinsing solvent and frequency. This study provides further insight into the use of fecal samples to evaluate and promote fish health during farming and supports the application of this and similar analyses to study the effects of environmental fluctuations and/or contamination.

Characterization of fermented seaweed sauce prepared from nori (Pyropia yezoensis).

High-salt content seaweed sauces were prepared for the first time using nori (Pyropia yezoensis) by fermentation and characterized. Components and taste of the two nori sauces (NSs) prepared separately were compared with those of soy and fish sauces. The NSs were rich in total nitrogen compounds (1.5 g N/100 ml on average) and potassium (880 mg/100 g), and had a unique free amino acid composition (e.g., taurine 617 mg/100 g), explaining their unique taste as evaluated by a taste sensing system. As for their food function, inhibitory activity of angiotensin-converting enzyme was observed. As for their food safety, arsenic was detected at a 0.8 mg/100 g level in total, but inorganic arsenic was not detected (<0.05 mg/100 g) and not regarded as a problem. Allergy-causing substances contained in wheat, soy beans, and crustaceans were not detected (<0.1 mg/100 g) with NSs. These results suggest that the nori sauce has a high potential as a novel nutritional source for humans.

Production of 16.5% v/v ethanol from seagrass seeds.

Ethanol fermentation on seeds of seagrass Zostera marina was studied. The seeds were collected from the annual plant colony of Z. marina at Hinase Bay, Okayama. The seeds contained 83.5% carbohydrates including 48.1% crude starch on a dry weight basis, which is comparable to cereals such as wheat flour and corns. The seeds were saccharified with glucoamylase (50°C, 96 h) and 103.4 g/l concentration of glucose juice was obtained. The glucose juice was further fermented (23°C-35°C, 15 days) with Saccharomyces cerevisiae strains NBRC10217(T) and Kyokai 7-go, and ethanol was obtained at a 65.0 g/l (82.3 ml/l) level by monographic double-fermentation and at a 130.4 g/l (165.1 ml/l) level by parallel double-fermentation. Fermented products of seagrass seeds containing such a high ethanol concentration as the present study have potential to be utilized not only for biofuel but also for foods and beverages in the future. Culturing of seagrass seeds as a crop may enable development of a new marine fermentation industry.

Structural features of N-glycans of seaweed glycoproteins: predominant occurrence of high-mannose type N-glycans in marine plants.

We analyzed structural features of N-glycans linked to glycoproteins expressed in various seaweeds to identify new sources of biologically-important N-glycans or N-glycopeptides. Structural analysis of the N-glycans of glycopeptides prepared from pepsin digests of 15 species of seaweed revealed that only high-mannose type N-glycans occur in seaweed glycoproteins, and the Man9GlcNAc2 structure predominates in Sargassum fulvellum and Zostera marina, while no typical plant complex type N-glycans bearing ß1-2 xylosyl and α1-3 fucosyl residues present in either algae or seagrass. These results indicate that seaweeds lack the activities of several of the glycosyltransferases required for the biosynthesis of the complex type N-glycans found in terrestrial plants, and that the context of N-glycan processing in seaweeds is different from that in terrestrial plant cells.

Analysis of extracellular alginate lyase and its gene from a marine bacterial strain, Pseudoalteromonas atlantica AR06.

Pseudoalteromonas atlantica AR06 is a marine bacterial strain that can utilize alginate as a sole source of carbon and energy. The extracellular protein fraction prepared from the AR06 cultivation media exhibited alginate lyase activity to depolymerize the alginate molecules having homopolymeric and heteropolymeric forms of mannuronate and guluronate so as to mainly convert into the dimer to tetramer. A DNA fragment encoding a portion of alginate lyase was amplified from AR06 genomic DNA by PCR using a set of degenerated primers, and then the whole alginate lyase gene, named alyA, and its flanking regions were obtained from a cosmid library of AR06 genomic DNA. The alyA mutant of AR06 showed (1) the loss of alginate depolymerization activity on alginate agar plate and (2) significant growth defects in alginate minimal medium; these defects were complemented by the introduction of the alyA gene. Furthermore, zymography and biochemical analyses revealed that three extracellular protein bands of AR06 had alginate lyase activities and that all three protein bands were derived from the nascent alyA gene product. These results clearly indicated that the alyA gene greatly contributes to the assimilation of alginate in AR06. The transcription of the alyA gene was induced by the presence of alginate in minimal medium, but its obvious induction was not observed in rich medium even in the presence of alginate.

Fish protein stimulated the fibrinolysis in rats.

OBJECTIVE: We hypothesized that fish protein affects blood coagulation and/or fibrinolysis, and compared the activity and amounts of factors involved in blood coagulation and fibrinolysis in rats fed the fish protein, which was treated to remove water-soluble and ethanol-soluble elements, from sardine (sardine protein). METHODS: In the first experiment, rats were fed for 21 days an AIN-93G-based control diet, and diets in which the casein of the control diet was exchanged for sardine protein at 5, 10 and 20% levels. In the second experiment, rats were fed an AIN-93G control diet and diets containing 5% fish oil, 10% sardine protein or both (5% fish oil + 10% sardine protein) for 21 days. At the end of the experiments, blood coagulation time, hemostatic parameters and fibrinolysis parameters were measured. RESULTS: The activated partial thromboplastin time (APTT), which is an assay for blood coagulation time in the intrinsic blood coagulation pathway, of rats fed the 20% sardine protein diet was significantly prolonged compared to that of rats fed the control diet. The prolonged APTT by dietary sardine protein was due to a significant decrease of the activities of plasma blood coagulation factors VIII, IX, XI and XII. On the other hand, dietary sardine protein significantly increased the activity of tissue-type plasminogen activator, and the amount of plasma plasmin-alpha(2)-plasmin inhibitor complex, which are markers of activated plasmin. Moreover, we observed that the 20% sardine protein diet increased the amount of plasma D-dimer, which is a degraded product of the fibrin polymer by plasmin. In the second experiment, the APTT and PT of rats fed the F diet were prolonged compared to those of rats fed the control diet, however the concentration and amount of fibrinolytic parameters in the plasma were almost the same as those of rats fed the control diet. In contrast, the F+S diet not only prolonged APTT and PT, but also increased the concentration and amount of fibrinolytic parameters in plasma. CONCLUSIONS: We consider that the beneficial effects to health and amelioration of cardiovascular and cerebrovascular diseases by fish consumption are caused by a combination of the suppressing effect on blood coagulation of n-3 polyunsaturated fatty acids and the promoting effect on fibrinolysis of fish protein.

Blood coagulation and fibrinolysis of rats fed fish oil: reduced coagulation factors especially involved in intrinsic pathway and increased activity of plasminogen activator inhibitor.

Differences in the coagulation and fibrinolytic system of rats fed a fish oil based diet (fish oil diet) and fed a soybean oil based diet (control diet) were determined. Concentrations of plasma lipids were depressed in rats fed the fish oil diet. Prothrombin time (PT) and activated partial thromboplastin time (APTT) of rats fed the fish oil diet were longer than for the rats fed the control diet. Fish oil intake lowered the activities of most of the blood coagulation factors, and strongly depressed the factors involved in the intrinsic pathway. Fish oil also affected the fibrinolysis of rats. Plasminogen activator inhibitor (PAI) activity was elevated in rats fed the fish oil diet. In this study, both blood coagulation and fibrinolysis were down-regulated by feeding the fish oil diet.

Dietary fish oil and Undaria pinnatifida (wakame) synergistically decrease rat serum and liver triacylglycerol.

Japanese eating habits are characterized by the consumption of various food materials such as cereals, vegetables, fish, shellfish, marine algae and meat. Therefore, properties of functional substances in food materials may be enhanced or lessened by the combination of various food materials. In the present study, we examined how the combination of wakame and fish containing polyunsaturated fatty acids, which are typical Japanese food materials, affected rat lipid metabolism. Rats were fed one of four diets [control diet (C), AIN-76 diet with 5 g/100 g rapeseed oil; wakame diet (W) containing 19.1 g/100 g Undaria pinnatifida (wakame) dried powder in the C diet; fish oil diet (FO), AIN-76 diet with 4.1 g/100 g fish oil; wakame-fish oil diet (W + FO), the FO diet containing 19.1 g/100 g dried wakame powder] for 4 wk. We measured the concentration of lipids in serum and liver and hepatic activities of enzymes involved in fatty acid metabolism. The W diet, FO diet and W + FO diet significantly reduced the concentration of triacylglycerols in the serum and liver compared with the C diet. This decrease in the concentration of hepatic triacylglycerol was greatest in rats fed the W + FO diet. The activity of glucose-6-phosphate dehydrogenase, which is involved in fatty acid synthesis in the liver, of rats fed the W, FO and W + FO diets was lower than that in rats fed the C diet. However, the activities of malic enzyme and fatty acid synthetase did not differ among the four groups. In contrast, the W diet and W + FO diet increased the serum concentration of beta-hydroxybutyrate. Further, the activity of 3-hydroxyacyl-CoA dehydrogenase, which is involved in fatty acid beta-oxidation in the liver, was greater in rats fed the W diet (42%), the FO diet (154%) and the W + FO diet (381%) than in those fed the C diet. Because the decrease in the concentration of triacylglycerol in the liver was greatest when rats were fed wakame and fish oil at the same time (W + FO diet), we conclude that there was a synergistic process affecting fatty acid beta-oxidation in the liver. These results suggest that the simultaneous consumption of fish (fish oil) and wakame decreases the concentration of triacylglycerol in the serum and liver.