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Koji is commonly used in manufacturing Japanese fermented products and promotes enzymatic degradation. In recent years, a seaweed koji has been developed by culturing Aspergillus oryzae on nori Pyropia yezoensis. In the present study, we prepared nori koji by culturing A. oryzae on high- and low-quality noris and then mixing this preparation with additional dried nori. Suitable fermentation conditions for increasing the taste-active components of the nori mixed with nori koji were investigated. The mixture of nori and koji prepared from high-quality nori, with added water, cultured for 120 days at 10 °C, provided the greatest increase (7 times) in free amino acids. The changes in taste after culturing the nori and koji mixture were evaluated using a taste-sensing system. The sourness score of the nori and koji mixture increased significantly, but the scores for other attributes such as bitterness and umami richness did not increase after culture. The present study has demonstrated a clear increase in the free amino acid content of nori and a modification in the taste score by aging the culture with nori koji. These results will encourage the development of ‘aged seaweed,’ a novel value-added product with nutritional and taste elements modified using seaweed koji.

The present study tested processes to manufacture fermented sauce from low-quality nori (dried and fresh fronds of Pyropia yezoensis). The nori sauce was prepared using three tanks with fresh or dried nori cultured in different conditions. In the present study enzymes were not added for the promotion of the degradation of nori, while in a previous study they were. The supernatants of culture mashes obtained from the three tanks were combined, and this low-quality nori sauce (LNS) was characterized and compared with sauces manufactured from high-quality nori, soy, and fish. The LNS showed low concentrations of total nitrogen compounds (0.20 g/100 ml) and free amino acids, and its taste showed a high sourness score as evaluated by a taste-sensing system. On the other hand, the LNS was rich in polysaccharides, which were observed to be readily degraded to lower molecular weight size sugars by heat treatment. The LNS showed little risk for heavy metal or allergen contamination. The obtained sauce product is expected to be commercially utilized as a component of low allergen-risk sauce products after blending with other seasonings without wheat or soy elements.

A novel strain Vibrio aphrogenes sp. nov. strain CA-1004T isolated from the surface of seaweed collected on the coast of Mie Prefecture in 1994 [1] was characterized using polyphasic taxonomy including multilocus sequence analysis (MLSA) and a genome based comparison. Both phylogenetic analyses on the basis of 16S rRNA gene sequences and MLSA based on eight protein-coding genes (gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA, and topA) showed the strain could be placed in the Rumoiensis clade in the genus Vibrio. Sequence similarities of the 16S rRNA gene and the multilocus genes against the Rumoiensis clade members, V. rumoiensis, V. algivorus, V. casei, and V. litoralis, were low enough to propose V. aphrogenes sp. nov. strain CA-1004T as a separate species. The experimental DNA-DNA hybridization data also revealed that the strain CA-1004T was separate from four known Rumoiensis clade species. The G+C content of the V. aphrogenes strain was determined as 42.1% based on the genome sequence. Major traits of the strain were non-motile, halophilic, fermentative, alginolytic, and gas production. A total of 27 traits (motility, growth temperature range, amylase, alginase and lipase productions, and assimilation of 19 carbon compounds) distinguished the strain from the other species in the Rumoiensis clade. The name V. aphrogenes sp. nov. is proposed for this species in the Rumoiensis clade, with CA-1004T as the type strain (JCM 31643T = DSM 103759T).

[This corrects the article DOI: 10.1371/journal.pone.0180053.].

The cell wall of the red alga Bangia atropurpurea is composed of three unique polysaccharides (β-1,4-mannan, β-1,3-xylan, and porphyran), similar to that in Porphyra. In this study, we visualized β-mannan in the regenerating cell walls of B. atropurpurea protoplasts by using a fusion protein of a carbohydrate-binding module (CBM) and green fluorescent protein (GFP). A mannan-binding family 27 CBM (CBM27) of β-1,4-mannanase (Man5C) from Vibrio sp. strain MA-138 was fused to GFP, and the resultant fusion protein (GFP-CBM27) was expressed in Escherichia coli. Native affinity gel electrophoresis revealed that GFP-CBM27 maintained its binding ability to soluble β-mannans, while normal GFP could not bind to β-mannans. Protoplasts were isolated from the fronds of B. atropurpurea by using three kinds of bacterial enzymes. The GFP-CBM27 was mixed with protoplasts from different growth stages, and the process of cell wall regeneration was observed by fluorescence microscopy. Some protoplasts began to excrete β-mannan at certain areas of their cell surface after 12 h of culture. As the protoplast culture progressed, β-mannans were spread on their entire cell surfaces. The percentages of protoplasts bound to GFP-CBM27 were 3%, 12%, 17%, 29%, and 25% after 12, 24, 36, 48, and 60 h of culture, respectively. Although GFP-CBM27 bound to cells at the initial growth stages, its binding to the mature fronds was not confirmed definitely. This is the first report on the visualization of β-mannan in regenerating algal cell walls by using a fluorescence-labeled CBM.

Two test diets with and without 5% Porphyra spheroplasts (PS) were formulated using white fishmeal as the main protein source. Red sea bream Pagrus major (mean body weight 15.4 ± 0.1 g) were maintained in a flow-through system (100 L) of thermo-controlled sea water (salinity 32-34, 25°C, 8 L/min) with ordinary aeration (400-600 mL/min) under laboratory light conditions (light-dark 12 h:12 h). Fish were fed three times a day at 10:00, 14:00 and 18:00 hours by hand for 42 days at 6% body weight on each experimental diet. Studies revealed that growth performance, survival and nutrient retention were significantly (P < 0.05) higher in the groups fed a diet containing spheroplasts (PS diet). Further, the fish fed the PS diet showed significantly (P < 0.05) lower feed conversion rates. Both groups of the PS and control diets had similar levels of body nutritional profile in terms of proximate compositions and fatty acids without compromising blood serum related parameters. From these experimental results, thus, it is comprehensible that a supplementary diet containing Porphyra spheroplasts can be used for maximizing not only growth of P. major but also for utilization of the feed ingredients.

A 56-day feeding trial was conducted to evaluate the effects of Porphyra spheroplasts (PS) as a feed additive on growth, carcass composition and feed utilization of black sea bream. Four experimental diets with or without PS (0, 1, 3, 5% inclusion level) were formulated to determine the proper incorporation level for best growth and utilization using white fishmeal as the principal protein source. Three replicate groups of fish averaging 1.28 ± 0.05 g (mean ± SD) were maintained in flow-through thermo-controlled sea water (32-34 psu, 25°C) aquaria (100 L) under the laboratory conditions (L12 : D12) and fed three times a day at 09:00, 13:00 and 17:00 hours by hand at approximately 3% body weight on each diet. The rearing trial revealed that growth performance (in terms of weight gain, specific growth rate) are almost the same level in all the dietary groups, nevertheless the survival, nutrient utilizations and retentions (feed efficiency, protein efficiency ratio, protein retention rate, lipid retention rate) were significantly higher (P < 0.05) in a group fed on the diet containing 3% spheroplasts. Further, the fish fed on the diet containing PS had significantly higher (P < 0.05) crude lipid level in their dorsal muscles and viscera. The fish fed a PS-based diet showed higher level of eicosapentaenoic acid and docosahexaenoic acid (DHA) in viscera and DHA in dorsal muscles. The results obtained in this feeding trial suggest that the optimum dietary PS supplementation level up to 3% for black sea bream can be considered as improved consequence on growth performance, nutrient utilization or body composition were noticed.

The xylA gene from a marine bacterium, Vibrio sp. strain XY-214, encoding D: -xylose isomerase (XylA) was cloned and expressed in Escherichia coli. The xylA gene consisted of 1,320-bp nucleotides encoding a protein of 439 amino acids with a predicted molecular weight of 49,264. XylA was classified into group II xylose isomerases. The native XylA was estimated to be a homotetramer with a molecular mass of 190 kDa. The purified recombinant XylA exhibited maximal activity at 60°C and pH 7.5. Its apparent K m values for D: -xylose and D: -glucose were 7.93 and 187 mM, respectively. Furthermore, we carried out D: -xylulose production from β-1,3-xylan, a major cell wall polysaccharide component of the killer alga Caulerpa taxifolia. The synergistic action of β-1,3-xylanase (TxyA) and β-1,3-xylosidase (XloA) from Vibrio sp. strain XY-214 enabled efficient saccharification of β-1,3-xylan to D: -xylose. D: -Xylose was then converted to D: -xylulose by using XylA from the strain XY-214. The conversion rate of D: -xylose to D: -xylulose by XylA was found to be approximately 40% in the presence of 4 mM sodium tetraborate after 2 h of incubation. These results demonstrated that TxyA, XloA, and XylA from Vibrio sp. strain XY-214 are useful tools for D: -xylulose production from β-1,3-xylan. Because D: -xylulose can be used as a source for ethanol fermentation by yeast Saccharomyces cerevisiae, the present study will provide a basis for ethanol production from β-1,3-xylan.

The agaA gene encoding β-agarase-a (AgaA) was cloned from the chromosomal DNA of a marine bacterium, Vibrio sp. strain PO-303. The nucleotide sequence of the agaA gene consists of 2,958 bp and encodes a protein of 985 amino acids with a molecular mass of 106,062 Da. The deduced enzyme protein contains a typical N-terminal signal peptide of 29 amino acid residues, followed by a 266 amino acid sequence that is homologous to catalytic module of family 16 glycoside hydrolases, a bacterial immunoglobulin group 2 (Big-2)-like domain of 52 amino acid residues, two carbohydrate-binding modules of family 6 separated from Big-2-like domain by nine times repeated GDDTDP amino acid sequence. AgaA is the first agarase that was identified to possess a Big-2-like domain. The recombinant AgaA (rAgaA) expressed in Escherichia coli exhibited maximal activity around 40°C and pH 7.5, with a specific activity of 16.4 units mg-¹, a K m of 1.10 mg ml-¹, and a V max of 22.5 μmol min-¹ mg-¹ for agarose. The rAgaA hydrolyzed neoagarohexaose, but did not act on neoagarotetraose and neoagarobiose.

Sequence analysis of β-1,3-xylanase (TxyA) from a marine bacterium, Alcaligenes sp. strain XY-234 implied that an xylan-binding module belonging to carbohydrate-binding module family 31 (TxyA-CBM) is separated from a catalytic module belonging to glycosyl hydrolase family 26 (TxyA-CM) by a putative glycine-rich linker [Okazaki, F., et al. (2002) J. Bacteriol. 184: 2399–2403]. In order to reveal the role of these structural features of TxyA, two modules, TxyA-CBM and TxyA-CM, were constructed, and those modules and full-length TxyA were characterized by thermodynamic studies. TxyA-CBM and TxyA-CM showed full reversible folding from denaturant-induced unfolded forms, exhibited higher thermodynamic stabilities. The conformational stability of both truncated modules is industrially desirable, as well as aiding the understanding of the enzymatic characterization of the two modules of β-1,3-xylanase separated by a long linker.