JP6090838B2 - Fluorescent substance and method for producing the same - Google Patents
Fluorescent substance and method for producing the same Download PDFInfo
- Publication number
- JP6090838B2 JP6090838B2 JP2013019971A JP2013019971A JP6090838B2 JP 6090838 B2 JP6090838 B2 JP 6090838B2 JP 2013019971 A JP2013019971 A JP 2013019971A JP 2013019971 A JP2013019971 A JP 2013019971A JP 6090838 B2 JP6090838 B2 JP 6090838B2
- Authority
- JP
- Japan
- Prior art keywords
- fluorescent substance
- strain
- fluorescent
- culture
- alcohol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000000126 substance Substances 0.000 title claims description 78
- 238000004519 manufacturing process Methods 0.000 title claims description 19
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- 239000002904 solvent Substances 0.000 claims description 18
- 125000003118 aryl group Chemical group 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 241000589774 Pseudomonas sp. Species 0.000 claims description 7
- KCDXJAYRVLXPFO-UHFFFAOYSA-N syringaldehyde Chemical compound COC1=CC(C=O)=CC(OC)=C1O KCDXJAYRVLXPFO-UHFFFAOYSA-N 0.000 claims description 6
- COBXDAOIDYGHGK-UHFFFAOYSA-N syringaldehyde Natural products COC1=CC=C(C=O)C(OC)=C1O COBXDAOIDYGHGK-UHFFFAOYSA-N 0.000 claims description 6
- 239000000049 pigment Substances 0.000 description 24
- 239000000463 material Substances 0.000 description 21
- 239000000306 component Substances 0.000 description 14
- 238000000605 extraction Methods 0.000 description 14
- 239000002609 medium Substances 0.000 description 10
- 238000001228 spectrum Methods 0.000 description 10
- 239000012141 concentrate Substances 0.000 description 8
- 229920005610 lignin Polymers 0.000 description 8
- 238000002523 gelfiltration Methods 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 238000005160 1H NMR spectroscopy Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 5
- 238000001157 Fourier transform infrared spectrum Methods 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000002994 raw material Substances 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 239000002028 Biomass Substances 0.000 description 3
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- 150000001299 aldehydes Chemical class 0.000 description 3
- 238000005102 attenuated total reflection Methods 0.000 description 3
- 238000012937 correction Methods 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- KWCCUYSXAYTNKA-UHFFFAOYSA-N 3-O-methylgallic acid Chemical compound COC1=CC(C(O)=O)=CC(O)=C1O KWCCUYSXAYTNKA-UHFFFAOYSA-N 0.000 description 2
- 238000004566 IR spectroscopy Methods 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- -1 aromatic organic compounds Chemical class 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 238000004007 reversed phase HPLC Methods 0.000 description 2
- 239000013535 sea water Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- JMSVCTWVEWCHDZ-UHFFFAOYSA-N syringic acid Chemical compound COC1=CC(C(O)=O)=CC(OC)=C1O JMSVCTWVEWCHDZ-UHFFFAOYSA-N 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- 125000006615 aromatic heterocyclic group Chemical group 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical group 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229920001429 chelating resin Polymers 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229940074391 gallic acid Drugs 0.000 description 1
- 235000004515 gallic acid Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000006325 marine broth Substances 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000011403 purification operation Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- YIBXWXOYFGZLRU-UHFFFAOYSA-N syringic aldehyde Natural products CC12CCC(C3(CCC(=O)C(C)(C)C3CC=3)C)C=3C1(C)CCC2C1COC(C)(C)C(O)C(O)C1 YIBXWXOYFGZLRU-UHFFFAOYSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Description
本発明は、新規の蛍光物質及びその製造方法に関する。 The present invention relates to a novel fluorescent material and a method for producing the same.
リグニンは、植物細胞壁の１５〜３０％を占める主要な成分であり、複雑な構造を有し、未利用バイオマスの一つである。微生物機能を利用して、このようなリグニンを有用性が高い物質に変換する方法の開発は、未利用バイオマスの有効利用という観点から非常に重要である。
このような中、本発明者らは、リグニン由来の成分であるシリングアルデヒドを含有する培地を用いて、特定の微生物を培養することにより、水溶性の黒色色素が得られることを見出している（非特許文献１参照）。
Lignin is a major component that occupies 15-30% of the plant cell wall, has a complex structure, and is one of the unused biomass. Development of a method for converting such lignin into a highly useful substance by utilizing a microbial function is very important from the viewpoint of effective utilization of unused biomass.
Under such circumstances, the present inventors have found that a water-soluble black pigment can be obtained by culturing a specific microorganism using a medium containing shilling aldehyde which is a component derived from lignin ( Non-patent document 1).
一方、有機発光材料は、その多彩で鮮やかな発色性、優れた加工性に加え、検出感度が高く、分子設計により種々の機能付加が可能であり、発光素子、トレーサー、診断薬、試薬等、種々の分野への利用が見込まれることから、優れた化学素材として大きく期待されている。このような有機発光材料のうち、例えば、芳香族環を有する有機蛍光物質については、リグニン由来の成分以外のものを原料として用い、シュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）属の微生物により産生されることが知られている（非特許文献２参照）。 On the other hand, organic light-emitting materials have high detection sensitivity in addition to their colorful and vivid color developability and excellent processability, and various functions can be added by molecular design. Light-emitting elements, tracers, diagnostic agents, reagents, etc. Since it is expected to be used in various fields, it is highly expected as an excellent chemical material. Among such organic light-emitting materials, for example, organic fluorescent materials having an aromatic ring are known to be produced by microorganisms belonging to the genus Pseudomonas using materials other than lignin-derived components as raw materials. (See Non-Patent Document 2).
しかしながら、リグニン由来の成分を原料として用いて新規の蛍光物質を得る試みは、その蛍光物質の有用性の高さに比して、未だ十分に為されたとはいえないのが実情である。 However, in reality, attempts to obtain a new fluorescent material using a component derived from lignin as a raw material have not yet been made sufficiently compared to the high usefulness of the fluorescent material.
本発明は上記事情に鑑みて為されたものであり、リグニン由来の成分を原料として得られる新規の蛍光物質、及びその製造方法を提供することを課題とする。 This invention is made | formed in view of the said situation, and makes it a subject to provide the novel fluorescent material obtained by using the component derived from lignin as a raw material, and its manufacturing method.
上記課題を解決するため、
本発明は、シュードモナス・エスピー（Ｐｓｅｕｄｏｍｏｎａｓ ｓｐ．）ＩＴＨ−ＳＡ−１株（ＮＩＴＥ Ｐ−１５２１）を、シリングアルデヒド共存下で培養して得られた蛍光物質を提供する。
To solve the above problem,
The present invention provides a fluorescent substance obtained by culturing Pseudomonas sp. ITH-SA-1 strain (NITE P-1521 ) in the presence of syringaldehyde.
本発明の蛍光物質は、シュードモナス・エスピー（Ｐｓｅｕｄｏｍｏｎａｓ ｓｐ．）ＩＴＨ−ＳＡ−１株（ＮＩＴＥ Ｐ−１５２１）を、シリングアルデヒド共存下で培養し、得られた培養物から、アルコール又はアルコール及び水を含有する溶媒で抽出して得られたものが好ましい。
本発明の蛍光物質は、芳香族環を有しないものが好ましい。
本発明の蛍光物質は、分子量が２〜９ｋＤａであるものが好ましい。
本発明の蛍光物質は、前記アルコールがメタノールであるものが好ましい。
The fluorescent substance of the present invention is obtained by culturing Pseudomonas sp. ITH-SA-1 strain (NITE P-1521 ) in the presence of syringaldehyde, and alcohol or alcohol and water are obtained from the obtained culture. What was obtained by extracting with the solvent to contain is preferable.
The fluorescent substance of the present invention preferably has no aromatic ring.
The fluorescent substance of the present invention preferably has a molecular weight of 2 to 9 kDa.
The fluorescent substance of the present invention is preferably such that the alcohol is methanol.
また、本発明は、シュードモナス・エスピー（Ｐｓｅｕｄｏｍｏｎａｓ ｓｐ．）ＩＴＨ−ＳＡ−１株（ＮＩＴＥ Ｐ−１５２１）を、シリングアルデヒド共存下で培養する工程を有する蛍光物質の製造方法を提供する。
本発明の蛍光物質の製造方法は、前記培養する工程で得られた培養物から、アルコール又はアルコール及び水を含有する溶媒で前記蛍光物質を抽出する工程をさらに有することが好ましい。
The present invention also provides a method for producing a fluorescent substance comprising a step of culturing Pseudomonas sp. ITH-SA-1 strain (NITE P-1521 ) in the presence of syringaldehyde.
The method for producing a fluorescent substance of the present invention preferably further includes a step of extracting the fluorescent substance from a culture obtained in the culturing step with a solvent containing alcohol or alcohol and water.
本発明によれば、リグニン由来の成分を原料として得られる新規の蛍光物質、及びその製造方法が提供される。 ADVANTAGE OF THE INVENTION According to this invention, the novel fluorescent material obtained by using the component derived from lignin as a raw material, and its manufacturing method are provided.
本発明に係る蛍光物質は、シュードモナス・エスピー（Ｐｓｅｕｄｏｍｏｎａｓ ｓｐ．）ＩＴＨ−ＳＡ−１株（ＮＩＴＥ Ｐ−１５２１）（以下、「本菌株」と略記することがある）を、シリングアルデヒド（以下、「ＳＹＡＬ」と略記することがある）共存下で培養して得られたものである。
本発明者らは、本菌株をＳＹＡＬ共存下で培養することにより、黒色色素が得られることを確認しているが、本発明に係る蛍光物質は、前記黒色色素とは異なる成分である。
The fluorescent substance according to the present invention comprises Pseudomonas sp. ITH-SA-1 strain (NITE P-1521 ) (hereinafter sometimes abbreviated as “the present strain”), shilling aldehyde (hereinafter “the strain”). (It may be abbreviated as “SYAL”).
The present inventors have confirmed that a black pigment can be obtained by culturing this strain in the presence of SYAL, but the fluorescent substance according to the present invention is a component different from the black pigment.
本菌株は、岩手県釜石市の生海水から取得でき、例えば、この生海水を微生物源として、ＳＹＡＬと共に無機塩培地又は完全培地に添加して培養を行い、蛍光物質を産生する菌株を単離することにより、取得できる。本菌株は、蛍光物質産生時に黒色色素も産生するため、この黒色色素の産生の有無により、本菌株を識別することも可能である。 This strain can be obtained from raw seawater in Kamaishi City, Iwate Prefecture. For example, this raw seawater is used as a microbial source and added to an inorganic salt medium or complete medium together with SYAL and cultured to isolate a fluorescent substance-producing strain. Can be obtained. Since this strain also produces a black pigment at the time of fluorescent substance production, it is possible to identify this strain by the presence or absence of production of this black pigment.
本菌株は、２０１３年２月１日付けで、受託番号ＮＩＴＥ Ｐ−１５２１として、独立行政法人製品評価技術基盤機構特許微生物寄託センターに受託されている。 The strain, on February 1, 2013, as accession number NITE P-1521, that has been entrusted to the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center.
本菌株の培養は、ＳＹＡＬ共存下で行えばよいが、培地中のＳＹＡＬの初期濃度（培養開始時の濃度）は、０．０１〜２０ｍｇ／ｍＬであることが好ましく、このようにすることで、蛍光物質の産生量がより増大する。
本菌株の培養は、ＳＹＡＬ以外に本菌株の培養を促進する成分を含有する培地で行ってもよい。ただし、培地中のグルコースの濃度は、０．３５質量％以下であることが好ましく、このようにすることで、蛍光物質の産生量がより増大する。
The strain may be cultured in the presence of SYAL, but the initial concentration of SYAL in the medium (concentration at the start of cultivation) is preferably 0.01 to 20 mg / mL. The production amount of the fluorescent substance is further increased.
You may culture | cultivate this strain by the culture medium containing the component which accelerates | stimulates culture | cultivation of this strain other than SYAL. However, the concentration of glucose in the medium is preferably 0.35% by mass or less, and by doing so, the production amount of the fluorescent substance is further increased.
本菌株の培養方法は、振とう培養、静置培養等、公知のいずれの方法でもよい。
本菌株の培養温度は、４〜３０℃であることが好ましい。また、培養時間は、蛍光物質の産生量が十分となるように適宜調節すればよく、特に限定されない。例えば、培養温度が４〜３０℃である場合、本菌株の培養時間は４８〜１２０時間であることが好ましい。
The culture method of this strain may be any known method such as shaking culture or stationary culture.
The culture temperature of the strain is preferably 4-30 ° C. In addition, the culture time is not particularly limited, and may be appropriately adjusted so that the production amount of the fluorescent substance is sufficient. For example, when the culture temperature is 4 to 30 ° C., the culture time of this strain is preferably 48 to 120 hours.
本発明に係る蛍光物質は、上記の方法による本菌株の培養（本菌株をＳＹＡＬ共存下で培養する工程）後に、例えば、本菌株の菌体外に産出された培養物から得られる。そして、前記蛍光物質は、例えば、この培養物からアルコール又はアルコール及び水を含有する溶媒（抽出溶媒）で抽出すること（蛍光物質を抽出する工程）により、高純度で得られる。このとき、黒色色素は抽出溶媒に溶解せず、固形物となるが、蛍光物質は抽出溶媒に溶解するため、ろ過や遠心分離等の固液分離操作を行い、必要に応じて洗浄して得られた液体成分から、溶媒を除去することにより、容易に蛍光物質を分離できる。 The fluorescent substance according to the present invention can be obtained from, for example, a culture produced outside the cells of the present strain after the strain is cultured by the above-described method (step of culturing the strain in the presence of SYAL). And the said fluorescent substance is obtained with high purity by extracting with the solvent (extraction solvent) containing alcohol or alcohol and water from this culture (extraction process of a fluorescent substance), for example. At this time, the black pigment does not dissolve in the extraction solvent and becomes a solid, but the fluorescent substance dissolves in the extraction solvent, so it is obtained by performing solid-liquid separation operations such as filtration and centrifugation, and washing as necessary. By removing the solvent from the obtained liquid component, the fluorescent material can be easily separated.
抽出溶媒に用いる前記アルコールは、水酸基を有し、常温・常圧で液状のものであればよいが、メタノールであることが好ましい。 The alcohol used for the extraction solvent may be any alcohol that has a hydroxyl group and is liquid at room temperature and pressure, but is preferably methanol.
抽出溶媒中のアルコール及び水の総含有量は、９５質量％以上であることが好ましく、９８質量％以上であることがより好ましく、１００質量％であることが特に好ましい。このような範囲とすることで、蛍光物質の分離（回収）効率がより向上する。抽出溶媒中のアルコール及び水以外の溶媒成分の種類は、本発明の効果を損なわない限り、特に限定されない。
また、抽出溶媒中のアルコールの含有量は、アルコールの種類に応じて適宜調節すればよいが、８５質量％以上であることが好ましく、９０質量％以上であることがより好ましく、９５質量％以上であることが特に好ましく、１００質量％であってもよい。このような範囲とすることで、得られる蛍光物質の純度がより向上する。
The total content of alcohol and water in the extraction solvent is preferably 95% by mass or more, more preferably 98% by mass or more, and particularly preferably 100% by mass. By setting it as such a range, the isolation | separation (recovery) efficiency of a fluorescent substance improves more. The kind of solvent components other than alcohol and water in the extraction solvent is not particularly limited as long as the effects of the present invention are not impaired.
The content of the alcohol in the extraction solvent may be appropriately adjusted according to the type of alcohol, but is preferably 85% by mass or more, more preferably 90% by mass or more, and 95% by mass or more. It is particularly preferable that it may be 100% by mass. By setting it as such a range, the purity of the obtained fluorescent substance improves more.
蛍光物質の抽出は、例えば、培養物を濃縮し、好ましくは乾固させた後、得られた濃縮物に対して抽出溶媒を添加して撹拌することにより、行うことができる。ここで、「乾固」とは、常温及び常圧下で濃縮物を静置した状態において、この濃縮物の質量が変動しない程度にまで濃縮されることを意味する。そして、上記の抽出操作を繰り返し行うことで、蛍光物質の分離（回収）効率がより向上する。 Extraction of the fluorescent substance can be performed, for example, by concentrating the culture, preferably drying, and then adding an extraction solvent to the resulting concentrate and stirring. Here, “drying” means that the concentrate is concentrated to such an extent that the mass of the concentrate does not fluctuate in a state where the concentrate is allowed to stand at normal temperature and normal pressure. And the separation (recovery) efficiency of the fluorescent substance is further improved by repeating the above extraction operation.
抽出溶媒の使用量は、濃縮物に対して１〜１００質量倍であることが好ましく、３〜３０質量倍であることがより好ましい。抽出溶媒の使用量が前記下限値以上であることで、得られる蛍光物質の純度がより向上し、前記上限値以下であることで、蛍光物質の分離（回収）効率がより向上する。 It is preferable that the usage-amount of an extraction solvent is 1-100 mass times with respect to a concentrate, and it is more preferable that it is 3-30 mass times. When the amount of the extraction solvent used is not less than the lower limit, the purity of the obtained fluorescent substance is further improved, and when it is not more than the upper limit, the separation (recovery) efficiency of the fluorescent substance is further improved.
蛍光物質は、前記培養物からの抽出以外に、必要に応じてその他の操作を適宜追加して分離してもよい。そして、分離した蛍光物質は、さらに各種クロマトグラフィー等の公知の精製操作に供して、純度を向上させることもできる。 In addition to extraction from the culture, the fluorescent substance may be separated by appropriately adding other operations as necessary. The separated fluorescent substance can be further subjected to known purification operations such as various chromatography to improve the purity.
本発明に係る蛍光物質は、核磁気共鳴分光法（ＮＭＲ）、赤外分光法（ＩＲ）等の分析によって、有機化合物であるものの、芳香族環を有しないことが確認されている。ここで「芳香族環」とは、芳香族炭化水素環及び芳香族複素環の両方を含む概念である。従来知られている有機蛍光物質は芳香族環を有しており、本発明に係る蛍光物質は、有機非芳香族化合物である点で、従来のものとは全く相違するものである。 Although the fluorescent substance according to the present invention is an organic compound by analysis such as nuclear magnetic resonance spectroscopy (NMR) and infrared spectroscopy (IR), it has been confirmed that it does not have an aromatic ring. Here, the “aromatic ring” is a concept including both an aromatic hydrocarbon ring and an aromatic heterocyclic ring. Conventionally known organic fluorescent materials have an aromatic ring, and the fluorescent materials according to the present invention are completely different from conventional ones in that they are organic non-aromatic compounds.
本発明に係る蛍光物質は、ゲルろ過等の分析によって、分子量が２〜９ｋＤａ、数平均分子量が６〜８ｋＤａの高分子化合物であることが確認されている。 The fluorescent substance according to the present invention is confirmed to be a polymer compound having a molecular weight of 2 to 9 kDa and a number average molecular weight of 6 to 8 kDa by analysis such as gel filtration.
本発明に係る蛍光物質は、例えば、極大波長が３６５ｎｍの光で励起され、極大波長が４９８ｎｍの蛍光を発生する。すなわち、前記蛍光物質は、紫外線又は可視光線により励起され、青緑色の蛍光を発生する。 The fluorescent substance according to the present invention is excited with light having a maximum wavelength of 365 nm, for example, and generates fluorescence having a maximum wavelength of 498 nm. That is, the fluorescent material is excited by ultraviolet light or visible light to generate blue-green fluorescence.
本発明に係る蛍光物質は、本菌株の培養時における代謝産生物の解析から、以下の化学式で表される経路で本菌株により産生されると推測される。
ＳＹＡＬ共存下で本菌株の培養を開始すると、培養の進行と共に培地中のＳＹＡＬの濃度が低下する。次いで、シリンガ酸（以下、「ＳＹＡＣ」と略記することがある）の産生が確認され、培地中でのＳＹＡＬの濃度の低下に伴い、ＳＹＡＣの濃度が上昇する。次いで、３−Ｏ−メチルガリック酸（以下、「３ＭＧＡ」と略記することがある）の産生が確認され、ＳＹＡＣの濃度の上昇よりも遅れる形で、３ＭＧＡの濃度が上昇する。次いで、前記蛍光物質及び黒色色素の産生が確認され、以降、ある時点を境にＳＹＡＣ及び３ＭＧＡの濃度は低下していくが、前記蛍光物質及び黒色色素の濃度が上昇していく。なお、前記蛍光物質及び黒色色素の産生に伴い、微量のガリック酸（以下、「ＧＡ」と略記することがある）の産生が確認されることがある。このように、本菌株の培養の進行と共に、ＳＹＡＬは酸化されてＳＹＡＣとなり、ＳＹＡＣは２個のメトキシ基のうち、一方において脱メチル化されて３ＭＧＡとなり、この３ＭＧＡが何らかの作用を受けて重合することにより、前記蛍光物質及び黒色色素が産生されると推測される。前記蛍光物質以外のこれら代謝産生物（ＳＹＡＣ、３ＭＧＡ、ＧＡ、黒色色素）は、いずれも蛍光活性を有していない。
ここで挙げた各成分はすべて、例えば、逆相高速液体クロマトグラフィー（逆相ＨＰＬＣ）により分離して検出可能である。
The fluorescent substance according to the present invention is presumed to be produced by the present strain by the pathway represented by the following chemical formula from the analysis of metabolites during the cultivation of the present strain.
When the culture of this strain is started in the presence of SYAL, the concentration of SYAL in the medium decreases as the culture progresses. Next, production of syringic acid (hereinafter sometimes abbreviated as “SYAC”) is confirmed, and as the concentration of SYAL in the medium decreases, the concentration of SYAC increases. Next, production of 3-O-methylgallic acid (hereinafter sometimes abbreviated as “3MGA”) is confirmed, and the concentration of 3MGA increases in a manner delayed from the increase in the concentration of YAAC. Next, the production of the fluorescent material and the black pigment is confirmed. Thereafter, the concentrations of YAC and 3MGA decrease at a certain point in time, but the concentrations of the fluorescent material and the black pigment increase. In addition, with the production of the fluorescent substance and the black pigment, production of a small amount of gallic acid (hereinafter sometimes abbreviated as “GA”) may be confirmed. Thus, with the progress of the culture of this strain, SYAL is oxidized to SYAC, and SYAC is demethylated at one of two methoxy groups to become 3MGA, and this 3MGA undergoes some action to polymerize. Thus, it is estimated that the fluorescent substance and the black pigment are produced. None of these metabolites (SYAC, 3MGA, GA, black pigment) other than the fluorescent substance has fluorescent activity.
All the components listed here can be separated and detected by, for example, reverse phase high performance liquid chromatography (reverse phase HPLC).
本発明に係る蛍光物質は、芳香族環を有しないため、従来の蛍光物質のように芳香族環を有していることによる制約を受けない。したがって、本発明に係る蛍光物質は、従来の蛍光物質では実現できなかった新規の利用法の開発に有望なものである。
また、本発明に係る蛍光物質は、従来とは異なり、リグニン由来の成分であるシリングアルデヒドを原料として製造されるため、未利用バイオマスの有効利用という点においても優れたものである。
Since the fluorescent substance according to the present invention does not have an aromatic ring, it is not restricted by having an aromatic ring unlike conventional fluorescent substances. Therefore, the fluorescent substance according to the present invention is promising for the development of a new utilization method that could not be realized by the conventional fluorescent substance.
Further, unlike the conventional case, the fluorescent substance according to the present invention is manufactured using shilling aldehyde, which is a component derived from lignin, as a raw material, and thus is excellent in terms of effective utilization of unused biomass.
以下、具体的実施例により、本発明についてさらに詳しく説明する。ただし、本発明は、以下に示す実施例に何ら限定されるものではない。
なお、本実施例における「数平均分子量」は、特に断りの無い限り、ＨＰＬＣ分析でのピーク面積値のデータを用いて算出したものである。
Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples.
The “number average molecular weight” in this example is calculated using peak area data in HPLC analysis unless otherwise specified.
＜蛍光物質の製造＞
［実施例１］
マリンブロスに、初期濃度が１０ｍｇ／ｍＬとなるようにＳＹＡＬを添加した培地を作製し、本菌株をこの培地に添加して、２５℃で９６時間振とう培養を行った。
次いで、培養によって得られ、菌体外に産出された黒色溶液状の培養物を４０℃で減圧乾固させ、黒色の固形状の濃縮物を得た。
次いで、この濃縮物１５ｇに、メタノール（和光純薬社製、特級メタノール、以下、同様）８０ｇを添加し、２５℃で１分間撹拌することにより、黒色の不溶物を含み、液相部分が薄青緑色に蛍光発色する液体を得た。そして、この液体から上澄みを回収し、残った前記不溶物に対して、メタノールを添加してから上澄みを回収するまでの上記操作を３回繰り返し、回収した上澄みをすべてあわせて３００ｍＬの抽出液を得た。
次いで、この抽出液から、エバポレーターを用いてメタノールを除去し、得られた濃縮物に水を添加して溶解させた後、この水溶液を凍結乾燥させることにより、目的物である蛍光物質を得た。
<Manufacture of fluorescent substances>
[Example 1]
A medium in which SYAL was added to marine broth to an initial concentration of 10 mg / mL was prepared, and this strain was added to this medium, followed by shaking culture at 25 ° C. for 96 hours.
Subsequently, the black solution-like culture obtained by culture and produced outside the cells was dried at 40 ° C. under reduced pressure to obtain a black solid concentrate.
Next, 80 g of methanol (special grade methanol manufactured by Wako Pure Chemical Industries, hereinafter the same) is added to 15 g of this concentrate, and stirred at 25 ° C. for 1 minute to contain black insoluble matter and the liquid phase portion is thin. A liquid that fluoresces blue-green is obtained. Then, the supernatant is recovered from this liquid, and the above operation from the addition of methanol to the recovery of the supernatant is repeated three times for the remaining insoluble matter, and all of the recovered supernatant is combined to obtain 300 mL of the extract. Obtained.
Next, methanol was removed from the extract using an evaporator, water was added to the resulting concentrate to dissolve it, and then the aqueous solution was freeze-dried to obtain the target fluorescent substance. .
一方、固液分離で得られた黒色の前記不溶物１０ｇには、水１００ｇを添加し、２５℃で１分間撹拌することにより、前記不溶物が溶解した水溶液を得た。
次いで、得られた水溶液を、膨潤させたイオン交換樹脂（Ａｍｂｅｒｌｉｔｅ ＸＡＤ２０００）（カラム：３．０×３８ｃｍ）に充填し、水及び２０質量％メタノール水溶液で順次洗浄した後、８０質量％メタノール水溶液を用いて溶出させることにより、黒色色素含有画分を得た後、溶媒を除去することにより、黒色色素を得た。
On the other hand, 100 g of water was added to 10 g of the black insoluble matter obtained by solid-liquid separation, and stirred at 25 ° C. for 1 minute to obtain an aqueous solution in which the insoluble matter was dissolved.
Next, the obtained aqueous solution was filled in a swollen ion exchange resin (Amberlite XAD2000) (column: 3.0 × 38 cm), washed sequentially with water and a 20% by mass aqueous methanol solution, and then an 80% by mass aqueous methanol solution was added. The black pigment-containing fraction was obtained by elution, and then the solvent was removed to obtain a black pigment.
得られた蛍光物質及び黒色色素について、１Ｈ−ＮＭＲ、１３Ｃ−ＮＭＲ及びＦＴ−ＩＲによる構造解析を行った。このとき取得したスペクトルデータを図１〜３に示す。図１中、（ａ）が黒色色素の、（ｂ）が蛍光物質の１Ｈ−ＮＭＲスペクトルであり、図２中、（ａ）が黒色色素の、（ｂ）が蛍光物質の１３Ｃ−ＮＭＲスペクトルである。また、図３は蛍光物質のＦＴ−ＩＲスペクトルであり、図４は黒色色素のＦＴ−ＩＲスペクトルであり、それぞれにおいて、ａはＫＢｒ錠剤法でのスペクトルであり、ｂはＡＴＲ（Ａｔｔｅｎｕａｔｅｄ Ｔｏｔａｌ Ｒｅｆｌｅｃｔｉｏｎ）補正を行って得られたスペクトルである。なお、図３及び４において、縦軸は吸光度（Ａｂｓ）であり、横軸は波数（ｃｍ−１）である。ＦＴ−ＩＲでは、試料の屈折率を１．５、プリズムの屈折率を２．４とし、入射光の入射角度を４５°に設定して解析を行った。 About the obtained fluorescent substance and black pigment | dye, the structural analysis by < 1 > H-NMR, < 13 > C-NMR, and FT-IR was performed. The spectrum data acquired at this time are shown in FIGS. In FIG. 1, (a) is a black dye, (b) is a 1 H-NMR spectrum of a fluorescent substance, (a) is a black dye, and (b) is a 13 C-NMR of a fluorescent substance in FIG. It is a spectrum. 3 is an FT-IR spectrum of a fluorescent substance, FIG. 4 is an FT-IR spectrum of a black pigment, a is a spectrum in the KBr tablet method, and b is an ATR (Attenuated Total Reflection). It is a spectrum obtained by performing correction. 3 and 4, the vertical axis represents absorbance (Abs), and the horizontal axis represents wave number (cm −1 ). In FT-IR, the analysis was performed by setting the refractive index of the sample to 1.5, the refractive index of the prism to 2.4, and the incident angle of incident light to 45 °.
図１及び２に示すように、１Ｈ−ＮＭＲ及び１３Ｃ−ＮＭＲにおいて、蛍光物質は黒色色素と概ね類似のスペクトルを示したが、１Ｈ−ＮＭＲにおいては２．８ｐｐｍに、１３Ｃ−ＮＭＲにおいては４０ｐｐｍに、それぞれ特徴的なシグナルが見られた。これらのシグナルは、−Ｃ（＝Ｏ）−ＣＨ３のメチル基（ＣＨ３）、又は−Ｃ（＝Ｏ）−ＣＨ２−Ｃ（＝Ｏ）−のメチレン基（ＣＨ２）に対応するものと推測された。そして、１Ｈ−ＮＭＲ及び１３Ｃ−ＮＭＲにおいて、蛍光物質及び黒色色素はいずれも、カルボニル基（Ｃ＝Ｏ）、メチン基（ＣＨ）、メチレン基（ＣＨ２）、メチル基（ＣＨ３）の各基に相当するシグナルが多数見られたのに対し、図１に示すように１Ｈ−ＮＭＲにおいては、６〜８ｐｐｍ辺りの領域に通常見られる、芳香族環に特有のシグナルは見られず、蛍光物質及び黒色色素が非芳香族有機化合物であることを示していた。 As shown in FIGS. 1 and 2, in 1 H-NMR and 13 C-NMR, the fluorescent substance showed a spectrum almost similar to that of the black pigment, but in 1 H-NMR, it was 2.8 ppm, 13 C-NMR. , Each had a characteristic signal at 40 ppm. These signals, -C (= O) methyl group (CH 3) a -CH 3, or -C (= O) -CH 2 -C (= O) - those which correspond to the methylene group (CH 2) It was speculated. In 1 H-NMR and 13 C-NMR, the fluorescent substance and the black pigment are all carbonyl groups (C═O), methine groups (CH), methylene groups (CH 2 ), and methyl groups (CH 3 ). While many signals corresponding to each group were observed, as shown in FIG. 1, in 1 H-NMR, a signal peculiar to an aromatic ring, which is usually observed in a region around 6 to 8 ppm, was not observed. The fluorescent substance and the black pigment were non-aromatic organic compounds.
また、図３及び４に示すように、蛍光物質及び黒色色素は、いずれもＦＴ−ＩＲにおいて、Ｃ＝Ｏと、Ｃ−Ｈ、Ｏ−Ｈ又はＮ−Ｈの各結合に相当するシグナルが見られたのに対し、５００〜１０００ｃｍ−１辺りの領域に通常見られる、芳香族環に特有のシグナルが見られず（図３及び４において、この領域に見られるピークらしきものは、ベースラインのせり上がりによるノイズであり、何らかのシグナルを示すものではない）、ＮＭＲの場合と同様に、蛍光物質及び黒色色素が非芳香族有機化合物であることを示していた。 As shown in FIGS. 3 and 4, both the fluorescent substance and the black dye have signals corresponding to C = O and C—H, O—H, or N—H bonds in FT-IR. In contrast, the signal typical of the aromatic ring, which is usually seen in the region around 500 to 1000 cm −1 , was not seen (in FIGS. 3 and 4, the peak-like signal seen in this region is This is noise due to uplifting and does not show any signal). As in the case of NMR, it indicates that the fluorescent substance and the black pigment are non-aromatic organic compounds.
蛍光物質は、極大波長が３５６ｎｍの励起光の照射によって、極大波長が４９８ｎｍの蛍光を発生した。このときの励起光及び蛍光のスペクトルを図５に示す。 The fluorescent material generated fluorescence having a maximum wavelength of 498 nm when irradiated with excitation light having a maximum wavelength of 356 nm. The excitation light and fluorescence spectra at this time are shown in FIG.
蛍光物質は、ＦＴ−ＩＲスペクトルが硫酸カルシウムのものと類似していた。なお、原子吸光による解析の結果、蛍光物質はカルシウム塩を多く含んでいたが、これは培地中の塩化カルシウムに由来する可能性がある。 The fluorescent material had an FT-IR spectrum similar to that of calcium sulfate. As a result of analysis by atomic absorption, the fluorescent substance contained a large amount of calcium salt, which may be derived from calcium chloride in the medium.
上記で得られた蛍光物質を、さらに下記条件でゲルろ過に供した。
（ゲルろ過の条件）
カラム：ＴＳＫｇｅｌ Ｇ３０００ＳＷＸＬ（７．８ｍｍ ｉ．ｄ．×３００ｍｍ、東ソー社製）
移動相：０．２モル／Ｌ塩化ナトリウム含有０．１モル／Ｌリン酸緩衝液（ｐＨ ７．０）
流速：１．２ｍＬ／分
カラム温度：室温
検出波長：２１０ｎｍ、２５４ｎｍ
The fluorescent material obtained above was further subjected to gel filtration under the following conditions.
(Conditions for gel filtration)
Column: TSKgel G3000SWXL (7.8 mm id × 300 mm, manufactured by Tosoh Corporation)
Mobile phase: 0.1 mol / L phosphate buffer (pH 7.0) containing 0.2 mol / L sodium chloride
Flow rate: 1.2 mL / min Column temperature: Room temperature Detection wavelength: 210 nm, 254 nm
その結果、得られた蛍光物質は数平均分子量が７．２ｋＤａであった。ゲルろ過で得られた、蛍光物質を含む画分を、さらにアミコンウルトラ−遠心式フィルターユニット（３Ｋ）（ミリポア社製）を用いて、４０００×ｇで５０分間遠心処理した後、得られた上清を、さらにアミコンウルトラ−遠心式フィルターユニット（１０Ｋ）（ミリポア社製）を用いて、４０００×ｇで５０分間遠心処理することにより、合計で３つの試料を得た。そして、これら試料をさらにゲルろ過に供して解析した結果、最初のゲルろ過で得られた蛍光物質は、複数種の蛍光物質の混合物であり、分子量が２．１ｋＤａ、５．２ｋＤａ、８．５ｋＤａの３種のものに大別され、８．５ｋＤａのものが主たる成分であった。 As a result, the obtained fluorescent substance had a number average molecular weight of 7.2 kDa. The fraction obtained by gel filtration and containing a fluorescent substance was further centrifuged at 4000 × g for 50 minutes using Amicon Ultra-centrifugal filter unit (3K) (manufactured by Millipore). The sample was further centrifuged at 4000 × g for 50 minutes using Amicon Ultra-centrifugal filter unit (10K) (Millipore) to obtain a total of three samples. And as a result of further subjecting these samples to gel filtration and analyzing them, the fluorescent substance obtained by the first gel filtration is a mixture of a plurality of kinds of fluorescent substances, and the molecular weights are 2.1 kDa, 5.2 kDa, 8.5 kDa. The main component was 8.5 kDa.
これに対して、上記で得られた蛍光物質を別途、ＯＤＳカラムを用いたＨＰＬＣ解析に供したところ、複数のピークが分離されて観測されることはなかった。 On the other hand, when the fluorescent substance obtained above was separately subjected to HPLC analysis using an ODS column, a plurality of peaks were not separated and observed.
蛍光物質の場合と同じ方法で、黒色色素もゲルろ過及び遠心処理で解析した結果、黒色色素は数平均分子量が１２．８ｋＤａであり、これは分子量が５．８ｋＤａ、８．５ｋＤａ、１６．８ｋＤａの３種のものに大別され、１６．８ｋＤａのものが主たる成分であった。 As a result of analyzing the black pigment by gel filtration and centrifugation in the same manner as in the case of the fluorescent substance, the black pigment has a number average molecular weight of 12.8 kDa, which has a molecular weight of 5.8 kDa, 8.5 kDa, 16.8 kDa. The main component was 16.8 kDa.
＜蛍光物質の安定性試験＞
［試験例１］
実施例１で得られた蛍光物質を、ｐＨ１〜１３の緩衝液に溶解させたところ、ｐＨ１の場合には、得られた溶液は蛍光を発生しなかったが、ｐＨ２〜１３の場合には、得られた溶液は蛍光を発生した。一方、ｐＨ１の場合には、得られた溶液にさらにアルカリを加えてｐＨを７に調整したところ、蛍光を発生するようになり、前記蛍光物質は、ｐＨ依存性の光吸収と蛍光発色を示すことが確認された。また、いずれのｐＨの場合も、得られた溶液をＨＰＬＣで分析すると、全く同じクロマトグラムを示し、いずれのｐＨでも蛍光物質の分解は確認されなかった。また、蛍光物質をｐＨ７の緩衝液に溶解させて９５℃で３日間加熱しても、蛍光は変化しなかった。このように、前記蛍光物質はアルカリ性条件下及び酸性条件下のいずれにおいても安定であり、またその水溶液は９５℃での加熱に対しても安定であることが確認できた。
<Fluorescent substance stability test>
[Test Example 1]
When the fluorescent substance obtained in Example 1 was dissolved in a buffer solution having a pH of 1 to 13, when the pH was 1, the obtained solution did not generate fluorescence, but when the pH was 2 to 13, The resulting solution generated fluorescence. On the other hand, in the case of pH 1, when alkali is further added to the obtained solution and the pH is adjusted to 7, fluorescence is generated, and the fluorescent substance exhibits pH-dependent light absorption and fluorescence coloring. It was confirmed. Further, when the obtained solution was analyzed by HPLC at any pH, the same chromatogram was shown, and the decomposition of the fluorescent substance was not confirmed at any pH. Further, even when the fluorescent substance was dissolved in a pH 7 buffer and heated at 95 ° C. for 3 days, the fluorescence did not change. Thus, it was confirmed that the fluorescent material was stable under both alkaline and acidic conditions, and that the aqueous solution was stable against heating at 95 ° C.
Claims (7)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2013019971A JP6090838B2 (en) | 2013-02-04 | 2013-02-04 | Fluorescent substance and method for producing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2013019971A JP6090838B2 (en) | 2013-02-04 | 2013-02-04 | Fluorescent substance and method for producing the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2014166143A JP2014166143A (en) | 2014-09-11 |
| JP6090838B2 true JP6090838B2 (en) | 2017-03-08 |
Family
ID=51616255
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2013019971A Active JP6090838B2 (en) | 2013-02-04 | 2013-02-04 | Fluorescent substance and method for producing the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP6090838B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6344647B2 (en) * | 2014-08-20 | 2018-06-20 | 学校法人日本大学 | Fluorescent substance and method for producing the same |
-
2013
- 2013-02-04 JP JP2013019971A patent/JP6090838B2/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| JP2014166143A (en) | 2014-09-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bhunia et al. | Characterization, genetic regulation and production of cyanobacterial exopolysaccharides and its applicability for heavy metal removal | |
| CN104845612B (en) | Polystyrene mercury ion fluorescence recognition materials and preparation method thereof | |
| CN103342697A (en) | Difunctional near-infrared fluorescence molecular probe for detecting hypochlorous acid and preparation method for same | |
| Bai et al. | Novel pyrano and vinylphenol adducts of deoxyanthocyanidins in sorghum sourdough | |
| JP6090838B2 (en) | Fluorescent substance and method for producing the same | |
| Sum et al. | Antimicrobial and cytotoxic cyathane-xylosides from cultures of the basidiomycete Dentipellis fragilis | |
| CN115747275A (en) | Method for increasing yield of pyrroloquinoline quinone fermented by methylobacterium | |
| Zuo et al. | Identification of pterins as characteristic humic-like fluorophores released from cyanobacteria and their behavior and fate in natural and engineered water systems | |
| CN114014848A (en) | RNA fluorescent probe and preparation method and application thereof | |
| US20150126727A1 (en) | Novel process | |
| CN106279092B (en) | A kind of double 1,4-benzoquinone class compounds and its extracting method | |
| Park et al. | Enantioseparation using sulfated cyclosophoraoses as a novel chiral additive in capillary electrophoresis | |
| CN105949104B (en) | A kind of purposes of the chain diterpene-kind compound of haematinic acid esterification | |
| CN111548445A (en) | Optical activity AIE fluorescent material and preparation method of aqueous polymer thereof | |
| CN106119117A (en) | The toxiferous algae of a kind of Azaspiracid toxin and the preparation and application of standard sample thereof | |
| CN104804071B (en) | A kind of depside peptides and its preparation method and application | |
| KR101756123B1 (en) | Manufacturing method of anthocyanin oligomer using crude enzyme from aspergilus sp. | |
| CN1255409C (en) | Fluorescent compounds | |
| Zhang et al. | Application of cross‐flow ultrafiltration for isolating exopolymeric substances from a marine diatom (Amphora sp.) | |
| Wiesemeier et al. | Direct quantification of dimethylsulfoniopropionate (DMSP) in marine micro-and macroalgae using HPLC or UPLC/MS | |
| CN113912579A (en) | Rhodamine compound, preparation method thereof and application thereof in detection of microcystins | |
| JP6813885B2 (en) | Manufacturing method of fluorescent substance | |
| CN107686491B (en) | Alkaloid compound prepared from stenotrophomonas maltophilia and preparation method thereof | |
| JP6344647B2 (en) | Fluorescent substance and method for producing the same | |
| Kwon et al. | Chiral separation of catechin by capillary electrophoresis with α-cyclosophorooctadecaose isolated from Rhodobacter sphaeroides as chiral selectors |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20160121 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20170124 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20170202 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6090838 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313113 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |