Details of a Researcher - HAYASHI Masahiro

Fed-batch cultivation of Euglena gracilis for the high-yield production and GPC-assisted molecular weight determination of paramylon Reviewed

The Utilization of Heterotrophic Cultivation for Large Scale Production of　Microalgae Invited Reviewed

HAYASHI Masahiro

31 ( 1 ) 4 - 7 2024.4

Authorship：Lead author,　Corresponding author Language：Japanese Publishing type：Research paper (scientific journal)

Fukuda M., Kinkawa M., Hayashi M.

Bioscience, Biotechnology and Biochemistry 88 ( 2 ) 206 - 211 2024.4

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Bioscience, Biotechnology and Biochemistry

Paramylon, a starch-like carbohydrate accumulated in Euglena gracilis cells, could be a potential source as a raw material for chemical products; its high-yield production would thus be highly desired. Although the molecular weight and polymerization degree of paramylon are important properties for its use as a raw material for chemical products, the available information about paramylon molecular weight remains insufficient. Therefore, in this study, we investigated a high-density E. gracilis culture approach and how culture conditions affect paramylon molecular weight. The nitrogen source, cultivation temperature, and nutrient feeding were optimized for maximum biomass and paramylon productivity. The maximum dry cell weight and paramylon content yields reached 108.9 g/L and 87.2%, respectively. Paramylon molecular weight was in the range of 220 000–320 000 Da. Our gel permeation chromatography analysis showed that the cells with a higher paramylon content tended to contain paramylon of a higher molecular weight.

DOI： 10.1093/bbb/zbad152

Characterization of thraustochytrid-specific sterol O-acyltransferase: modification of DGAT2-like enzyme to increase the sterol production in Aurantiochytrium limacinum mh0186 Reviewed

石橋洋平, 定光翔平, 深堀義朝, タノガシラリン, 渡邉昂, 林雅弘, 伊東信, 沖野望

Applied and Environmental Microbiology 89 ( 11 ) 2023.11

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society for Microbiology

Thraustochytrids are marine microorganisms expected to produce useful lipids. They synthesize polyunsaturated fatty acids and sterols and store them in lipid droplets as a form of triacylglycerol (TG) and sterol ester (SE), respectively. TG is synthesized by diacylglycerol O-acyltransferase (DGAT). There are several DGAT2 homologs in Aurantiochytrium limacinum. This study indicated that DGAT2C and DGAT2D are SE synthase and TG synthase, respectively, by disrupting their corresponding genes in A. limacinum mh0186. DGAT2C is revealed as thraustochytrid-specific acyl-CoA:sterol- O-acyltransferase by performing in vivo and in vitro assays after heterologous expression in Saccharomyces cerevisiae. DGAT2C and DGAT2D localized mainly to the endoplasmic reticulum (ER) and the lipid droplet, respectively, and the two of the N-terminal domains unique to DGAT2C were essential for ER localization and SE synthesis in A. limacinum mh0186. Interestingly, the study also found that deletion of the first eight transmembrane domains in the unique N-terminal region of DGAT2C increased SE and total sterol productivity when expressed in DGAT2C-deficient A. limacinum mh0186. In addition, intermediates such as Δ7-cholesterol (provitamin D3) also accumulated in SE by expression of the N-terminal-truncated DGAT2C, along with cholesterol and Δ7-stigmasterol, a major phytosterol in A. limacinum. This study proves that DGAT2C is an ER-localized SE synthase and that its suitable N-terminal deletion can increase sterol production in thraustochytrids.

Fed-batch cultivation of Euglena gracilis for the high-yield production and GPC-assisted molecular weight determination of paramylon Reviewed

Mizuki Fukuda, Manato Kinkawa and Masahiro Hayashi

Bioscience, Biotechnology and Biochemistry 88 ( 2 ) 206 - 211 2023.11

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Bioscience, Biotechnology and Biochemistry

Comparative metabolic profiling analysis was performed to investigate light- and aeration-dependent regulation of central metabolism in Euglena gracilis. The metabolic profiles of E. gracilis were significantly altered in response to changes in aeration conditions. While many glycolytic intermediates and amino acids accumulated in aerobically grown E. gracilis, a significant reduction in these metabolites was observed for cells under anaerobic conditions, which resulted in elevated production of wax ester.

DOI： https://doi.org/10.1093/bbb/zbad152

The Utilization of Heterotrophic Cultivation for Large Scale Production of　Microalgae Invited Reviewed

HAYASHI Masahiro, MATSUDA Ayako

20 ( 6 ) 37 - 42 2023.9

Authorship：Lead author,　Corresponding author Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Japan Oil Chemists' Society

In various industries, practical utilizations of many microalgae are investigated recently. Many problems are recognized for a practical realization of microalgal industry, such as, a choice of cultivation method, autotroph or heterotroph, an effect of sunlight cycle on algal growth, a balance between cell density and efficacy of photosynthesis, an effects of carbon concentration on the growth, a shape and function of culture tank, practical use of extraction residue, molecular breeding and sterilization of the strains.Euglena has unique properties for the cultivation among various microalgae. Euglena can grow autotrophically, heterotrophically, or photoheterotrophically. Although each cultivation method has some merit and demerit, the authors focused on the heterotrophy cultivation, Euglena is industrially cultivated in large scale tanks in the dark.

The Utilization of Heterotrophic Cultivation for Large Scale Production of Euglena gracilis Invited Reviewed

HAYASHI Masahiro, MATSUDA Ayako, TOKAI Shouta, UKEGAWA Yuino, UDAKA Takaki

Oleoscience 23 ( 2 ) 65 - 72 2023.2

Authorship：Lead author,　Corresponding author Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Japan Oil Chemists' Society

DOI： 10.5650/oleoscience.23.65

Characterization of thraustochytrid-specific sterol O-acyltransferase: modification of DGAT2-like enzyme to increase the sterol production in Aurantiochytrium limacinum mh0186 Reviewed

Ishibashi Y., Sadamitsu S., Fukahori Y., Yamamoto Y., Tanogashira R., Watanabe T., Hayashi M., Ito M., Okino N.

Applied and Environmental Microbiology 89 ( 11 ) 2023

Publishing type：Research paper (scientific journal) Publisher：Applied and Environmental Microbiology

Thraustochytrids are marine microorganisms expected to produce useful lipids. They synthesize polyunsaturated fatty acids and sterols and store them in lipid droplets as a form of triacylglycerol (TG) and sterol ester (SE), respectively. TG is synthesized by diacylglycerol O-acyltransferase (DGAT). There are several DGAT2 homologs in Aurantiochytrium limacinum. This study indicated that DGAT2C and DGAT2D are SE synthase and TG synthase, respectively, by disrupting their corresponding genes in A. limacinum mh0186. DGAT2C is revealed as thraustochytrid-specific acyl-CoA:sterolO-acyltransferase by performing in vivo and in vitro assays after heterologous expression in Saccharomyces cerevisiae. DGAT2C and DGAT2D localized mainly to the endoplasmic reticulum (ER) and the lipid droplet, respectively, and the two of the N-terminal domains unique to DGAT2C were essential for ER localization and SE synthesis in A. limacinum mh0186. Interestingly, the study also found that deletion of the first eight transmembrane domains in the unique N-terminal region of DGAT2C increased SE and total sterol productivity when expressed in DGAT2C-deficient A. limacinum mh0186. In addition, intermediates such as Δ7-cholesterol (provitamin D3) also accumulated in SE by expression of the N-terminal-truncated DGAT2C, along with cholesterol and Δ7-stigmasterol, a major phytosterol in A. limacinum. This study proves that DGAT2C is an ER-localized SE synthase and that its suitable N-terminal deletion can increase sterol production in thraustochytrids.

DOI： 10.1128/aem.01001-23

PUFA synthase-independent DHA synthesis pathway in Parietichytrium sp. and its modification to produce EPA and n-3DPA Reviewed

有用新奇海洋微生物の探索：ラビリンチュラ Invited Reviewed

林雅弘, 松田綾子

遺伝 76 ( 6 ) 428 - 433 2022.11

Authorship：Lead author,　Corresponding author Language：Japanese Publishing type：Research paper (scientific journal) Publisher：公益社団法人日本油化学会

様々な微細藻類の社会実装が検討される中で，独立栄養か従属栄養かの選択，太陽光による光合成への日周サイクルの影響，培養密度と光合成効率のバランス，培養液中の炭素源濃度の影響，培養槽の形状や機能の問題，目的物の抽出残渣処理，株の育種や無菌化など，実用化に向けてクリアすべき問題は多い。微細藻類の中でもユーグレナは特徴的な培養特性を持ち，光合成による独立栄養培養，光合成に加えて有機炭素源を利用した光従属栄養培養，光を利用せず培地中の有機炭素源を利用する従属栄養培養のいずれの培養による増殖も可能である。いずれの培養様式もそれぞれ特徴を持ち，目的や規模に応じて使い分ける必要があるが筆者らのグループは従属栄養培養に焦点を絞り，ユーグレナの工業レベルでの大量生産を行っている。本稿では筆者らが行っているユーグレナの工業レベルの大量培養を1つの題材に，微細藻類の社会実装に向けた大量培養技術について考える。

海洋微生物ラビリンチュラの食品素材利用 Invited Reviewed

林雅弘, 松田綾子、親泊政二三

アグリバイオ 6 ( 10 ) 897 - 901 2022.9

Authorship：Lead author,　Corresponding author Language：Japanese Publishing type：Research paper (scientific journal) Publisher：公益社団法人日本油化学会

微細藻類の産業利用ー従属栄養培養の活用ー Invited Reviewed

林雅弘, 松田綾子

アクアネット 25 ( 8 ) 32 - 37 2022.8

Authorship：Lead author,　Corresponding author Language：Japanese Publishing type：Research paper (scientific journal) Publisher：公益社団法人日本油化学会

Ishibashi Yohei, Goda Hatsumi, Hamaguchi Rie, Sakaguchi Keishi, Sekiguchi Takayoshi, Ishiwata Yuko, Okita Yuji, Mochinaga Seiya, Ikeuchi Shingo, Mizobuchi Takahiro, Takao Yoshitake, Mori Kazuki, Tashiro Kosuke, Okino Nozomu, Honda Daiske, Hayashi Masahiro, Ito Makoto

Communications Biology 4 ( 1 ) 1378 2021.12

Language：English Publishing type：Research paper (scientific journal)

The demand for n-3 long-chain polyunsaturated fatty acids (n-3LC-PUFAs), such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), will exceed their supply in the near future, and a sustainable source of n-3LC-PUFAs is needed. Thraustochytrids are marine protists characterized by anaerobic biosynthesis of DHA via polyunsaturated fatty acid synthase (PUFA-S). Analysis of a homemade draft genome database suggested that Parietichytrium sp. lacks PUFA-S but possesses all fatty acid elongase (ELO) and desaturase (DES) genes required for DHA synthesis. The reverse genetic approach and a tracing experiment using stable isotope-labeled fatty acids revealed that the ELO/DES pathway is the only DHA synthesis pathway in Parietichytrium sp. Disruption of the C20 fatty acid ELO (C20ELO) and ∆4 fatty acid DES (∆4DES) genes with expression of ω3 fatty acid DES in this thraustochytrid allowed the production of EPA and n-3docosapentaenoic acid (n-3DPA), respectively, at the highest level among known microbial sources using fed-batch culture.

DOI： 10.1038/s42003-021-02857-w

PubMed

C4-monomethylsterol β-glucoside and its synthase in Aurantiochytrium limacinum mh0186 Reviewed

Endo I., Watanabe T., Miyamoto T., Monjusho-Goda H., Ohara J., Hayashi M., Hama Y., Ishibashi Y., Okino N., Ito M.

Glycobiology 31 ( 10 ) 1350 - 1363 2021.10

Language：English Publishing type：Research paper (scientific journal) Publisher：Glycobiology

Thraustochytrids, unicellular marine protists, synthesize polyunsaturated fatty acids (PUFAs) and PUFA-containing phospholipids; however, little is known about their glycolipids and their associated metabolism. Here, we report two glycolipids (GL-A, B) and their synthases in Aurantiochytrium limacinum mh0186. Two glycolipids were purified from A. limacinum mh0186, and they were determined by gas chromatography, mass spectrometry and 2D nuclear magnetic resonance to be 3-O-β-D-glucopyranosyl-stigmasta-5,7,22-triene (GL-A) and 3-O-β-D-glucopyranosyl-4α-methyl-stigmasta-7,22-diene (GL-B), both of which are sterol β-glucosides (β-SGs); the structure of GL-B has not been reported thus far. Seven candidate genes responsible for the synthesis of these β-SGs were extracted from the draft genome database of A. limacinum using the yeast sterol β-glucosyltransferase (SGT; EC 2.4.1.173) sequence as a query. Expression analysis using Saccharomyces cerevisiae revealed that two gene products (AlSGT-1 and 2) catalyze the transfer of glucose from uridine diphosphate (UDP)-glucose to sterols, generating sterylglucosides (SGs). Compared to AlSGT-1, AlSGT-2 exhibited wide specificity for sterols and used C4-monomethylsterol to synthesize GL-B. The disruption of alsgt-2 but not alsgt-1 in strain mh0186 resulted in a decrease in the total SG and an almost complete loss of GL-B, indicating that AlSGT-2 is responsible for the synthesis of β-SGs in A. limacinum mh0186, especially GL-B, which possesses a unique sterol structure.

DOI： 10.1093/glycob/cwab070

A thraustochytrid-specific lipase/phospholipase with unique positional specificity contributes to microbial competition and fatty acid acquisition from the environment Reviewed

Ishibashi Y., Aoki K., Okino N., Hayashi M., Ito M.

Scientific Reports 9 ( 1 ) 2019.12

Language：English Publishing type：Research paper (scientific journal) Publisher：Scientific Reports

© 2019, The Author(s). Thraustochytrids are heterotrophic marine protists that are considered as important decomposers in the marine ecosystem; however, how they digest and uptake lipid nutrients from the environment is largely unknown. Genomic clustering analysis using thraustochytrid draft genome databases revealed that novel proteins with a Lipase_3 domain are commonly present in thraustochytrids, including Aurantiochytrium limacinum. After heterologous expression and His tag-based purification, protein ID: 145138 was identified as lipase/phospholipase capable of hydrolyzing triacylglycerol (TG) and phosphatidylcholine (PC). 145138 was secreted into the medium, and deletion of the 145138 gene in A. limacinum reduced the degradation of extracellular lipids. Fatty acids generated by 145138 were reused for the biosynthesis of PC and TG, and 145138 allowed A. limacinum to survive in the medium containing TG as a sole carbon source. 145138 hydrolyzed all the acyl-ester linkages of TG; however, the enzyme showed strict positional specificity toward phospholipids, generating 2-acyl lysophospholipids. The 2-acyl lysophospholipids showed stronger antimicrobial activity compared with 1-acyl lysophospholipids. These results suggested that 145138 is a bifunctional enzyme that contributes to the acquisition of lipid nutrients from the environment, as well as to generate antimicrobial lysophospholipids that are beneficial for competition with bacteria over lipid nutrients in the marine environment.

DOI： 10.1038/s41598-019-52854-7

The glycerol-3-phosphate acyltransferase PLAT2 functions in the generation of DHA-rich glycerolipids in Aurantiochytrium limacinum F26-b Reviewed

バイオ燃料の社会実装に向けた技術的課題とおの実現可能性 Invited Reviewed

平川拓弥、林　雅弘

応用細胞生物学研究 32 ( 1 ) 11 - 34 2019.5

Authorship：Corresponding author Language：Japanese Publishing type：Research paper (scientific journal)

物質生産ツールとしてのユーグレナの可能性および生産性向上を目指した高密度培養技術 Invited Reviewed

山嵜真彩、林　雅弘

応用細胞生物学研究 32 ( 1 ) 35 - 54 2019.5

Authorship：Corresponding author Language：Japanese Publishing type：Research paper (scientific journal)

Eri Nutahara, Eriko Abe, Shinya Uno, Yohei Ishibashi, Takashi Watanabe, Masahiro Hayashi, Nozomu Okino, Makoto Ito

PLOS ONE ( 1 ) 2019.4

Language：English Publishing type：Research paper (scientific journal)

食糧資源としてのユーグレナの可能性を広げるために Invited Reviewed

長岡　綾、林　雅弘

応用細胞生物学研究 30 ( 1 ) 7 - 12 2018.4

Authorship：Corresponding author Language：Japanese Publishing type：Research paper (scientific journal)

Regulation of TG accumulation and lipid droplet morphology by the novel TLDP1 in Aurantiochytrium limacinum F26-b Reviewed

Takashi Watanabe, Ryo Sakiyama, Yuya Iimi, Satomi Sekine, Eriko Abe, Kazuko H. Nomura, Kazuya Nomura, Yohei Ishibashi, Nozomu Okino, Masahiro Hayashi, Makoto Ito

Journal of Lipid Research 58 2334 - 2347 2017.10

Language：English Publishing type：Research paper (scientific journal)

Degradation of distillery lees (Shochu kasu) by cellulase-producing thraustochytrids Reviewed

Taoka Y., Nagano N., Kai H., Hayashi M.

Journal of Oleo Science 66 ( 1 ) 31 - 40 2017.5

Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Oleo Science

© 2017 by Japan Oil Chemists’ Society. Single cell oils produced by oleaginous microorganisms have attracted increasing interests as a petroleum alternative energy. Marine eukaryotes, thraustochytrids were heterotrophic, and can grow rapidly and accumulate large amount of lipids containing functional fatty acids, such as docosahexaenoic acid (DHA) in their cells body. In this investigation, thraustochytrids isolated from marine environment were cultured in the medium containing an industrial waste and an unused resource, distillery lees (Shochu kasu) to produce biofuel or functional fatty acids by microorganisms. Sixty-nine thraustochytrids and Schizochytrium aggregatum ATCC 28209 were screened for cellulase production, and the activities were detected using sodium carboxymethyl cellulose (CMC) as a substrate. Based on the screening test, strain TM02Bc identified to Schizochytrium sp. was selected for the Shochu kasu degradation test and compared with S. aggregatum ATCC 28209 previously known as a cellulase-producing thraustochytrid. Strains TM02Bc and ATCC 28209 were cultured in artificial seawater containing Shochu kasu for 15 days. The two strains could degrade Schochu kasu, especially that from sweet potato Shochu (Imo Shochu). Cellulase (CMCase) and protease activities were detected in culture supernatant of both strains, and the ratio of polyunsaturated fatty acids (PUFAs) significantly increased as a result of incubation of Shochu kasu with two strains. This preliminary study indicated that strain TM02Bc was a potent candidate for Shochu kasu treatment and fatty acid production.

Preparation of transparent self-standing thin films made from acetylated euglenoid β-1,3-glucans Reviewed

Lipid Droplet-Related Protein 1 (LDRP1) Regulates the Triacylglycerol Accumulation and Lipid Droplet Morphology in a Thraustochytrid, Aurantiochytrium limacinum F26-b Reviewed

Takashi Watanabe1, Ryo Sakiyama1, Yuya Iimi, Satomi Sekine1, Eriko Abe, Kazuko H. Nomura, Kazuya Nomura, Yohei Ishibashi, Nozomu Okino, Masahiro Hayashi, Makoto Ito

Journal of Biological Chemistry 2017.1

Language：English Publishing type：Research paper (scientific journal)

微細藻類プラスチックの開発 Invited Reviewed

林　雅弘、芝上基成

化学工学 80 ( 5 ) 2 - 4 2016.12

Authorship：Lead author Language：Japanese Publishing type：Research paper (scientific journal)

Synthesis of nanofiber-formable carboxymethylated Euglena-derived beta-1,3-glucan Reviewed

Motonari Shibakami, Gen Tsubouchi, Mitsugu Sohma, Masahiro Hayashi

Carbohydrate Polymers 152 468 - 478 2016.6

Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)

微細藻類とホワイトバイオテクノロジー Reviewed

末永智幸、林　雅弘

応用細胞生物学研究 28 ( 1 ) 10 - 14 2015.12

Authorship：Corresponding author Language：Japanese Publishing type：Research paper (scientific journal)

Water-soluble jack-knife prawn extract inhibits 5-hydroxytryptamine-induced vasoconstriction and platelet aggregation in humans Reviewed

Shuji Gamoh, Tasuku Kanai, Naoko Tanaka-Totoribe, Masamichi Ohkura, Masachika Kuwabara, Eisaku Nakamura, Atsuko Yokota, Tetsuo Yamasaki, Akiko Watanabe, Masahiro Hayashi, Shouichi Fujimoto and Ryuichi Yamamoto

Food & Function 6 444 - 449 2015.12

Language：English Publishing type：Research paper (scientific journal)

油糧海洋微生物ラビリンチュラの産業利用 Invited Reviewed

林　雅弘、松田綾子、松田高宜、泉　可也

酵素工学ニュース ( 74 ) 6 - 9 2015.10

Authorship：Lead author Language：Japanese Publishing type：Research paper (scientific journal)

Shibakami M., Tsubouchi G., Sohma M., Hayashi M.

Carbohydrate Polymers 133 421 - 428 2015.8

Authorship：Corresponding author Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Carbohydrate Polymers

© 2015 Elsevier Ltd. All rights reserved. The feasibility of using acetylparamylon to prepare self-standing optical films was investigated. Its thermal stability, represented by the glass transition temperature, and 5% weight loss temperature are equivalent to those of acetylcellulose. The mechanical properties (maximum stress, elongation at break, and elastic modulus) of acetylparamylon films prepared by solution casting were comparable to those of acetylcellulose films. The degrees of their transparency and birefringence were the same as those of the acetylcellulose film. These results, together with the finding that acetylparamylon with a higher degree of acetylation formed a thin film with fewer micrometer-sized defects and that acetylparamylon with a lower degree of acetylation formed a thin film with a nanometer-level flat surface, suggest that acetylparamylon is a feasible material for self-standing optical film.

DOI： 10.1016/j.carbpol.2015.06.104

油糧微生物ラビリンチュラ Invited Reviewed

林　雅弘

生物工学 93 ( 7 ) 426 - 429 2015.7

Authorship：Lead author,　Corresponding author Language：Japanese Publishing type：Research paper (scientific journal)

One-pot synthesis of thermoplastic mixed paramylon esters using trifluoroacetic anhydride Reviewed

Motonari Shibakami, Gen Tsubouchi, Masahiro Hayashi

Carbohydrate Polymers 119 1 - 7 2015.4

Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)

油糧微生物ラビリンチュラ(生物材料インデックス)

林雅弘

生物工学会誌 : seibutsu-kogaku kaishi 93 ( 7 ) 426 - 429 2015

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：日本生物工学会

Novel lysophospholipid acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b responsible for generation of palmitate-docosahexaenoate- phosphatidylcholine and phosphatidylethanolamine Reviewed

Abe E., Ikeda K., Nutahara E., Hayashi M., Yamashita A., Taguchi R., Doi K., Honda D., Okino N., Ito M.

PLoS ONE 9 ( 8 ) 2014.8

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：PLoS ONE

N-3 polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid (DHA, 22:6n-3), have been reported to play roles in preventing cardiovascular diseases. The major source of DHA is fish oils but a recent increase in the global demand of DHA and decrease in fish stocks require a substitute. Thraustochytrids, unicellular marine protists belonging to the Chromista kingdom, can synthesize large amounts of DHA, and, thus, are expected to be an alternative to fish oils. DHA is found in the acyl chain(s) of phospholipids as well as triacylglycerols in thraustochytrids; however, how thraustochytrids incorporate DHA into phospholipids remains unknown. We report here a novel lysophospholipid acyltransferase (PLAT1), which is responsible for the generation of DHA-containing phosphatidylcholine and phosphatidylethanolamine in thraustochytrids. The PLAT1 gene, which was isolated from the genomic DNA of Aurantiochytrium limacinum F26-b, was expressed in Saccharomyces cerevisiae, and the FLAG-tagged recombinant enzyme was characterized after purification with anti-FLAG affinity gel. PLAT1 shows wide specificity for donor substrates as well as acceptor substrates in vitro, i.e, the enzyme can adopt lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine and lysophosphatidylinositol as acceptor substrates, and 15:0/16:0-CoA and DHA-CoA as donor substrates. In contrast to the in vitro experiment, only lysophosphatidylcholine acyltransferase and lysophosphatidylethanolamine acyltransferase activities were decreased in plat1-knockout mutants, resulting in a decrease of 16:0-DHA-phosphatidylcholine (PC) [PC(38:6)] and 16:0-DHA-phosphatidylethanolamine (PE) [PE(38:6)] , which are two major DHA-containing phospholipids in A. limacinum F26-b. However, the amounts of other phospholipid species including DHA-DHA-PC [PC(44:12)] and DHA-DHA-PE [PE(44:12)] were almost the same in plat-knockout mutants and the wild-type. These results indicate that PLAT1 is the enzyme responsible for the generation of 16:0-DHA-PC and 16:0-DHA-PE in the thraustochytrid. © 2014 Abe et al.

DOI： 10.1371/journal.pone.0102377

Thermoplasticization of euglenoid beta-1,3-glucans by mixed esterification Reviewed

Motonari Shibakami, Gen Tsubouchi, Masahiro Hayashi

Carbohydrate Polymers 105 90 - 96 2014.5

Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)

ミドリムシを主原料としたバイオプラスチックの開発 Invited Reviewed

芝上基成、林　雅弘

プラスチックエージ ( 12 ) 92 - 96 2013.12

Authorship：Lead author Language：Japanese Publishing type：Research paper (other academic)

Effect of graded levels of dietary DHA included in rotifers Brachionus plicatilis on laviculture performance of red sea bream Pagrus major Reviewed

Kotani, T., H. Fushimi, Y. Ohta, A. Miyashita, K. Sudoh, M. Hayashi, N. Satoh, S. Satoh

Aquaculture Science 61 ( 4 ) 321 - 330 2013.12

Language：English Publishing type：Research paper (scientific journal)

藻類を活用する食品素材開発 Invited Reviewed

林　雅弘

生物工学 91 ( 11 ) 621 - 624 2013.11

Authorship：Lead author,　Corresponding author Language：Japanese Publishing type：Research paper (scientific journal)

Food material development to take advantage of algae Invited Reviewed

Hayashi M.

Seibutsu-kogaku Kaishi 91 ( 11 ) 621 - 624 2013.11

Authorship：Lead author,　Corresponding author Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Seibutsu-kogaku Kaishi

ミドリムシプラスチックとミドリムシナノファイバーの開発 Invited Reviewed

芝上基成、林　雅弘

自動車技術 67 ( 9 ) 88 - 89 2013.9

Authorship：Corresponding author Language：Japanese Publishing type：Research paper (other academic)

Effect of trace elements on growth of marine eukaryotes, tharaustochytrids Reviewed

Nagano Naoki, Taoka Yousuke, Honda Daiske, Hayashi Masahiro

Journal of bioscience and bioengineering 116 ( 3 ) 337 - 339 2013.9

Authorship：Corresponding author Language：Japanese Publishing type：Research paper (scientific journal) Publisher：公益社団法人日本生物工学会

We determined the effect of trace elements on the growth of thraustochytrids. The growth of the strains cultured with the trace elements was much higher than that of the strains cultured without any trace element. Iron and zinc were particularly important to obtaining the optimum growth of thraustochytrids.

藻類を活用する食品素材開発(<特集>美味しい健康生活は微生物が作る～作物生産,食品素材開発,健康支援～)

ミドリムシプラスチックの開発 Invited Reviewed

芝上基成、林　雅弘

機能材料 33 ( 8 ) 9 - 13 2013.8

Authorship：Corresponding author Language：Japanese Publishing type：Research paper (other academic)

Polysaccharide nanofiber made from euglenoid alga Reviewed

Motonari Shibakami, Gen Tsubouchi, Makoto Nakamura, Masahiro Hayashi

Carbohydrate Polymers 98 ( 3 ) 499 - 505 2013.4

Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)

毎洋性微生物資源を活用するオメガ3脂防酸素材の生産 Invited Reviewed

林　雅弘

Bioindustry 30 ( 2 ) 25 - 31 2013.2

Authorship：Lead author,　Corresponding author Language：Japanese Publishing type：Research paper (scientific journal)

Preparation of carboxylic acid-bearing polysaccharide nanofiber made from euglenoid beta-1,3-glucans Reviewed

Motonari Shibakami, Gen Tsubouchi, Makoto Nakamura, Masahiro Hayashi

Carbohydrate Polymers 98 ( 1 ) 95 - 101 2013.1

Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)

林雅弘

生物工学会誌 : seibutsu-kogaku kaishi 91 ( 11 ) 621 - 624 2013

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：日本生物工学会

シオミズツボワムシ中の DHA 量がマダイ種苗生産飼育成績に与える効果

小谷知也, 伏見浩, 太田有香, 宮嶋暁, 須藤健介, 林雅弘, 佐藤信光, 佐藤秀一

水産増殖 61 ( 4 ) 321 - 330 2013

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：水産増殖談話会

本研究はマダイ種苗飼育成績改善を目的として，ワムシ栄養強化剤中の DHA 量について検討した。DHA 量の異なる4種類の栄養強化剤（0～60%）および市販強化剤を用いてワムシを強化した。強化剤中の総脂肪酸中 DHA 量が20%以上の時，ワムシ単独給餌期の仔魚の成長を改善できたが，アルテミアの給餌が始まった後，強化剤中 DHA 量が60%であった試験区の魚の成長が低下した。したがって，マダイ種苗生産ではワムシ強化剤中の DHA 量は総脂肪酸中20あるいは40%が適していると考えられる。

DOI： 10.11233/aquaculturesci.61.321

Analysis of Δ12-fatty acid desaturase function revealed that two distinct pathways are active for the synthesis of PUFAs in T. aureum ATCC 34304

Matsuda T., Sakaguchi K., Hamaguchi R., Kobayashi T., Abe E., Hama Y., Hayashi M., Honda D., Okita Y., Sugimoto S., Okino N., Ito M.

Journal of Lipid Research 53 ( 6 ) 1210 - 1222 2012.6

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Lipid Research

Thraustochytrids are known to synthesize PUFAs such as docosahexaenoic acid (DHA). Accumulating evidence suggests the presence of two synthetic pathways of PUFAs in thraustochytrids: the polyketide synthase-like (PUFA synthase) and desaturase/elongase (standard) pathways. It remains unclear whether the latter pathway functions in thraustochytrids. In this study, we report that the standard pathway produces PUFA in Thraustochytrium aureum ATCC 34304. We isolated a gene encoding a putative Δ12-fatty acid desaturase (TauΔ12des) from T. aureum. Yeasts transformed with the tauΔ12des converted endogenous oleic acid (OA) into linoleic acid (LA). The disruption of the tauΔ12des in T. aureum by homologous recombination resulted in the accumulation of OA and a decrease in the levels of LA and its downstream PUFAs. However, the DHA content was increased slightly in tauΔ12des -disruption mutants, suggesting that DHA is primarily produced in T. aureum via the PUFA synthase pathway. The transformation of the tauΔ12des -disruption mutants with a tauΔ12des expression cassette restored the wild-type fatty acid profiles. These data clearly indicate that TauΔ12des functions as Δ12-fatty acid desaturase in the standard pathway of T. aureum and demonstrate that this thraustochytrid produces PUFAs via both the PUFA synthase and the standard pathways. Copyright © 2012 by the American Society for Biochemistry and Molecular Biology, Inc.

DOI： 10.1194/jlr.M024935

Versatile transformation system that is applicable to both multiple transgene expression and gene targeting for thraustochytrids

Sakaguchi K., Matsuda T., Kobayashi T., Ohara J., Hamaguchi R., Abe E., Nagano N., Hayashi M., Ueda M., Honda D., Okita Y., Taoka Y., Sugimoto S., Okino N., Ito M.

Applied and Environmental Microbiology 78 ( 9 ) 3193 - 3202 2012.5

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Applied and Environmental Microbiology

A versatile transformation system for thraustochytrids, a promising producer for polyunsaturated fatty acids and fatty acid-derived fuels, was established. G418, hygromycin B, blasticidin, and zeocin inhibited the growth of thraustochytrids, indicating that multiple selectable marker genes could be used in the transformation system. A neomycin resistance gene (neo r ), driven with an ubiquitin or an EF-1α promoter-terminator from Thraustochytrium aureum ATCC 34304, was introduced into representatives of two thraustochytrid genera, Aurantiochytrium and Thraustochytrium. The neo r marker was integrated into the chromosomal DNA by random recombination and then functionally translated into neo r mRNA. Additionally, we confirmed that another two genera, Parietichytrium and Schizochytrium, could be transformed by the same method. By this method, the enhanced green fluorescent protein was functionally expressed in thraustochytrids. Meanwhile, T. aureum ATCC 34304 could be transformed by two 18S ribosomal DNA-targeting vectors, designed to cause single- or double-crossover homologous recombination. Finally, the fatty acid Δ5 desaturase gene was disrupted by double-crossover homologous recombination in T. aureum ATCC 34304, resulting in an increase of dihomo-γ-linolenic acid (C 20:3n-6 ) and eicosatetraenoic acid (C 20:4n-3 ), substrates for Δ5 desaturase, and a decrease of arachidonic acid ( C20:4n-6 ) and eicosapentaenoic acid (C 20:5n-3 ), products for the enzyme. These results clearly indicate that a versatile transformation system which could be applicable to both multiple transgene expression and gene targeting was established for thraustochytrids. © 2012, American Society for Microbiology.

DOI： 10.1128/AEM.07129-11

Comparative profiling analysis of central metabolites in euglena gracilis under various cultivation conditions

Fabrication of Doughnut-Shaped Particles from Spheroidal Paramylon Granules of Euglena gracilis using Acetylation Reaction Reviewed

Motonari Shibakami, Mitsugu Sohma, Masahiro Hayashi

Carbohydrate Polymers 87 452 - 456 2012.4

Language：English Publishing type：Research paper (scientific journal)

Analysis of delta-12-fatty acid desaturase function revealed that two distinct pathways are active for the synthesis of PUFAs in T. aureum ATCC 34304 Reviewed

Takanori Matsuda , Keishi Sakaguchi , Rie Hamaguchi , Takumi Kobayashi , Eriko Abe , Yoichiro Hama , Masahiro Hayashi , Daiske Honda ,Yuji Okita , Shinichi Sugimoto, Nozomu Okino,Makoto Ito

Journal of Lipid Research 53 1210 - 1222 2012.4

Language：English Publishing type：Research paper (scientific journal)

Matsuda F., Hayashi M., Kondo A.

Bioscience, Biotechnology and Biochemistry 75 ( 11 ) 2253 - 2256 2011.11

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Bioscience, Biotechnology and Biochemistry

DOI： 10.1271/bbb.110482

Molecular cloning of a Pinguiochrysis pyriformis oleate-specific microsomal Δ12-fatty acid desaturase and functional analysis in yeasts and thraustochytrids

Matsuda Takanori, Sakaguchi Keishi, Kobayashi Takumi, ABE Eriko, KURANO Norihide, SATO Akira, OKITA Yuji, SUGIMOTO Shinichi, HAMA Yoichiro, HAYASHI Masahiro, OKINO Nozomu, ITO Makoto

The journal of biochemistry 150 ( 4 ) 375 - 383 2011.10

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Japanese Biochemical Society

Detection of genes involved in fatty acid elongation and desaturation in thraustochytrid marine eukaryotes

Nagano N., Sakaguchi K., Taoka Y., Okita Y., Honda D., Ito M., Hayashi M.

Journal of Oleo Science 60 ( 9 ) 475 - 481 2011.8

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Oleo Science

Heterotrophic marine protists known as thraustochytrids can synthesize polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA). The biosynthetic pathways of PUFAs in thraustochytrids are poorly understood, however. In this study, we attempted to reveal the enzymes involved in DHA synthesis in thraustochytrids. Nine thraustochytrid strains representing 3 genera (Aurantiochytrium, Schizochytrium, and Thraustochytrium) were used for PCR-based detection of the genes encoding Δ5-elongase and Δ4-desaturase and for fatty acid analysis. The degenerate primers were designed to amplify the Δ5-elongase and Δ4-desaturase genes, and the partial sequences of the enzymes were obtained from the genera Thraustochytrium and Schizochytrium. These fragments were identical to those of known Δ5-elongase and Δ4-desaturase. Neither Δ5-elongase nor Δ4-desaturase was detected in the strains belonging to the genus Aurantiochytrium, however, suggesting that this group likely synthesizes DHA not via the elongation/desaturation pathway but via an alternate pathway such as the polyketide synthase pathway. The fatty acid profiles of thraustochytrids were consistent with the presence of genes involved in PUFA biosynthesis in thraustochytrid genera. Thus, our fi ndings suggest that two biosynthetic pathways for PUFAs exist in these organisms. © 2011 by Japan Oil Chemists' Society.

DOI： 10.5650/jos.60.475

Increase of eicosapentaenoic acid in thraustochytrids through thraustochytrid ubiquitin promoter-driven expression of a fatty acid Δ5 desaturase gene

ラビリンチュラ類の脂質蓄積関連タンパク質の単離と機能解析 Reviewed

山元悠樹、小林巧、安部英理子、坂口圭史、沖野望、林雅弘、沖田裕司、杉本愼一、伊東信

脂質生化学論文集 2011.7

Language：Japanese Publishing type：Research paper (scientific journal)

The Distribution of Extracellular Cellulase Activity in Marine Eukaryotes, Thraustochytrids Reviewed

Naoki Nagano, Yousuke Taoka, Yuji Okita, Hitoshi Izumida, Shinichi Sugimoto, Masahiro Hayashi

Marine Biotechnology 13 133 - 136 2011.6

Language：English Publishing type：Research paper (scientific journal)

Molecular cloning of a Pinguiochrysis pyriformis oleate-specific microsomal Δ12-fatty acid desaturase and functional analysis in yeasts and thraustochytrids Reviewed

Matsuda T, Sakaguchi K, Kobayashi T, Abe E, Kurano N, Sato A, Okita Y, Sugimoto S, Hama Y, Hayashi M, Okino N, Ito M.

Journal of Biochemistry 150 2011.6

Language：English Publishing type：Research paper (scientific journal)

Kobayashi T., Sakaguchi K., Matsuda T., Abe E., Hama Y., Hayashi M., Honda D., Okita Y., Sugimoto S., Okino N., Ito M.

Applied and Environmental Microbiology 77 ( 11 ) 3870 - 3876 2011.6

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Applied and Environmental Microbiology

Thraustochytrids, marine protists known to accumulate polyunsaturated fatty acids (PUFAs) in lipid droplets, are considered an alternative to fish oils as a source of PUFAs. The major fatty acids produced in thraustochytrids are palmitic acid (C 16:0 ), n - 6 docosapentaenoic acid (DPA) (C 22:5n - 6 ), and docosahexaenoic acid (DHA) (C 22:6n - 3), with eicosapentaenoic acid (EPA) (C 20:5n - 3) and arachidonic acid (AA) (C 20:4n - 6 ) as minor constituents. We attempted here to alter the fatty acid composition of thraustochytrids through the expression of a fatty acid Δ5 desaturase gene driven by the thraustochytrid ubiquitin promoter. The gene was functionally expressed in Aurantiochytrium limacinum mh0186, increasing the amount of EPA converted from eicosatetraenoic acid (ETA) (C 20:4n - 3 ) by the Δ5 desaturase. The levels of EPA and AA were also increased by 4.6and 13.2-fold in the transgenic thraustochytrids compared to levels in the mock transfectants when ETA and dihomo-γ-linolenic acid (DGLA) (C 20:3n - 6) were added to the culture at 0.1 mM. Interestingly, the amount of EPA in the transgenic thraustochytrids increased in proportion to the amount of ETA added to the culture up to 0.4 mM. The rates of conversion and accumulation of EPA were much higher in the thraustochytrids than in baker's yeasts when the desaturase gene was expressed with the respective promoters. This report describes for the first time the finding that an increase of EPA could be accomplished by introducing the Δ5 desaturase gene into thraustochytrids and indicates that molecular breeding of thraustochytrids is a promising strategy for generating beneficial PUFAs. © 2011, American Society for Microbiology.

DOI： 10.1128/AEM.02664-10

The Distribution of Extracellular Cellulase Activity in Marine Eukaryotes, Thraustochytrids

Naoki Nagano, Yousuke Taoka, Yuji Okita, Hitoshi Izumida, Shinichi Sugimoto, Masahiro Hayashi

Marine Biotechnology 13 133 - 136 2011.6

Language：Japanese Publishing type：Research paper (scientific journal)

Effect of Tween 80 on the growth, lipid accumulation and fatty acid composition of Thraustochytrium aureum ATCC 34304

Effects of cold shock teratment on total lipid content and fatty acid composition of Aurantiochytrium limacinum strain mh0186 Reviewed

Taoka, Y., Nagano, N., Okita, Y., Izumida, H., Sugimoto, S., Hayashi, M.

Journal of Oleo Science 60 ( 5 ) 217 - 220 2011.5

Language：English Publishing type：Research paper (scientific journal)

Taoka Yousuke, Nagano Naoki, Okita Yuji, IZUMIDA Hitoshi, SUGIMOTO Shinichi, HAYASHI Masahiro

Journal of bioscience and bioengineering 111 ( 4 ) 420 - 424 2011.4

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：公益社団法人日本生物工学会

Thraustochytrium aureum ATCC 34304 was grown in the presence and absence of polyoxyethylene sorbitan monooleate (Tween 80). The aim of this work was to obtain basic knowledge about the effect of Tween 80 on growth, lipid accumulation and fatty acid composition in T. aureum. The addition of Tween 80 to a culture medium significantly enhanced the growth of T. aureum, and the biomass increased with an increase of Tween 80 content. Total lipid content and total fatty acid content were significantly higher in 1.0% Tween 80 in comparison with the control (absence of Tween 80). The fatty acid profile showed that the content of C18:1n-9 (oleicacid) significantly increased as a result of the addition of Tween 80. These results indicated that part of the Tween 80 added to the medium was utilized as a carbon source or that the oleate included in Tween 80 was directly incorporated into T. aureum cells as a fatty acid. Neither the DHA content nor the percentage of DHA did not change in spite of the addition of Tween 80. However, the DHA yield significantly increased because the biomass increased due to the addition of Tween 80.

Increase of EPA in Thraustochytrids through Expression of a Fatty Acid 5 Desaturase Gene driven by the Thraustochytrid Ubiquitin Promoter

Takumi Kobayashi1, Keishi Sakaguchi1, Takanori Matsuda1, Eriko Abe1, Yoichiro Hama2, Masahiro Hayashi3, Daisuke Honda4, Yuji Okita5, Shinichi Sugimoto5,Nozomu Okino1 and Makoto Ito1,6,

Journal of Biochemistry 2011.1

Language：Japanese Publishing type：Research paper (scientific journal)

Use of an antifungal drug, amphotericin B for isolation of thraustochytrids

TAOKA Yousuke, NAGANO Naoki, OKITA Yuji, IZUMIDA Hitoshi, SUGIMOTO Shinichi, HAYASHI Masahiro

Journal of bioscience and bioengineering 110 ( 6 ) 720 - 723 2010.12

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：The Society for Biotechnology, Japan

The inhibitory effect of amphotericin B (AMPH) on the growth of fungi during the isolation of thraustochytrids was examined. The growth of fungi was significantly inhibited by addition of AMPH, and therefore colonies of thraustochytrids were not overlaid with fungal mycelia, which resulted in increased efficiency of thraustochytrids isolation.

Effect of nutritional enrichment method on fatty acid contents of rotifer Brachionus plicatilis

Kotani T., Genka T., Tanabe M., Miyashima A., Fushimi H., Hayashi M.

Journal of the World Aquaculture Society 41 ( 6 ) 884 - 892 2010.12

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of the World Aquaculture Society

Nutritional enrichment for rotifer is necessary for the stable production in larviculture. Although manufacturers recommend feeding amount for enrichment diets, the effects of these recommended amounts on nutritional enrichment of rotifer are unknown. We assessed the effects of normal and overdose amount of enrichment diets on nutritional enrichment of rotifer using batch and continuous culture methods. Rotifer populations in two culture systems were enriched using four enrichment conditions, matched two diet levels, and two enrichment durations: 0.25 g/L for 8 h (normal enrichment) 0.25 g/L for 24 h (longer enrichment), 0.75 g/L for 8 h (overdose enrichment), and 0.75 g/L for 24 h (overdose + longer enrichment). When the batch-cultured rotifer populations were enriched, longer and overdose enrichments could not make the fatty acid contents differ from the normal one. Overdose enrichment was most effective of all enrichment conditions when continuous-cultured rotifer populations were used. In both culture methods, the docosahexaenoic acid (DHA) content under overdose + longer enrichment conditions was two to three times as high as normal enrichment conditions and it resulted in two- to threefold increase in DHA/eicosapentaenoic acid ratio by overdose + longer enrichment. Accordingly, it is possible to alter the fatty acid composition of rotifer by changing the enrichment method. © by the World Aquaculture Society 2010.

DOI： 10.1111/j.1749-7345.2010.00431.x

物質生産ツールとしての微細藻 : 電子指向型バイオテクノロジー(e-バイオ)を起点とする藻類工学の夜明け(第62回大会シンポジウム報告)

石井正治, 林雅弘

生物工学会誌 : seibutsu-kogaku kaishi 88 ( 12 ) 2010

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：日本生物工学会

Optimization of culture conditions for growth and docosahexaenoic acid production by a marine thraustochytrid, Aurantiochytrium limacinum mh0186

Nagano N., Taoka Y., Honda D., Hayashi M.

Journal of Oleo Science 58 ( 12 ) 623 - 628 2009.11

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Oleo Science

The effects of carbon sources, seawater concentration and seawater component in a culture medium were investigated to optimize culture conditions for growth by a marine thraustochytrid Aurantiochytrium limacinum strain mh0186. Strain mh0186 could utilize D-glucose, D-fructose and D- mannose as carbon sources. Seawater concentrations between 12.5 - 200% were required for good growth, and a single omission of magnesium sulfate from the seawater reduced the growth of the cells. Jar fermentor trials were carried out for the purpose of docosahexaenoic acid (DHA) production by strain mh0186. The total fatty acid content of the cell was 466.5 mg/g dry cells, and biomass and DHA yield were estimated as 23.1 g/L and 4.3 g/L, respectively, at 26 h. The daily production of DHA by the strain was 4.0 g/L/d, suggesting that the higher DHA production rate of our strain mh0186 should be appropriate for industrial production of DHA. © 2009 by Japan Oil Chemists' Society.

Effect of cultivation methods on nutritional enrichment of euryhaline rotifer Brachionus plicatilis

KOTANI Tomonari, GENKA Teruhisa, FUSHIMI Hiroshi, HAYASHI Masahiro, DIERCKENS Kristof, SORGELOOS Patrick

Fisheries science : FS 75 ( 4 ) 975 - 984 2009.7

Language：Japanese Publishing type：Research paper (scientific journal)

Extracellular enzymes produced by marine eukaryotes, thraustochytrids

Influence of Culture Temperature on the Growth, Lipid Content and Fatty Acid Composition of Aurantiochytrium sp. Strain mh0186 Reviewed

Yousuke Taoka, Naoki Nagano, Yuji Okita, Hitoshi Izumida, Shinichi Sugimoto, Masahiro Hayashi

Marine Biotechnology 11 368 - 374 2009.6

Language：English Publishing type：Research paper (scientific journal)

Taoka Y., Nagano N., Okita Y., Izumida H., Sugimoto S., Hayashi M.

Bioscience, Biotechnology and Biochemistry 73 ( 1 ) 180 - 182 2009.2

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Bioscience, Biotechnology and Biochemistry

Extracellular enzymes produced by six strains of thraustochytrids, Thraustochytrium, Schizochytrium, and Aurantiochytrium, were investigated. These strains produced 5 to 8 kinds of the extracellular enzymes, depending on the species. Only the genus Thraustochytrium produced amylase. When insoluble cellulose was used as substrate, cellulase was not detected in the six strains of thraustochytrids. This study indicates that marine eukaryotes, thraustochytrids, produced a wide variety of extracellular enzymes.

DOI： 10.1271/bbb.80416

Effect of Addition of Tween 80 and Potassium Dihydrogenphosphate to Basal Medium on the Isolation of Marine Eukaryotes, Thraustochytrids

Taoka Yousuke, Nagano Naoki, Okita Yuji, IZUMIDA Hitoshi, SUGIMOTO Shinichi, HAYASHI Masahiro

Journal of bioscience and bioengineering 105 ( 5 ) 562 - 565 2008.5

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：公益社団法人日本生物工学会

Tween 80, KH_2PO_4 and tomato juice were added to basal medium for the isolation of thraustochytrids. By the addition of Tween 80 and KH_2PO_4, the number of thraustochytrids isolated from seawater increased. KH_2PO_4 and Tween 80 were considered to be useful for isolating thraustochytrids.

Effect of primary cultivation method and population growth phase on nutritional enrichment of euryhaline rotifer Brachionus plicatilis(Asian-Pacific Aquaculture 2007,Abstracts of Conference)

Kotani Tomonari, Genka Teruhisa, Fushimi Hiroshi, Hayashi Masahiro, Dierckens Kristof, Sorgeloos Patrick

福山大学内海生物資源研究所報告 18 11 - 12 2008.2

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：福山大学

Occurrence of 5′-deoxyadenosylcobalamin and its physiological function as the coenzyme of methylmalonyl-CoA mutase in a marine eukaryotic microorganism, Schizochytrium limacinum SR21

Miyamoto E., Tanioka Y., Yukino T., Hayashi M., Watanabe F., Nakano Y.

Journal of Nutritional Science and Vitaminology 53 ( 6 ) 471 - 475 2007.12

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Nutritional Science and Vitaminology

A marine eukaryotic microorganism, Schizochytrium limacinum SR21, had the ability to absorb and accumulate exogenous cobalamin, which was converted to the cobalamin coenzymes 5′-deoxyadenosylcobalamin (20.1%) and methylcobalamin (29.6%). A considerably high activity (about 38 mU/mg protein) of 5′-deoxyadenosylcobalamin-dependent methylmalonyl-CoA mutase (EC 5.4.99.2) involved in amino acid and odd-chain fatty acid metabolism was found in the cell homogenate of S. limacinum SR21. The enzyme was purified to homogeneity and characterized.

DOI： 10.3177/jnsv.53.471

Effect of primary cultivation method and population growth phase on nutritional enrichment of euryhaline rotifer Brachionus plicatilis

Kotani Tomonari, Genka Teruhisa, Fushimi Hiroshi, Hayashi Masahiro

Annual report of Faculty of Life Science and Biotechnology, Fukuyama University 6 35 - 49 2007.9

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Fukuyama University

It is important to evaluate the effect of the primary cultivation method of rotifers on the secondary cultivation as nutritional enrichment. So far, various methods of rotifer cultivation have been developed. Recently, mainly two methods are performed, batch or continuous culture. This study aimed to clarify the fatty acid contents after the nutritional enrichment in order to evaluate the quality of rotifers cultured with different methods. Two primary rotifer cultures were performed using batch or continuous methods. From the batch culture, three experimental populations were used; they were taken from the culture one, 24 and 48 hour after inoculation of the rotifers. The continuous culture was performed with two tanks; one was for cultivation with continuous feeding and water supply (cultivation tank), and another was for stocking from cultivation tank by over flow (harvest tank). From the continuous culture, two experimental populations were used from the cultivation and harvest tanks. Secondary cultures were performed after each primary culture and each rotifer population was nutritionally enriched with Nannochloropsis oculata and a commercial nutritional enrichment diet. After secondary culture, the fatty acid contents of each population were analyzed using gas chromatography. Although there was no significant difference of lipid quantity among primary rotifer cultures, in both cases of secondary culture, total n-3 HUFA quantity from both continuous culture populations was higher than that from batch culture population one and 48 hour after inoculation. When the enrichment was performed with N. oculata, rotifer populations from the two tanks of continuous culture and the batch culture tank 24 hour after inoculation contained a higher quantity of ARA and EPA than those from the two other samples taken from the batch culture. When the enrichment was performed with enrichment diet, populations from the two tanks of continuous culture and the sample taken 24 hour after inoculation from the batch culture tank contained higher quantities of ARA, EPA and DHA than those from the two other samples taken from the batch culture.

Effect of vitamin B12-enriched thraustochytrids on the population growth of rotifers

Hayashi M., Yukino T., Watanabe F., Miyamoto E., Nakano Y.

Bioscience, Biotechnology and Biochemistry 71 ( 1 ) 222 - 225 2007.1

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Bioscience, Biotechnology and Biochemistry

Newly isolated thraustochytrids showed uptake of vitamin B12 from the culture into the cells. Cultivation of thraustochytrids in a medium containing 1 μg/ml of vitamin B12 greatly increased the contents of vitamin B12 in the cells. Similarly to Schizochytrium limacinum, odd numbered fatty acids decreased in the cells of new isolates cultivated with vitamin B12. Vitamin B12-enriched thraustochytrids, strain mh0186, enhanced the population growth of rotifers fed on the cells as sole feed.

DOI： 10.1271/bbb.60308

Rotifers fed with n-3 highly unsaturated fatty acid-enriched Chlorella vulgaris are suitable for the rearing of larval red sea bream Pagrus major. Reviewed

Maruyama, I., Yamamoto, S., Hayashi, M., Murata, O.

Aquaculture Science 54 229 - 230 2006.11

Language：English Publishing type：Research paper (scientific journal)

Hemolytic suppression roles of taurine in yellowtail Seriola quinqueradiata fed non-fishmeal diet based on soybean protein

Takagi, S., Murata, H., Goto, T., Hayashi, M., Hatate, H., Endo, M., Yamashita, M., Ukawa, M.

Fisheries Science 72 546 - 555 2006.10

Language：Japanese Publishing type：Research paper (scientific journal)

A Novel Phosphatidylcholine Which Contains Pentadecanoic Acid at sn-1 and Docosahexaenoic Acid at sn-2 in Schizochytrium sp. F26-b

Abe Eriko, Hayashi Yasuhiro, Hama Yoichiro, HAYASHI Masahiro, INAGAKI Masanori, ITO Makoto

The journal of biochemistry 140 ( 2 ) 247 - 253 2006.8

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Japanese Biochemical Society

n-3高度不飽和脂肪酸強化クロレラChlorella vulgarisで培養したシオミズツボワムシによるマダイPagrus major仔魚の飼育

MARUYAMA Isao, YAMAMOTO Shinji, HAYASHI Masahiro, MURATA Osamu

水産増殖 54 ( 2 ) 229 - 230 2006.6

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：水産増殖談話会

Rotifers Fed with n-3 Highly Unsaturated Fatty Acid-enriched Chlorella vulgaris are Suitable for the Rearing of Larval Red Sea Bream Pagrus major

MARUYAMA Isao, YAMAMOTO Shinji, HAYASHI Masahiro, MURATA Osamu

水産増殖 54 ( 2 ) 229 - 230 2006

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：日本水産増殖学会

Rearing of larval red sea bream Pagrus major was performed by feeding with rotifers cultured in Chlorella vulgaris K-22 enriched with n-3HUFAs (mainly DHA) . Results for larval growth, survival rate, activity, and air-bladder opening rate suggested the successful rearing of the red sea bream. The total content of n-3HUFAs in the larvae at the end of the rearing was even higher than the level immediately after hatching. It can therefore be concluded that the enhanced n-3HUFAs in C. vulgaris were effective via rotifer for the rearing of red sea bream larvae. The n-3HUFA-enriched C. vulgaris makes it possible to realize the rearing of red sea bream without a secondary rotiferculture process for enhancement of n-3HUFAs.

DOI： 10.11233/aquaculturesci1953.54.229

2・3 ワムシの栄養強化方法(2.集中課題「ワムシ-その生物学、培養法から餌料価値-」,第三回因島種苗生産技術交流会,学会講演要旨)

Effect of dietary laurate on lipid accumulation and vitality in juvenile Pagrus major

共著者：Hong, J., Om, A. D., Umino, T., Yoshimatsu, T., Hayashi, M., Nakagawa, H., Asano, M., Nakagawa, A.

Journal of Fisheries of ChinaVol. 29, 804-810 2005.12

林雅弘

福山大学内海生物資源研究所報告 16 25 - 26 2005.12

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：福山大学

Developmental Stage and Morphogenesis of Finfish Larvae, with special reference to Improvement of Larval Health(larvi 2005 (4th fish & shellfish larviculture symposium),Abstracts of Conference)

伏見浩, 小谷知也, 鈴木久英, Hayashi M.

福山大学内海生物資源研究所報告 16 18 - 20 2005.12

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：福山大学

Purification and characterization of a corrinoid compound from a Japanese salted and fermented salmon kidney "mefun"

Adachi S., Miyamoto E., Watanabe F., Enomoto T., Kuda T., Hayashi M., Nakano Y.

Journal of Liquid Chromatography and Related Technologies 28 ( 16 ) 2561 - 2569 2005.10

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Liquid Chromatography and Related Technologies

Significant amounts of vitamin B 12 (about 116.3 ∼ 556.3 μg/100 g) were determined in a Japanese salted and fermented salmon kidney "Mefun." A corrinoid compound was purified to homogeneity from Mefun and partially characterized. TLC and HPLC patterns of the purified corrinoid compound were identical to those of authentic vitamin B 12 , but not to inactive corrinoids. The vitamin B 12 found in Mefun was not derived from concomitant vitamin B 12 -synthesizing bacteria, but had accumulated in the salmon kidney. Gel filtration experiments demonstrated that most of the vitamin B 12 found in Mefun was recovered in the free vitamin B 12 fractions. These results indicate that Mefun would be an excellent vitamin B 12 (free form) source for elderly persons with food-bound vitamin B 12 malabsorption. Copyright © Taylor & Francis, Inc.

DOI： 10.1080/10826070500189778

Preparation of docosahexaenoic acid fortified Spirulina platensis and its lipid and fatty acid compositions

ドコサヘキサエン酸富化スピルリナSpilurina platensis の調製と脂質特性

共著者：雪野繼代,　林　雅弘, 井上良計, 今村純子, 長野直樹, 村田　寿

日本水産学会誌71巻 1号74-79頁 2005.1

Incorporation of exogenous docosahexaenoic acid into triacylglycerols and polar lipids of Chlorella vulgaris

共著者：T. Yoshimatsu, T. Higuchi, D. Zhang, Y. Hamasaki, K. Tanaka, T. Yukino, M. Hayashi

Journal of OleoscienceVol. 54, No. 115-19 2005.1

YUKINO TUGIYO, HAYASHI MASAHIRO, INOUE YOSHIKAZU, IMAMURA JUNKO, NAGANO NAOKI, MURATA HISASHI, TSUGIYO YUKINO, MASAHIRO HAYASHI, YOSHIKAZU INOUE, JUNKO IMAMURA, NAOKI NAGANO, HISASHI MURATA, United Graduate School of Agriculture Kagoshima University:(Present address)University of Miyazaki, Department of Biological Production and Environmental Science Faculty of Agriculture University of Miyazaki, CycloChem Co. Ltd, Bizen Chemical Co. Ltd., Department of Biological Production and Environmental Science Faculty of Agriculture University of Miyazaki, Department of Biological Production and Environmental Science Faculty of Agriculture University of Miyazaki

NSUGAF 71 ( 1 ) 74 - 79 2005.1

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：The Japanese Society of Fisheries Science

Nutritional fortification of Spirulina with docosahexaenoic acid (DHA) was attempted. Fish oil, DHA-ethyl and free DHA were supplemented into the culture of Spirulina as a source of DHA. Exogenous DHA in triglycerides and ethyl ester were not taken up into Spirulina cells. However, the cells took up the DHA in free form from the culture media. The cells without supplementation with DHA did not contain DHA in the cellular fatty acids. The DHA content of the cells was greatly increased by supplementation with DHA into the culture media. It reached to 70.2% of the total fatty acid in the cells. DHA was mainly incorporated into the neutral lipids such as monoglycerides and diglycerides in the cells. Free fatty acids were slightly detected in the DHA-fortified cells. A small amount of DHA was also incorporated into glycolipid and phospholipid fractions. Sulfoquinovosyldiacylglycerol in the DHA-fortified Spirulina contained 9.1% of DHA in the total fatty acid. However, only 1-2% of DHA were incorporated into the other polar lipids.

DOI： 10.2331/suisan.71.74

Incorporation of exogenous docosahexaenoic acid into triacylglycerols and polar lipids of Chlorella vulgaris

共著者：T. Yoshimatsu, T. Higuchi, D. Zhang, Y. Hamasaki, K. Tanaka, T. Yukino, M. Hayashi

Journal of OleoscienceVol. 54, No. 115-19 2005.1

Language：Japanese Publishing type：Research paper (scientific journal)

ドコサヘキサエン酸富化スピルリナSpilurina platensis の調製と脂質特性

共著者：雪野繼代,　林　雅弘, 井上良計, 今村純子, 長野直樹, 村田　寿

日本水産学会誌71巻 1号74-79頁 2005.1

Language：Japanese Publishing type：Research paper (scientific journal)

Incorporation of Exogenous Docosahexaenoic Acid into Triacylglycerols and Polar Lipids of Chlorella vulgaris

Yukino T., Hayashi M., Maruyama I., Murata H.

Journal of Oleo Science 54 ( 1 ) 15 - 19 2005

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Oleo Science

Docosahexaenoic acid (DHA)-enriched Chlorella vulgaris is commonly used as a feed in fisheries and as a dietary supplement. The time course DHA uptake into the Chlorella cells and incorporation of DHA into the triacylglycerols (TAG) and polar lipids (PL) were examined. After the addition of DHA into the Chlorella culture, DHA was rapidly taken up by the Chlorella cells and was mainly incorporated into TAG. However, some DHA was also incorporated into PL molecules. Although the percentage of DHA in the total fatty acids of total cellular lipids reached at 67%, the percentage in the total fatty acids of PL was 27.5%. As for the effects of pH, exogenous DHA was mainly incorporated into TAG molecules at pH 5-7. At pH 3-4 and 8, the DHA accumulated as free fatty acid in the cells. The exogenous DHA was rapidly incorporated into TAG molecules within 24 hours at pH 5-8. The findings should contribute profitably to production of better fishery feed and food supplement by using DHA-enriched Chlorella. © 2005, Japan Oil Chemists' Society. All rights reserved.

DOI： 10.5650/jos.54.15

DHA強化クロレラ Chlorella vulgaris K-22 によるワムシ培養と栄養強化

雪野繼代, 林雅弘, 吉松隆夫, 丸山功, 村田寿

水産増殖 52 ( 4 ) 381 - 386 2004.12

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：水産増殖談話会

ドコサヘキサエン酸（DHA）を強化したクロレラ Chlorella vulgaris K-22によるワムシ培養の効果を検討した。各種濃度でDHAを強化したクロレラ細胞には主として中性脂質にDHAが取り込まれており、総脂質中のDHA含有率は最高82.9％に達した。DHA強化クロレラを給餌したワムシ中には6時間で乾物中3.3％のDHAが蓄積し、短時間でワムシの栄養強化が可能であった。また、3日間によるワムシ培養では通常のクロレラ給餌より高い増殖が得られた。培養期間中、ワムシ中のDHA含有量は安定して高い値を示し、DHA強化クロレラ給餌によるワムシ培養で、栄養強化処理の必要ないレベルのDHAを含有するワムシを安定して得ることができた。

Effect of conjugated linoleic and docosahexaenoic acids on growth of juvenile tilapia Oreochromis niloticus

Nutritional enrichment and cultivation of rotifers by feeding of docosahexaenoic acid enriched Chlorella vulgaris K-22

共著者：Yukino, T., Hayashi, M., Yoshimatsu, T., Maruyama, I., Murata, H.

SuisanzoushokuVol. 52, No. 4381-386 2004.8

Yasmin Afsana, Takeuchi Toshio, Hayashi Masahiro, HIROTA TETSUYA, ISHIZUKA WAICHI, ISHIDA SHUZO

Fisheries science : FS 70 ( 3 ) 473 - 481 2004.6

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Japanese Society of Fisheries Science

A Technique to Enrich α-tocopherol in Clilorella and Marine Rotifers

NAGANO Naoki, HAYASHI Masahiro, YUKINO Tsugiyo, MURATA Hisashi

水産増殖 = The aquiculture 51 ( 4 ) 459 - 460 2003.12

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：日本水産増殖学会

α-Tocopherol (TOC) and various fatty acids were supplemented into the Chlorella vulgaris strain K-22 culture at 28°C for 24 h, and TOC content of the Chlorella cells was determined. The addition of TOC alone into the Chlorella culture medium did not give an increase of the cellular TOC content. However, the TOC contents in the Chlorella cells supplemented with TOC and various fatty acids were much higher than those supplemented with TOC alone. Moreover, the TOC content in the cells supplemented with a mixture of TOC and oleic acid was higher than those supplemented with a mixture of TOC and docosahexaenoic acid (DHA) . The TOC content of the rotifers fed on the TOC-enriched Chlorella was much higher than rotifers fed on unenriched Chlorella. On the other hand, rotifers could be easily enriched with TOC by feeding the TOC-enriched Chlorella cells.

DOI： 10.11233/aquaculturesci1953.51.459

Effect of dietary vitamins C and E fortification on lipid metabolism in red sea bream Pagrus major and black sea bream Acanthopagrus schlegeli

Ji Hong, Om Ahmad Daud, Yoshimatsu Takao, HAYASHI MASAHIRO, UMINO TETSUYA, NAKAGAWA HEISUKE, ASANO MASAYA, NAKAGAWA ATSUSHI

Fisheries science : FS 69 ( 5 ) 1001 - 1009 2003.10

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Japanese Society of Fisheries Science

Effect of Sulfoquinovosyldiacylglycerol (SQDG) extracted from Sea alga (Porphyra yezoensis) on Morphological Differentiation and Apoptosis in Neuro2a Neuroblastoma Cells

NOGUCHI Satoko, AKIYAMA Jun, HADA Takahiro, INOUE Yoshikazu, ARAKI Shigeru, YUKINO Tsugiyo, HAYASHI Masahiro, TADA Mikiro, TAKAHATA Kyoya

Japanese journal of food chemistry 10 ( 2 ) 101 - 107 2003.8

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Japanese Society of Food Chemistry

About 70% of total lipids in porphyra yezoensis, which is processed for use as edible sea alga, is composed with three types of glycolipids : MGDG (monogalactosyldiacylglycerol), DGDG (digalactosyldiacylglycerol), and SQDG (sulfoquinovosylgalactosyldiacylglycerol). In these glycolipids, SQDG is reported as an inhibitor of DNA synthesis. In this paper, with the aim of studying more about the physiological functions of SQDG extracted from sea alga (A-SQDG), we investigate its effects on the growth and differentiation of murine neuroblastoma cells (Neuro2a). These glycolipids (MGDG, DGDG, and SQDG) extracted from sea alga inhibited the cell growth of Neuro2a, but this inhibition was most potent in A-SQDG, which was extracted from alga. We therefore compared this inhibitory effect of A-SQDG with S-SQDG or C-SQDG extracted from spinach or chlorella, respectively. It was found that A-SQDG and S-SQDG had an equivalent inhibitory effect on cell growth, but C-SQDG had none at all. Furthermore, we investigated the effects of these three types of SQDG on neuritogenesis in Neuro2a cells. We found that A-SQDG revealed a potent effect on neuritogenesis than that by retinoic acid (RA) known as a potent reagent of neuritogenesis. It was also found that S-SQDG revealed the same degree of neuritogenesis as RA, but no neuritogenesis could be found in C-SQDG. The different effects of these three types of SQDG are thought to be originated in their molecular compositions of acyl group. The caspase-3 activity was known as a maker enzyme of apoptosis. As a result, A-SQDG, S-SQDG and C-SQDG almost inhibited etoposide-induced apoptosis. These SQDG were expected to be reagents to prevent neuronal cell death in Alzheimer's disease.

Preparation and Utilization of the Chlorella Cells Enriched with Docosahexaenoic Acid

HAYASHI Masahiro

NIPPON SHOKUHIN KOGYO GAKKAISHI 50 ( 6 ) 289 - 292 2003.6

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Japanese Society for Food Science and Technology

DOI： 10.3136/nskkk.50.289

ドコサヘキサエン酸富化Chlorella vulgaris CK22株のヒトにおける安全性および血清脂質濃度に対する影響の予備的検討

共著者田中邦明，小西史子，丸山　功，雪野繼代，木柎久雄，熊谷多妙子，羽田尚彦，林　雅弘

日本栄養・食糧学会誌，55巻，6号，323-330頁 2002.12

Language：Japanese Publishing type：Research paper (scientific journal)

ドコサヘキサエン酸富化Chlorella vulgaris CK22株の脂質特性

共著者：雪野繼代，田中邦明，丸山　功，小西史子，熊谷多妙子，羽田尚彦，林　雅弘

日本栄養・食糧学会誌，55巻，6号，331-337頁 2002.12

Language：Japanese Publishing type：Research paper (scientific journal)

Isolation of highly DHA-accumulated Labyrinthulales and their utilization for nutritional enrichment of rotifers and Artemia

Distribution of docosahexaenoic acid in DHA-enriched Euglena gracilis

共著者：Hayashi, M., Yukino, T., Park, B. S.

Fisheries Science, Vol. 68, Supplement 1,1002-1003 2002.11

A preliminary experiment on the dietary value of vitamin E-fortified Chlorella for rotifer

共著者Yoshimatsu, T., Furusawa, T., Hayashi, M.：

Fisheries Science, Vol. 68, Supplemen1,1004-1005 2002.11

Effect of Cobalt Compound on Population Growth of Rotifer

共著者：Yoshimatsu, T., Higuchi, T., Shirakawa, K., Hayashi, M., Tanaka, K., Yoshimura, K.

Proceeding of the 5th Korea-Japan (Japan-Korea) Biannual Joint Meeting, Kunsan National University, Kunsan, Koreap68 2002.10

ドコサヘキサエン酸高蓄積性ラビリンチュラ類の分離と栄養強化飼料としての利用

共著者：林　雅弘，松本竜一，吉松隆夫，田中悟広，清水　昌

日本水産学会誌，68巻，5号，674-678頁 2002.9

Hayashi M., Matsumoto R., Yoshimatsu T., Tanaka S., Shimizu S.

Nippon Suisan Gakkaishi (Japanese Edition) 68 ( 5 ) 674 - 678 2002.9

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Nippon Suisan Gakkaishi (Japanese Edition)

Twelve strains of highly DHA-accumulated Labyrinthulales were isolated from coastal sea water in Japan. The lipid contents in the cells and the DHA contents in the total fatty acids of these isolates were 13.7-23.0% and 21.5-55.4%, respectively. For the utilization of these isolates as a feed for nutritional enrichment of rotifers and Artemia, dispersibility of the cells in the water was evaluated. The cells of several isolates were agglutinated in sea water or tap water, and cawsed high mortality of rotifers and Artemia during their nutritional enrichment. Strain KY-1, however, showed high DHA content and good dispersibility in the water. Moreover, the DHA in KY-1 cells was rapidly transferred into rotifer and Artemia fed on KY-1 cells during their nutritional enrichment. These results showed that the strain KY-1 would be a novel and profitable feed for nutritional enrichment of rotifers and Artemia.

ドコサヘキサエン酸高蓄積性ラビリンチュラ類の分離と栄養強化飼料としての利用

共著者：林　雅弘，松本竜一，吉松隆夫，田中悟広，清水　昌

日本水産学会誌，68巻，5号，674-678頁 2002.9

Language：Japanese Publishing type：Research paper (scientific journal)

A preliminary experiment on the dietary value of vitamin E-fortified Chlorella for rotifer

ドコサヘキサエン酸を富化したクロレラ・ブルガリス(Chlorella vulgaris) CK22株の投与による高コレステロール摂餌ラット血清脂質上昇抑制作用

共著者：小西史子，田中邦明，丸山　功，雪野繼代，熊谷多妙子，羽田尚彦，林　雅弘

日本栄養・食糧学会誌，55巻，4号，215-222頁 2002.8

YOSHIMATSU TAKAO, FURUSAWA TAKAYOSHI, HAYASHI MASAHIRO

Ｆｉｓｈｅｒｉｅｓ　ｓｃｉｅｎｃｅ 68 ( 1 ) 1004 - 1005 2002

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：公益社団法人日本水産学会

DOI： 10.2331/fishsci.68.sup1_1004

Foreword (Progress in Mass Production Technology of Rotifer and its Current Status)

YOSHIMATSU Takao, HAYASHI Masahiro, HINO Akinori, Takao Yoshimatsu, Masahiro Hayashi, Akinori Hino, Graduate School of Bioresource and Bioenvironmental Sciences, Faculty of Agriculture Miyazaki University, Graduate School of Agricultural and Life Sciences The University of Tokyo

67 ( 6 ) 2001.11

Language：Japanese Publishing type：Research paper (scientific journal)

Novel feed for nutritional enrichment of rotifers

高密度培養時代の新たなワムシ栄養強化餌料の探索

日本水産学会誌，67巻，6号，1146-1147頁 2001.11

Hayashi M.

Nippon Suisan Gakkaishi (Japanese Edition) 67 ( 6 ) 1146 - 1147 2001.11

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Nippon Suisan Gakkaishi (Japanese Edition)

Uptake and accumulation of exogenous docosahexaenoic acid by Chlorella

共著者：Hayashi, M., Yukino, T., Maruyama, I., Kitaoka, S.

Biosci. Biotechnol. Biochem.，Vol. 65, No.1, 202-204 2001.1

Language：Japanese Publishing type：Research paper (scientific journal)

New anthocyanins from purple pods of pea (Pisum spp.)

共著者：Terahara, N., Honda, T., Hayashi, M., Ishimaru, K

Biosci. Biotechnol. Biochem.，Vol. 64, No. 12, 2569-2574 2000.12

Language：Japanese Publishing type：Research paper (scientific journal)

豚内蔵に含まれるアラキドン酸含有リン脂質

共著者：井上良計，林　雅弘，恒吉雅治，工藤　寛

日本油化学会誌，49巻，7号，723-726頁 2000.7

Language：Japanese Publishing type：Research paper (scientific journal)

Suppressive action of docosahexaenoic acid enriched-Euglena on reduction of endothelium-dependent relaxation in stroke-prone spontaneously hypertensive rats (SHRSP)

共著者：Murakami, T., Ogawa, H., Hayashi, M., Yoshizumi, H.

J. Nutr. Sci. Vitaminol.，Vol. 43, No. 2，211-223 1997.4

Language：Japanese Publishing type：Research paper (scientific journal)

Preliminary results in improving essential fatty acids enrichment of rotifer cultured in high density

共著者：Yoshimatsu, T., Imoto, H., Hayashi, M., Toda, K., Yoshimura, K.

Hydrobiologia，Vol. 358，Sup. 153-157 1997.3

Language：Japanese Publishing type：Research paper (scientific journal)

Supplemental effects of Euglena gracilis in a casein diet for Penaeus japonicus

共著者：Hayashi, M., Toda, K., Kanazawa, A., Yamatoya, Y., Kitaoka, S.

Asian Fisheries Science，Vol. 8, No. 3-4，201-209 1995.12

メナダ仔魚の必須脂肪酸要求と飼育槽へのナンノクロロプシスの添加効果

共著者：吉松隆夫，林　雅弘，戸田享次，古市政幸，北島　力

日本水産学会誌，61巻，6号，912-918頁 1995.11

Language：Japanese Publishing type：Research paper (scientific journal)

ユーグレナ（Euglena gracilis Z）加水分解物の降圧作用

共著者：村上哲男，林　雅弘，吉栖　肇

日本栄養・食糧学会誌，48巻，5号，399-405頁 1995.10

Language：Japanese Publishing type：Research paper (scientific journal)

Effect of pH shift in the stationary phase of growth on the incorporation of exogenous docosahexaenoic acid into Euglena gracilis

Hayashi, M., Komatsu, R., Kitaoka, S.

J. Jpn. Oil Chem. Soc.(YUKAGAKU), 44 ( 9 ) 640 - 646 1995.9

Language：Japanese Publishing type：Research paper (scientific journal)

脳卒中易発症性高血圧自然発症ラットの脂質代謝に及ぼすＤＨＡ強化ユーグレナ（Euglena gracilis Z）の影響

村上哲男，小川　博，林　雅弘，吉栖　肇

日本栄養・食糧学会誌，48巻，4号，319-324頁 48 ( 4 ) 319 - 324 1995.8

Language：Japanese Publishing type：Research paper (scientific journal)

ユーグレナ（Euglena gracilis Z）飼料で飼育したSHRSP（脳卒中易発症性高血圧自然発症ラット）の血管組織アンギオテンシンⅠ-変換酵素活性の低下と血管壁の肥厚抑制 Reviewed

村上哲男，林　雅弘，吉栖　肇

日本栄養・食糧学会誌 48 ( 3 ) 203 - 208 1995.6

Language：Japanese Publishing type：Research paper (scientific journal)

脳卒中易発症性高血圧自然発症ラットの血圧，脳血管病変および寿命に及ぼすＤＨＡ強化ユーグレナ（Euglena gracilis Z）の影響

村上哲男，小川　博，林　雅弘，吉栖　肇

日本栄養・食糧学会誌 48 ( 3 ) 209 - 215 1995.6

Language：Japanese Publishing type：Research paper (scientific journal)

Preliminary Experiment on the Requirement of Larval Redlip Mullet for Essential Fatty Acids, and the Supplemental Effect of Nannochloropsis to Rearing Water

Yoshimatsu T., Furuichi M., Kitajima C., Hayashi M., Toda K.

Nippon Suisan Gakkaishi (Japanese Edition) 61 ( 6 ) 912 - 918 1995

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Nippon Suisan Gakkaishi (Japanese Edition)

A feeding experiment for redlip mullet larvae (4.58 ±0.25 mm TL) on the EFA requirement was conducted by using rotifers enriched with ethyl esters of four kinds of fatty acids: linoleic acid, LA; linolenic acid, LNA; eicosapentaenoic acid, EPA; docosahexaenoic acid, DHA. Larvae receiving rotifers enriched with LA and LNA showed typical symptoms of EFA deficiency syndromes, and obtained poor growth and high mortality. On the other hand, the larvae receiving rotifers enriched with EPA and/or DHA showed better growth, activity, and survival rates. These results suggested that the redlip mullet larvae required n-3HUFA like EPA and DHA as their essential fatty acids. Also, the supplemental effect of unicellular alga Nannochloropsis sp. to rearing water was investigated from the view point of EFA requirement. The n-3HUFA content in rotifer cultured with Nannochloropsis decreased linearly after they transferred into sea water. However, when 40–50 x 10 4 cells/m/ of Nannochloropsis was introduced to a rearing tank, the n-3HUFA content in rotifer remained without decreasing for 6 hours. The larvae reared in the tank with Nannochloropsis supplement showed better growth than those of non- or fewer-supplement groups. © 1995, The Japanese Society of Fisheries Science. All rights reserved.

DOI： 10.2331/suisan.61.912

Effects of shifting pH in thestationary phase of growth on the hemical composition of Euglena racilis Reviewed

Hayashi, M., Toda, K., Ishiko, H., Komatsu, R., Kitaoka, S.

Biosci. Biotechnol. Biochem., Vol. 58, No. 11, 1964-1967 58 ( 11 ) 1964 - 1967 1994.11

Language：English Publishing type：Research paper (scientific journal)

ユーグレナ Euglena gracilis による生物餌料の栄養強化とマダイ仔魚に対する餌料価値 Reviewed

林　雅弘，戸田享次，米司　隆，佐藤　修，北岡正三郎

日本水産学会誌 59 ( 6 ) 1051 - 1058 1993.6

Language：Japanese Publishing type：Research paper (scientific journal)

エイコサペンタエン酸およびドコサヘキサエン酸強化 Euglena gracilisの調製

林　雅弘，戸田享次，三澤嘉久，北岡正三郎

水産増殖 41 ( 2 ) 169 - 176 1993.6

Language：Japanese Publishing type：Research paper (scientific journal)

Dietary Value of Rotifers and Artemia Enriched with Euglena gracilis for Red Sea Bream

Enriching Euglena with unsaturated fatty acids Reviewed

Hayashi, M., Toda, K., Kitaoka, S.

Biosci. Biotechnol. Biochem., Vol. 57,No.2, 352-353 57 ( 2 ) 352 - 353 1993.2

Language：English Publishing type：Research paper (scientific journal)

Hayashi M., Toda K., Yoneji T., Sato O., Kitaoka S.

NIPPON SUISAN GAKKAISHI 59 ( 6 ) 1051 - 1058 1993

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：NIPPON SUISAN GAKKAISHI

Since Euglena gracilis incorporate and accumulate n-3 highly unsaturated fatty acids (n-3 HUFA), the fatty acid composition of the cells can be controlled by supplementation of the fatty acids into growth media. Rotifers and Artemia were enriched with Euglena that accumulated docosahexaenoic acid (DHA). The DHA content of the rotifers fed on more than 100000 cells/m/ of Euglena increased in 12 hours. Also, the feeding density of more than 100000 cells/m/ gave an increase in the DHA content in Artemia. As for Artemia, however, DHA was degraded fast after 6 hours of enrichment. A feeding experiment was conducted to evaluate the dietary value of living feed enriched with Euglena. Rotifers and Artemia enriched with Euglena contained much more DHA than those enriched with Nannochloropsis or methyl esters of n-3 HUFA. The larval red sea bream fed on living feed containing more DHA showed higher activity in the activity test. Thus, the dietary value of rotifers and Artemia to red sea bream was effectively improved by enrichment with Euglena. Euglena is expected to be profitable feed for the DHA enrichment of rotifers and Artemia. © 1993, The Japanese Society of Fisheries Science. All rights reserved.

DOI： 10.2331/suisan.59.1051

微生物によるワックスエステルの生産

奥村昌和,井伊茂,市川伸哉,林雅弘

油化学, 36巻, 10号, 802-807頁 36 ( 10 ) 802 - 807 1987.10

Language：Japanese Publishing type：Research paper (scientific journal)