JP5029877B2 - Method for producing icosapentaenoic acid and docosahexaenoic acid - Google Patents

Method for producing icosapentaenoic acid and docosahexaenoic acid Download PDF

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JP5029877B2
JP5029877B2 JP2007019483A JP2007019483A JP5029877B2 JP 5029877 B2 JP5029877 B2 JP 5029877B2 JP 2007019483 A JP2007019483 A JP 2007019483A JP 2007019483 A JP2007019483 A JP 2007019483A JP 5029877 B2 JP5029877 B2 JP 5029877B2
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伸夫 森
亮 鷲見
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Nikken Sohonsha Corp
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本発明は、ハプト植物門に属するパブロバ藻体を培養することにより、イコサペンタエン酸及びドコサヘキサエン酸を生産する方法に関する。   The present invention relates to a method for producing icosapentaenoic acid and docosahexaenoic acid by culturing Pavlova alga bodies belonging to the haptophyta.

従来、単細胞微細藻類を縦型偏平水槽中の無機液体培地に接種して、光照射しつつ、かつ、炭酸ガス含有空気を通気しつつ培養する方法において、ナンノクロロプシス藻体を、ＰＶＣ濃度を0 ．9 〜1 ．7ml ／l 接種して、水槽の前後面の何れかの片面から照度6000〜16000lx の光を照射しつつ、ＰＶＣ濃度が1 ．8 〜4 ．9ml ／l になるまで培養する第１工程と、当該ＰＶＣ濃度1 ．8 〜4 ．9ml ／l の培養液に、水槽の前後面の両面から照度11000 〜21000lx の光を照射しつつ、ＰＶＣ濃度が5 ．0 〜7 ．Oml ／l になるまで培養する第２工程と、当該ＰＶＣ濃度5 ．0 〜7 ．Oml ／l の培養液に、水槽の前後面の両面から照度10000 〜35000lx の光を照射しつつ、ＰＶＣ濃度が10．3 〜11．Oml ／l になるまで培養する第３工程とから成ることを特徴とするエイコサペンタエン酸を生産させることは、特許文献１に開示されており公知である。   Conventionally, in a method of inoculating a single-cell microalgae into an inorganic liquid medium in a vertical flat water tank and cultivating while irradiating with light and aeration with carbon dioxide-containing air, 0. 9-1. Inoculate 7 ml / l and irradiate light with illuminance of 6000 to 16000 lx from one of the front and rear surfaces of the aquarium. 8-4. A first step of culturing until 9 ml / l, and the PVC concentration 1. 8-4. While irradiating 9ml / l of the culture solution with light of illuminance 11000-21000lx from both the front and back of the water tank, the PVC concentration is 5. 0-7. A second step of culturing until Oml / l, and the PVC concentration 5. 0-7. While the Oml / l culture solution is irradiated with light having an illuminance of 10,000 to 35000 lx from both the front and rear surfaces of the aquarium, the PVC concentration is 10.3 to 11. The production of eicosapentaenoic acid, characterized by comprising a third step of culturing until Oml / l, is disclosed in Patent Document 1 and is known.

また、単細胞微細藻類を縦型偏平水槽中の無機液体培地に接種して、光照射しつつ、かつ、炭酸ガス含有空気を通気しつつ培養する方法において、円石藻体を、ＰＶＣ濃度を0 ．1 〜0 ．4ml ／l に接種して、水槽の前後面の何れかの片面から照度3000〜4000lxの光照射をしつつ、ＰＶＣ濃度が0 ．5 〜1ml ／l まで培養する第１工程と、該0 ．5 〜1ml ／l 濃度の培養液に、水槽の前後の両面から照度3000〜4000lxの光照射をしつつ、ＰＶＣ濃度が2 ．0 〜2 ．3ml ／l まで培養する第２工程と、該2 ．0 〜2 ．3ml ／l 濃度の培養液に、水槽の前後の両面から照度5000〜6000lxの光照射しつつ、ＰＶＣ濃度が5 ．0 〜5 ．5ml ／l まで培養する第３工程とから成ることを特徴とするドコサヘキサエン酸を生産させることは、特許文献２に開示されており公知である。
特開平９−２５２７６１号公報 特開平９−２５２７６２号公報 Further, in a method of inoculating a single-cell microalgae into an inorganic liquid medium in a vertical flat water tank and culturing while irradiating with light and aeration with carbon dioxide-containing air, the cobblestone algae is adjusted to have a PVC concentration of 0. . Ten . Inoculate 4 ml / l and irradiate light with an illuminance of 3000 to 4000 lx from either one of the front and rear surfaces of the water tank, while the PVC concentration is 0. A first step of culturing to 5 to 1 ml / l; While irradiating the culture solution having a concentration of 5 to 1 ml / l with an illuminance of 3000 to 4000 lx from both the front and rear sides of the water tank, the PVC concentration is 0-2. A second step of culturing up to 3 ml / l; 0-2. While the 3 ml / l concentration of the culture solution was irradiated with light having an illuminance of 5000 to 6000 lx from both the front and rear sides of the water tank, the PVC concentration was 5. 0-5. The production of docosahexaenoic acid, which comprises the third step of culturing up to 5 ml / l, is disclosed in Patent Document 2 and is known.
JP 9-252761 A Japanese Patent Laid-Open No. 9-252762

しかしながら、上記従来のナンノクロロプシス藻体の培養法では、（エ）イコサペンタエン酸だけしか生産させることができなかった。   However, only the (e) icosapentaenoic acid can be produced by the conventional culture method of the above-mentioned Nannochloropsis alga.

さらに、上記従来の円石藻体の培養法では、ドコサヘキサエン酸だけしか生産させることができなかった。   Furthermore, only the docosahexaenoic acid was able to be produced by the conventional method for cultivating cobble stones.

そこで、本発明者は、イコサペンタエン酸及びドコサヘキサエン酸を同時に大量に生産させるパブロバ藻体の培養法について研究した結果、本発明を達成したのである。   Therefore, the present inventor has achieved the present invention as a result of studying a culture method for Pavlova alga bodies that simultaneously produce a large amount of icosapentaenoic acid and docosahexaenoic acid.

本発明は、単細胞微細藻類を培養槽中の無機液体培地に接種して、光照射しつつ、かつ、炭酸ガス含有空気を通気しつつ培養する方法において、パブロバ藻体であるハプト植物部門(Haptophyta)に属するハプト藻網(Haptophyceae)のパブロバ目(Pavlovales)のパブロバ（Pavlova)を重量乾燥藻体濃度10〜40mg／l に接種して、培養槽に光強度75〜100 μE ／m ・s２ の光を照射しつつ、50〜100mg ／l まで培養する第１工程と、この50〜100mg ／l 濃度の培養液に、培養槽に光強度125 〜150 μE ／m ・s２ の光を照射しつつ、200 〜230mg ／l まで培養する第２工程と、この200 〜230mg ／l 濃度の培養液に、培養液のpHを6 ．8 〜7 ．2 の範囲に調整して、500 〜550mg ／l まで培養する第３工程とから成ることを特徴とするものである。 The present invention relates to a method of inoculating single-cell microalgae into an inorganic liquid medium in a culture tank, cultivating while irradiating with light and aeration of carbon dioxide-containing air, and a haptophyte department (Haptophyta ) that is a Pavlova alga. Haptophyceae Pavlova Pavlova belonging to) is inoculated to a dry dry alga concentration of 10 to 40 mg / l, and the culture tank has a light intensity of 75 to 100 μE / m · s 2 The first step of culturing to 50 to 100 mg / l while irradiating the light of 50 and the culture solution of 50 to 100 mg / l is irradiated with light with a light intensity of 125 to 150 μE / m · s 2 However, the second step of culturing up to 200 to 230 mg / l and the culture solution at a concentration of 200 to 230 mg / l are adjusted to pH 6. 8-7. And the third step of culturing up to 500 to 550 mg / l.

さらに、本発明は、単細胞微細藻類を培養槽中の無機液体培地に接種して、光照射しつつ、かつ、炭酸ガス含有空気を通気しつつ培養する方法において、パブロバ藻体であるハプト植物部門(Haptophyta)に属するハプト藻網(Haptophyceae)のパブロバ目(Pavlovales)のパブロバ（Pavlova)を重量乾燥藻体濃度10〜40mg／l に接種して、培養槽の片側から光強度75〜100 μE ／m ・s２ の光を照射しつつ、50〜100mg ／l まで培養する第１工程と、この50〜100mg ／l 濃度の培養液に、培養槽の両側から光強度125 〜150 μE ／m ・s２ の光を照射しつつ、200 〜230mg ／l まで培養する第２工程と、この200 〜230mg ／l 濃度の培養液に、培養液のpHを6 ．8 〜7 ．2 の範囲に調整して、500 〜550mg ／l まで培養する第３工程とから成ることを特徴とするものである。 Furthermore, the present invention relates to a haptophyte department that is a Pavlova alga body in a method of inoculating single-cell microalgae into an inorganic liquid medium in a culture tank, cultivating while irradiating with light and aeration of carbon dioxide-containing air. Pavlova of the Haptophyceae belonging to (Haptophyta) is inoculated at a dry dry algal body concentration of 10 to 40 mg / l and light intensity from one side of the culture tank is 75 to 100 μE / The first step of culturing to 50 to 100 mg / l while irradiating light of m · s 2 , and the light intensity of 125 to 150 μE / m The second step of culturing to 200 to 230 mg / l while irradiating the light of s 2 , and adding the pH of the culture solution to 6 to 200 to 230 mg / l concentration of the culture solution. 8-7. And the third step of culturing up to 500 to 550 mg / l.

本発明で使用する培養槽としては、例えば縦型偏平水槽が挙げられる。この縦型偏平水槽とは、好ましくは、前後の各面の幅が0 ．5 〜2m程度、高さが1 ．5 〜2 ．5m程度で、左右の各側面の幅が7 〜17cmの縦長の偏平の水槽で、上端の細い長方形の開口部には、周壁に適数個の通気孔が設けられ、かつ先端部が閉鎖されている。炭酸ガス含有空気を無機液体培地に送入する通気管が、その先端から挿入され、また、下端部に形成された船底形状の底部には、培養液の排出管が、開閉自在の弁部材を介して装着されているものである。そして、側面幅は、17cmを超えると、パブロバ藻体が光りを十分に受けることかできず、イコサペンタエン酸（以下、ＥＰＡという）及びドコサヘキサエン酸（以下、ＤＨＡという）の生産が低下するので好ましくない。また、船底形態の底部は、培養液が排出され易いように培養液排出管に向けて下方に傾斜されていることが好ましく、水槽を形成するガラス板またはアクリル板の厚さは３ｃｍ程度が望ましい。なお、本発明での光照射は、白色、昼色蛍光灯または白熱灯の照射をいう。   Examples of the culture tank used in the present invention include a vertical flat water tank. The vertical flat water tank preferably has a width of 0. About 5 to 2m, height is 1. 5-2. This is a vertically long flat water tank with a width of about 7 to 17 cm with a width of 7 to 17 cm on the left and right sides. An appropriate number of air holes are provided in the peripheral wall of the thin rectangular top and the tip is closed. ing. A vent pipe for feeding carbon dioxide-containing air into the inorganic liquid medium is inserted from the tip, and a culture medium discharge pipe is provided at the bottom of the bottom formed at the lower end of the openable valve member. Is attached. If the side width exceeds 17 cm, the Pavlova alga body cannot receive light sufficiently, and production of icosapentaenoic acid (hereinafter referred to as EPA) and docosahexaenoic acid (hereinafter referred to as DHA) is not preferable. . Moreover, it is preferable that the bottom of the ship bottom shape is inclined downward toward the culture medium discharge pipe so that the culture medium can be easily discharged, and the thickness of the glass plate or acrylic plate forming the water tank is preferably about 3 cm. . In addition, the light irradiation in this invention means irradiation of a white, daylight fluorescent lamp, or an incandescent lamp.

本発明でいうパブロバ藻体とは、ハプト植物部門(Haptophyta)に属するハプト藻網(Haptophyceae)のパブロバ目(Pavlovales)のパブロバ（Pavlova)が挙げられる。   Examples of the Pavlova alga used in the present invention include Pavlova of the order Pavlovales of the haptophyceae belonging to the haptophyta department.

ハプト植物部門に属するハプト藻網には、前記パブロバ目の他に、イソクリシス目(Isochrysidales)、コッコスフェラ目(Coccosphaerales) 、プリムネシウム目(Prymnesiales)が存在する。そして、パブロバ目には、前記パブロバの他に、ディアクロネマ(Diacronema)、エキサンテマクリシス(Exanthemachrysis)等が知られている。イソクリシス目には、アピストネマ(Apistonema)、クリソネマ(Chrysonema)、クリソティラ(Chrysotila)、クリコスフェラ(Cricosphaera)、ディクラテリア(Dicrateria)、エミリアニア(Emiliania) 、ゲフィロカプサ(Gephyrocapsa)、ヒメノモナス(Hymenomonas) 、イマントニア(Imantonia) 、イソクリシス(Isochrysis)、ペルロクリシス(Pleurochrysis) 、オクロスフェラ(Ochrosphaera)等が知られている。コッコスフェラ目には、アカントイカ(Acanthoica)、アノプロソレニア(Anoplosolenia) 、アントスフェラ(Anthosphaera)、ブラルドスフェラ(Braarudosphaera) 、カルシオパップス(Calciopappus)、カルシオソレニア(Calciosolenia) 、カリプトロスフェラ(Calyptrosphaera) 、コッコリタス(Coccolithus) 、コリスフェラ(Corisphaera) 、クリスタロリタス(Crystallolithus) 、シクロコッコリタス(Cyclococcolithus)、ドイツランディア(Deutschlandia) 、ディスコスフェラ(Discosphaera)、ヘリコスフェラ(Helicosphaera) 、ヘラドスフェラ(Helladosphaera)、ホモジゴスフェラ(Homozygosphaera) 、ミカエルサルシア(Michaelsarsia) 、オフィアスター(Ophiaster) 、パッポスフェラ(Papposphaera)、ポントスフェラ(Pontosphaera)、ラブドスフェラ(Rhabdosphaera) 、ラブドソラックス(Rhabdothorax)、シフォスフェラ(Scyphosphaera) 、スフェロカリテプトラ(Sphaerocalyptra) 、シラコリタス(Syracolithus)、シラコスフェラ(Syracosphaera) 、トラコスフェラ(Thoracosphaera)、ジゴスフェラ(Zygosphaera) 等が知られている。プリムネシウム目には、クリソクロムリナ(Chrysochromulina)、コリムベラス(Corymbellus) 、フェオシスティス(Phaeocystis) 、プラティクリシス(Platychrysis)、プリムネシウム(Prymnesium)等が知られている。   In addition to the Pavlova, there are Isochrysidales, Coccosphaerales, and Prymnesiales in the haptophyte net belonging to the haptophyte department. In addition to Pavlova, Diacronema, Exanthhemachrysis, and the like are known as Pavlova. In the isochrysis, Apistonema, Chrysonema, Chrysotila, Cricosphaera, Dicrateria, Emiliania, Gephyrocapsa, Hymenonas (Hymenonas) ), Isochrysis, perlechrysis, and Ochrosphaera are known. In the order of Cocosfera, Acanthoica, Anoplosolenia, Anthosphaera, Braarudosphaera, Calciopappus, Calciosolenia, Calyptosphaera, Calyptrosphaera (Coccolithus), Corisphaera, Crystallolithus, Cyclococcolithus, Deutschlandia, Discosphaera, Helicosphaera, Helladosphaera, Helladosphaera, Helladosphaera, Ella Sarcia (Ophiaster), Papposphaera, Pontosphaera, Rhabdosphaera, Rhabdothorax, Scyphosphaera, Spherocaritept (Sphaerocalyptra), Shirakoritasu (Syracolithus), Shirakosufera (Syracosphaera), Torakosu Blow (Thoracosphaera), Jigosufera (Zygosphaera) and the like are known. As the primumesium, Chrysochromulina, Corymbellus, Phaeocystis, Platychrysis, Primnesium and the like are known.

本発明では、これら多くのハプト植物部門に属するハプト藻網のうち、前記パブロバ目のパブロバが、ＥＰＡ及びＤＨＡを同時に大量に生産させることを見いだしたものである。   In the present invention, it has been found that among the haptoalgal nets belonging to many of these haptophyte departments, the Pavlova-type Pavlova simultaneously produces EPA and DHA in large quantities.

本発明でいう無機液体培地とは、培地100 リットルに対して、ＮａＣｌが2400〜3000g 、ＭｇＳＯ4 ・７Ｈ2 Ｏが600 〜700g、ＫＮＯ3 が30〜100g、ＣａＣｌ2 ・２Ｈ2 Ｏが10〜18g 、ＫＨ2 ＰＯ4 が5 〜9g、ＮａＨＣＯ3 が3 〜5g、Tris−ＨＣｌが220 〜260g、ビタミンＢ12が0 ．3 〜0 ．7mg 、チアミン−ＨＣｌが40〜60mg、ビオチンが0 ．1 〜1mg 、ＦｅＣｌ2 ・６Ｈ2 Ｏが200 〜300mg 、Ｎａ2 ＥＤＴＡが1500〜2500mg、ＺｎＣｌ2 が0 ．4 〜40mg、Ｈ3 ＢＯ3 が6 〜60mg、ＣｏＣｌ2 ・６Ｈ2 Ｏが0 ．15〜1 ．5mg 、ＣｕＣｌ2 ・２Ｈ2 Ｏが0 ．4 〜4mg 、ＭｎＣｌ2 ・４Ｈ2 Ｏが4 〜40mg、（ＮＨ4 ）6 ＭｏＯ24が0 ．37〜37mgの割合で含まれている培地をいう。 The inorganic liquid medium referred to in the present invention is 2400 to 3000 g of NaCl, 600 to 700 g of MgSO 4 .7H 2 O, 30 to 100 g of KNO 3 , and 10 to 10 of CaCl 2 · 2H 2 O per 100 liters of medium. 18g, KH 2 PO 4 is 5 ~9g, NaHCO 3 is 3 ~5g, Tris-HCl is 220 ~260g, vitamin B 12 is 0. 3-0. 7 mg, thiamine-HCl 40-60 mg, biotin 0. 1 to 1 mg, FeCl 2 · 6H 2 O 200 to 300 mg, Na 2 EDTA 1500 to 2500 mg, ZnCl 2 0. 4 to 40 mg, H 3 BO 3 6 to 60 mg, CoCl 2 · 6H 2 O 0. 15-1. 5 mg, CuCl 2 .2H 2 O is 0. 4 to 4 mg, MnCl 2 · 4H 2 O 4 to 40 mg, (NH 4 ) 6 MoO 24 0. A medium containing 37-37 mg.

本発明で、第１工程において、培養開始重量乾燥藻体濃度10〜40mg／l を接種し、培養槽の片側から光強度75〜100 μE ／m ・s2光を照射して培養するのは、好適に培養することができるからであり、また50〜100mg ／ lまで培養するのは、100mg ／l を超えてまで培養すると、照度が不足となり、培養液中のパブロバ藻体を凝集・黄変させ、ＥＰＡ及びＤＨＡの生産が低下するからである。よって、上記条件で培養することにより、ＥＰＡ及びＤＨＡの生産を低下させることなく、順調に第２工程の培養に移行することができるからである。 In the present invention, in the first step, inoculating with a culture start weight dry alga body concentration of 10-40 mg / l and irradiating with light intensity of 75-100 μE / m · s 2 light from one side of the culture tank This is because it can be suitably cultured, and the culture is carried out to 50 to 100 mg / l. If the culture exceeds 100 mg / l, the illuminance is insufficient, and the Pavlova alga in the culture is agglomerated / yellowed. This is because the production of EPA and DHA decreases. Therefore, by culturing under the above conditions, it is possible to smoothly shift to the second step culture without reducing the production of EPA and DHA.

第１工程で培養した重量乾燥藻体濃度50〜100mg ／l の培養液に、第２工程において、培養槽の両側から光強度125 〜150 μE ／m ・s2の光を照射して、200 〜230mg ／l まで培養するのは、パブロバ藻体を順調に培養してＥＰＡ及びＤＨＡを生産させるためであり、そして、230mg ／l を超えるとＥＰＡ及びＤＨＡの生産速度を低下させるからである。また、培養液中のパブロバ藻体を凝集体させたり、黄変させたりして、第３工程の培養に移行できないからである。 In the second step, the weight-dried algal body concentration cultured in the first step is irradiated with light having a light intensity of 125 to 150 μE / m · s 2 from both sides of the culture tank. The reason for culturing up to ˜230 mg / l is to cultivate Pavlova alga bodies smoothly to produce EPA and DHA, and when it exceeds 230 mg / l, the production rate of EPA and DHA is reduced. Moreover, it is because it cannot transfer to the culture | cultivation of a 3rd process by aggregating or yellowing the Pavlova alga body in a culture solution.

第２工程で培養した重量乾燥藻体濃度200 〜230mg ／l の培養液に、第３工程として、培養液のpHを6 ．8 〜7 ．2 の範囲に調整して500 〜550mg ／l まで培養するのは、550mg ／l まで培養した後の重量乾燥藻体濃度の増加は殆ど無くなり、従って、ＥＰＡ及びＤＨＡの生産も無くなるからである。   In the third step, the culture solution having a weight-dried algal body concentration of 200 to 230 mg / l cultured in the second step is adjusted to pH 6. 8-7. The reason for culturing from 500 to 550 mg / l after adjusting to the range of 2 is that there is almost no increase in the weight-dried algal body concentration after culturing to 550 mg / l, and therefore production of EPA and DHA is also eliminated.

本発明において、第１工程〜第３工程を通じ、温度10〜30℃で行うのが好ましく、10℃未満では重量乾燥藻体濃度の増加は殆どなく、30℃を超えればパブロバ藻体が凝集し、光照射時間が18〜24時間／日であればＥＰＡ及びＤＨＡの生産量の低下は来さず、また、炭酸ガス1 〜5 ％含有空気を5 〜7ml ／l の割合で通気、攪拌することにより、ＥＰＡ及びＤＨＡの生産量の低下を来すことがない。   In the present invention, it is preferable to carry out at a temperature of 10 to 30 ° C. through the first to third steps. When the temperature is less than 10 ° C., there is almost no increase in the weight-dried alga body concentration. If the light irradiation time is 18-24 hours / day, the production of EPA and DHA will not decrease, and air containing carbon dioxide 1-5% will be aerated and stirred at a rate of 5-7 ml / l. Therefore, the production amount of EPA and DHA does not decrease.

なお、確認試験として、パブロバ藻体を上記条件下において、10mg／l 培養（３０日間）したところ、図１に示したように、第１工程だけを行った場合は150mg ／l （２６日目）までしか増殖せず、第１工程、第２工程（６日目に開始）を行った場合は、287mg ／l （２４日目）までしか増殖しなかったが、第１工程、第２工程（６日目に開始）、第３工程（１８日目に開始）を行った場合は、503mg ／l （２６日目）まで増殖した。   As a confirmation test, Pavlova alga bodies were cultured at 10 mg / l (30 days) under the above conditions. As shown in FIG. 1, when only the first step was carried out, 150 mg / l (day 26) ), And when the first step and the second step (started on the 6th day) were performed, the growth increased only to 287 mg / l (the 24th day). When the third step (started on the 18th day) was performed (started on the 6th day), the cells grew to 503 mg / l (the 26th day).

本発明は、以上に述べたように構成されているので、パブロバ藻体を容易に大量培養することができ、ＥＰＡ及びＤＨＡを同時に効率よく生産できるものとなった。   Since the present invention is configured as described above, Pavlova alga bodies can be easily cultured in large quantities, and EPA and DHA can be efficiently produced at the same time.

以下、本発明を実施例に基づき詳細に説明する。
〔実施例１〕
培地100 リットルに対して、ＮａＣｌが2700g 、ＭｇＳＯ4 ・７Ｈ2 Ｏが660g、ＫＮＯ3 が36g 、ＣａＣｌ2 ・２Ｈ2 Ｏが15g 、ＫＨ2 ＰＯ4 が7g、ＮａＨＣＯ3 が4g、Tris−ＨＣｌが242g、ビタミンＢ12が0 ．5mg 、チアミン−ＨＣｌが60mg、ビオチンが0 ．1mg 、ＦｅＣｌ2 ・６Ｈ2 Ｏが276.9mg 、Ｎａ2 ＥＤＴＡが1860mg、ＺｎＣｌ2 が0 ．4 mg、Ｈ3 ＢＯ3 が6 mg、ＣｏＣｌ2 ・６Ｈ2 Ｏが0 ．15mg、ＣｕＣｌ2 ・２Ｈ2 Ｏが0 ．4mg 、ＭｎＣｌ2 ・４Ｈ2 Ｏが4mg 、（ＮＨ4 ）6 ＭｏＯ24が3.7mg の割合で含まれている無機液体培地（pH8 ．0 ）100 リットルを、高さ2 m 、前後の各面の幅1m、左右の各面の幅13cmのアクリル製の縦型偏平水槽に入れ、培養温度を26℃とし、照射の時間を18時間／日として、炭酸ガス1 ％を含む空気を5 l ／min の割合で通気しつつ、第１工程として、水槽中の無機液体培地に、パブロバを重量乾燥藻体濃度10mg／l の割合で接種し、水槽の前面片面に光強度75μE ／ｍ・ｓ2 の白色蛍光灯を照射し、培養して、50mg／l となった後、第２工程として、水槽の前後の両面に、光強度150 μE ／ｍ・ｓ2 の白色蛍光灯を照射し、培養して、230mg ／l となった後、第３工程として、培養液のpHを6 ．8 に調整して培養して、507mg ／l となった後、培養処理を止めて、得られた培養液をシャープレス遠心分離機（15000 回／分）で分別して得た藻体を蒸留水で洗浄してから凍結乾燥して、48g の乾燥藻体を得た。 Hereinafter, the present invention will be described in detail based on examples.
[Example 1]
For 100 liters of medium, 2700 g of NaCl, 660 g of MgSO 4 .7H 2 O, 36 g of KNO 3 , 15 g of CaCl 2 .2H 2 O, 7 g of KH 2 PO 4, 4 g of NaHCO 3 , Tris-HCl 242g, vitamin B 12 is 0. 5 mg, thiamine-HCl 60 mg, biotin 0. 1 mg, FeCl 2 .6H 2 O 276.9 mg, Na 2 EDTA 1860 mg, ZnCl 2 0. 4 mg, 6 mg of H 3 BO 3, 0 of CoCl 2 .6H 2 O. 15 mg, CuCl 2 .2H 2 O is 0. 100 liters of inorganic liquid medium (pH 8.0) containing 4 mg, 4 mg of MnCl 2 · 4H 2 O and 3.7 mg of (NH 4 ) 6 MoO 24 , 2 m in height, on each side Place in an acrylic vertical flat water tank with a width of 1 m and a width of 13 cm on each side, culture temperature of 26 ° C, irradiation time of 18 hours / day, and air containing 1% carbon dioxide at 5 l / min As a first step, pavlova is inoculated at a rate of 10 mg / l by weight dry algal body concentration into the inorganic liquid medium in the water tank, and the light intensity is 75 μE / m · s 2 on one side of the front surface of the water tank. After irradiating with white fluorescent light and cultivating to 50 mg / l, as a second step, irradiate both the front and back of the water tank with white fluorescent light with a light intensity of 150 μE / m · s 2 and culture. After reaching 230 mg / l, as a third step, the pH of the culture solution is adjusted to 6. After adjusting to 8 and culturing at 507 mg / l, the culture treatment was stopped, and the obtained culture broth was fractionated with a sharp press centrifuge (15000 times / min) to obtain the algal cells obtained in distilled water. After washing with lyophilization, 48 g of dry algal bodies were obtained.

得られた乾燥藻体中のＥＰＡ量及びＤＨＡ量を、５％の塩酸を含むメタノール溶媒に入れ、95℃で3 時間還流後、ヘキサン溶媒で脂肪酸メチルエステルを抽出し、トリコサン酸を内部標準によるガスタロマイグラフイー分析法で定量した結果、2 ．2gのＥＰＡ及び1 ．OgのＤＨＡを得た。   The EPA content and DHA content in the dried alga obtained are put in a methanol solvent containing 5% hydrochloric acid, refluxed at 95 ° C. for 3 hours, extracted with fatty acid methyl ester with hexane solvent, and tricosanoic acid according to the internal standard. As a result of quantification by gas taromy graphie analysis method, 2. 2 g EPA and 1. Og DHA was obtained.

〔実施例２〕
実施例１記載と同様に、無機液体培地100 リットルを縦型偏平水槽に入れ、培養温度を25℃とし、照射の時間を18時間／日として、炭酸ガス1 ％含有空気を5 l ／min の割合で通気しつつ、第１工程として、水槽中の100 リットルの無機液体培地に、パブロバを重量乾燥藻体濃度20mg／l の割合で接種して、水槽の前面片面に光強度75μE ／ｍ・ｓ2 の白色蛍光灯を照射し、培養して、100mg ／l となった後、第２工程として、水槽の前後の両面に、光強度125 μE ／ｍ・ｓ2 の白色蛍光灯を照射し、培養して、230mg ／l となった後、第３工程として、培養液のpHを7 ．2 に調整して515mg ／l となった後、培養処理を止めて、得られた100 リットルの培養液から分取した70リットルの培養液をシャープレス遠心分離機（15000 回転／分）で分取した藻体を蒸留水で洗浄し、凍結乾燥して32g の乾燥藻体を得た。そして、得られた乾燥藻体中のＥＰＡ量及びＤＨＡ量を実施例１記載と同様に定量した結果、1 ．6gのＥＰＡ及び0 ．6gのＤＨＡを得た。 [Example 2]
As described in Example 1, 100 liters of an inorganic liquid medium was placed in a vertical flat water tank, the culture temperature was 25 ° C., the irradiation time was 18 hours / day, and the air containing 1% carbon dioxide was 5 l / min. As a first step, inoculate 100 liters of inorganic liquid medium in the aquarium with pavlova at a rate of 20 mg / l by weight dry algal body concentration, and light intensity 75μE / m · After irradiating with white fluorescent lamp of s 2 and culturing to 100 mg / l, as a second step, irradiate both front and rear sides of the water tank with white fluorescent lamp with light intensity of 125 μE / m · s 2 After culturing to reach 230 mg / l, as a third step, the pH of the culture solution is set to 7. After adjusting to 2 to reach 515 mg / l, the culture treatment was stopped, and 70 liters of the culture solution taken from the 100 liters of culture solution obtained were separated using a Sharpless centrifuge (15000 rpm). The collected algal bodies were washed with distilled water and freeze-dried to obtain 32 g of dried algal bodies. And as a result of quantifying the amount of EPA and the amount of DHA in the obtained dry alga body as described in Example 1, 6 g EPA and 0. 6 g of DHA was obtained.

また、70リットルを分別した残余の、重量乾燥藻体濃度515mg ／l の培養液30リットルに、実施例１の無機液体培地を適量加えて、上記実施例２の第３工程と同様に光強度125 μE ／ｍ・ｓ2 の白色蛍光灯を18時間／日照射しつつ培養することを６回繰り返して得た培養液をシャープレス遠心分離機（15000 回転／分）で得た藻体を洗浄、凍結乾燥した乾燥藻体中のＥＰＡ量及びＤＨＡ量を定量した結果、9 ．2gのＥＰＡ及び3 ．8gのＤＨＡが得られた。 In addition, an appropriate amount of the inorganic liquid medium of Example 1 is added to the remaining 30 liters of the culture solution having a weight dry algal body concentration of 515 mg / l after separating 70 liters, and the light intensity is the same as in the third step of Example 2 above. Wash the algae obtained with a sharp press centrifuge (15000 rpm) by culturing 6 times with 125 μE / m · s 2 of white fluorescent light for 18 hours / day. 8. As a result of quantifying the amount of EPA and the amount of DHA in the freeze-dried dried algal bodies, 2 g EPA and 3. 8 g of DHA was obtained.

〔実施例３〕
培地200 リットルに対して、ＮａＣｌが5400g 、ＭｇＳＯ4 ・７Ｈ2 Ｏが1320g 、ＫＮＯ3 が72g 、ＣａＣｌ2 ・２Ｈ2 Ｏが30g 、ＫＨ2 ＰＯ4 が14g 、ＮａＨＣＯ3 が8g、Tris−ＨＣｌが482g、ビタミンＢ12が1mg 、チアミン−ＨＣｌが120mg 、ビオチンが0 ．2mg 、ＦｅＣｌ2 ・６Ｈ2 Ｏが553.8mg 、Ｎａ2 ＥＤＴＡが3730mg、ＺｎＣｌ2 が0 ．8 mg、Ｈ3 ＢＯ3 が12mg、ＣｏＣｌ2 ・６Ｈ2 Ｏが0 ．3mg 、ＣｕＣｌ2 ・２Ｈ2 Ｏが0 ．8mg 、ＭｎＣｌ2 ・４Ｈ2 Ｏが8mg 、（ＮＨ4 ）6 ＭｏＯ24が7.4mg の割合で含まれている無機液体培地（pH8 ．0 ）200 リットルを、高さ2 m 、前後面の各幅2m、左右側面の各幅13cmのアクリル製の縦型偏平水槽に入れ、培養温度を23℃とし、照射の時間を18時間／日として、炭酸ガス1 ％を含む空気を7 l ／min の割合で通気しつつ、第１工程として、水槽中の無機液体培地に、パブロバを重量乾燥藻体濃度10mg／l の割合で接種し、水槽の前面片面に光強度75μE ／ｍ・ｓ2 の昼色蛍光灯を照射し、培養して、70mg／l となった後、第２工程として、水槽の前後の両面に、光強度150 μE ／ｍ・ｓ2 の昼色蛍光灯を照射し、培養して、220mg ／l となった後、第３工程として、培養液のpHを6 ．8 に調整して培養して、520mg ／l となった後、培養処理を止めて、得られた培養液をシャープレス遠心分離機（15000 回／分）で分別して得た藻体を蒸留水で洗浄してから凍結乾燥して、101gの乾燥藻体を得た。 Example 3
For 200 liters of medium, 5400 g of NaCl, 1320 g of MgSO 4 .7H 2 O, 72 g of KNO 3 , 30 g of CaCl 2 .2H 2 O, 14 g of KH 2 PO 4 , 8 g of NaHCO 3 , and Tris-HCl 482 g, vitamin B 12 is 1 mg, thiamine -HCl is 120 mg, biotin 0. 2 mg, FeCl 2 .6H 2 O 553.8 mg, Na 2 EDTA 3730 mg, ZnCl 2 0. 8 mg, 12 mg of H 3 BO 3 , 0 of CoCl 2 .6H 2 O. 3 mg, CuCl 2 .2H 2 O is 0. 200 liters of inorganic liquid medium (pH 8.0) containing 8 mg, 8 mg of MnCl 2 .4H 2 O and 7.4 mg of (NH 4 ) 6 MoO 24 , 2 m in height and each width on the front and back sides Place in an acrylic vertical flat water tank with a width of 2m and a width of 13cm on each of the left and right sides, culture temperature of 23 ° C, irradiation time of 18 hours / day, and air containing 1% carbon dioxide at a rate of 7 l / min As a first step, pavlova is inoculated at a rate of 10 mg / l by weight of dry algal bodies into the inorganic liquid medium in the aquarium, and the daylight color with a light intensity of 75 μE / m · s 2 is applied to the front side of the aquarium. After irradiating with fluorescent light and cultivating to 70 mg / l, as a second step, irradiate with daylight fluorescent light with a light intensity of 150 μE / m · s 2 on both the front and rear sides of the aquarium. Then, after reaching 220 mg / l, as the third step, the pH of the culture solution is adjusted to 6. After adjusting to 8 and cultivating to 520 mg / l, the culture treatment was stopped, and the obtained culture solution was fractionated with a sharp press centrifuge (15000 times / min) to obtain the algal cells obtained in distilled water. After washing with lyophilized, 101 g of dried alga bodies were obtained.

得られた乾燥藻体中のＥＰＡ量及びＤＨＡ量を実施例１と同様にして定量した結果、ＥＰＡは4 ．4g、ＤＨＡは1 ．8gであった。   As a result of quantifying the amount of EPA and the amount of DHA in the obtained dry algal cells in the same manner as in Example 1, EPA was 4. 4g, DHA is 1. It was 8g.

〔実施例４〕
培地50リットルに対して、ＮａＣｌが1350g 、ＭｇＳＯ4 ・７Ｈ2 Ｏが330g、ＫＮＯ3 が18g 、ＣａＣｌ2 ・２Ｈ2 Ｏが7g、ＫＨ2 ＰＯ4 が3.5g、ＮａＨＣＯ3 が2g、Tris−ＨＣｌが121g、ビタミンＢ12が0.25mg、チアミン−ＨＣｌが30mg、ビオチンが0 ．05mg、ＦｅＣｌ2 ・６Ｈ2 Ｏが138.5mg 、Ｎａ2 ＥＤＴＡが930mg 、ＺｎＣｌ2 が0 ．2mg 、Ｈ3 ＢＯ3 が3mg 、ＣｏＣｌ2 ・６Ｈ2 Ｏが0 ．075mg 、ＣｕＣｌ2 ・２Ｈ2 Ｏが0 ．2mg 、ＭｎＣｌ2 ・４Ｈ2 Ｏが2mg 、（ＮＨ4 ）6 ＭｏＯ24が1.85mgの割合で含まれている無機液体培地（pH8 ．0 ）50リットルを、高さ2 m 、前後面の各幅0.5m、左右側面の各幅13cmのガラス製の縦型偏平水槽に入れ、培養温度を26℃とし、照射の時間を18時間／日として、炭酸ガス1 ％を含む空気を5 l ／min の割合で通気しつつ、第１工程として、水槽中の無機液体培地に、パブロバを重量乾燥藻体濃度13mg／l の割合で接種し、水槽の前面片面に光強度75μE ／ｍ・ｓ2 の白熱灯を照射し、培養して、50mg／l となった後、第２工程として、水槽の前後の両面に、光強度138 μE ／ｍ・ｓ2 の白熱灯を照射し、培養して、220mg ／l となった後、第３工程として、培養液のpHを7 ．0 に調整して培養して、510mg ／l となった後、培養処理を止めて、得られた培養液をシャープレス遠心分離機（15000 回／分）で分別して得た藻体を蒸留水で洗浄してから凍結乾燥して、26g の乾燥藻体を得た。 Example 4
For 50 liters of medium, NaCl is 1350 g, MgSO 4 · 7H 2 O is 330 g, KNO 3 is 18 g, CaCl 2 · 2H 2 O is 7 g, KH 2 PO 4 is 3.5 g, NaHCO 3 is 2 g, Tris-HCl 121 g, vitamin B 12 0.25 mg, thiamine-HCl 30 mg, biotin 0. 05 mg, FeCl 2 .6H 2 O 138.5 mg, Na 2 EDTA 930 mg, ZnCl 2 0. 2mg, H 3 BO 3 is 3mg, CoCl 2 · 6H 2 O is 0. 075 mg, CuCl 2 .2H 2 O is 0. 50 liters of inorganic liquid medium (pH 8.0) containing 2 mg, 2 mg of MnCl 2 .4H 2 O and 1.85 mg of (NH 4 ) 6 MoO 24 , 2 m in height, each width on the front and rear Place in a vertical flat water tank made of glass with a width of 0.5 m and a width of 13 cm on each side, culture temperature is 26 ° C, irradiation time is 18 hours / day, and air containing 1% carbon dioxide is 5 l / min. As a first step, aerial liquid medium in the aquarium is inoculated with Pavlova at a dry dry alga concentration of 13 mg / l, and the front surface of the aquarium is incandescent with a light intensity of 75 μE / m · s 2 . After irradiating with light and culturing to 50 mg / l, as a second step, incandescent light with a light intensity of 138 μE / m · s 2 is irradiated on both sides of the water tank before and after culturing to 220 mg / L After the third step, the pH of the culture solution is set to 7. After adjusting to 0 and culturing to 510 mg / l, the culture treatment was stopped, and the obtained culture broth was fractionated with a sharp press centrifuge (15000 times / min) to obtain the algal cells obtained in distilled water. After washing with lyophilization, 26 g of dry algae was obtained.

得られた乾燥藻体中のＥＰＡ量及びＤＨＡ量を実施例１と同様にして定量した結果、ＥＰＡは1 ．1g、ＤＨＡは0 ．5gであった。   As a result of quantifying the amount of EPA and the amount of DHA in the obtained dry algal cells in the same manner as in Example 1, EPA was 1. 1 g, DHA is 0. It was 5g.

〔実施例５〕
培地50リットルに対して、ＮａＣｌが1200g 、ＭｇＳＯ4 ・７Ｈ2 Ｏが330g、ＫＮＯ3 が15g 、ＣａＣｌ2 ・２Ｈ2 Ｏが6g、ＫＨ2 ＰＯ4 が2.5g、ＮａＨＣＯ3 が1.5g、Tris−ＨＣｌが110g、ビタミンＢ12が0.15mg、チアミン−ＨＣｌが20mg、ビオチンが0 ．05mg、ＦｅＣｌ2 ・６Ｈ2 Ｏが100mg 、Ｎａ2 ＥＤＴＡが750mg 、ＺｎＣｌ2 が0 ．2mg 、Ｈ3 ＢＯ3 が3mg 、ＣｏＣｌ2 ・６Ｈ2 Ｏが0 ．075mg 、ＣｕＣｌ2 ・２Ｈ2 Ｏが0 ．2mg 、ＭｎＣｌ2 ・４Ｈ2 Ｏが2mg 、（ＮＨ4 ）6 ＭｏＯ24が1.85mgの割合で含まれている無機液体培地（pH8 ．0 ）50リットルを、高さ1.5m、前後面の各幅0.5m、左右側面の各幅7cm のガラス製の縦型偏平水槽に入れ、培養温度を10℃とし、照射の時間を18時間／日として、炭酸ガス0.5 ％を含む空気を5 l ／min の割合で通気しつつ、第１工程として、水槽中の無機液体培地に、パブロバを重量乾燥藻体濃度10mg／l の割合で接種し、水槽の前面片面に光強度75μE ／ｍ・ｓ2 の白色蛍光灯を照射し、培養して、50mg／l となった後、第２工程として、水槽の前後の両面に、光強度148 μE ／ｍ・ｓ2 の白色蛍光灯を照射し、培養して、200mg ／l となった後、第３工程として、培養液のpHを7 ．1 に調整して培養して、507mg ／l となった後、培養処理を止めて、得られた培養液をシャープレス遠心分離機（15000 回／分）で分別して得た藻体を蒸留水で洗浄してから凍結乾燥して、24g の乾燥藻体を得た。 Example 5
For 50 liters of medium, 1200 g of NaCl, 330 g of MgSO 4 .7H 2 O, 15 g of KNO 3 , 6 g of CaCl 2 .2H 2 O, 2.5 g of KH 2 PO 4 , 1.5 g of NaHCO 3 , Tris− HCl is 110g, vitamin B 12 is 0.15 mg, thiamine -HCl is 20 mg, biotin 0. 05 mg, FeCl 2 .6H 2 O 100 mg, Na 2 EDTA 750 mg, ZnCl 2 0. 2mg, H 3 BO 3 is 3mg, CoCl 2 · 6H 2 O is 0. 075 mg, CuCl 2 .2H 2 O is 0. 50 liters of inorganic liquid medium (pH 8.0) containing 2 mg, 2 mg of MnCl 2 .4H 2 O and 1.85 mg of (NH 4 ) 6 MoO 24 , 1.5 m in height and each width on the front and rear surfaces Place in a vertical flat water tank made of glass with a width of 0.5 m and a width of 7 cm each on the left and right sides, culture temperature is 10 ° C, irradiation time is 18 hours / day, and air containing 0.5% carbon dioxide is 5 l / min. As a first step, inoculate the inorganic liquid medium in the aquarium with pavlova at a rate of 10 mg / l by weight dry algal body concentration, and on the front side of the aquarium, white with a light intensity of 75 μE / m · s 2 After irradiating and cultivating the fluorescent lamp to reach 50 mg / l, as a second step, irradiate the front and rear of the water tank with a white fluorescent lamp with a light intensity of 148 μE / m · s 2 and incubate. After 200 mg / l, as the third step, the pH of the culture solution is set to 7. After adjusting to 1 and culturing at 507 mg / l, the culture treatment was stopped, and the obtained culture broth was fractionated with a sharp press centrifuge (15000 times / min) to obtain the algal cells obtained in distilled water. After washing with lyophilization, 24 g of dry algae was obtained.

得られた乾燥藻体中のＥＰＡ量及びＤＨＡ量を実施例１と同様にして定量した結果、ＥＰＡは1 ．1g、ＤＨＡは0 ．6gであった。   As a result of quantifying the amount of EPA and the amount of DHA in the obtained dry algal cells in the same manner as in Example 1, EPA was 1. 1 g, DHA is 0. It was 6g.

〔実施例６〕
培地50リットルに対して、ＮａＣｌが1500g 、ＭｇＳＯ4 ・７Ｈ2 Ｏが350g、ＫＮＯ3 が20g 、ＣａＣｌ2 ・２Ｈ2 Ｏが9g、ＫＨ2 ＰＯ4 が4.5g、ＮａＨＣＯ3 が2.5g、Tris−ＨＣｌが130g、ビタミンＢ12が0.35mg、チアミン−ＨＣｌが30mg、ビオチンが0 ．5mg 、ＦｅＣｌ2 ・６Ｈ2 Ｏが150mg 、Ｎａ2 ＥＤＴＡが1250mg、ＺｎＣｌ2 が1mg 、Ｈ3 ＢＯ3 が15mg、ＣｏＣｌ2 ・６Ｈ2 Ｏが0 ．375mg 、ＣｕＣｌ2 ・２Ｈ2 Ｏが1mg 、ＭｎＣｌ2 ・４Ｈ2 Ｏが10mg、（ＮＨ4 ）6 ＭｏＯ24が9.25mgの割合で含まれている無機液体培地（pH8 ．0 ）50リットルを、高さ2.5m、前後面の各幅2m、左右側面の各幅16cmのアクリル製の縦型偏平水槽に入れ、培養温度を30℃とし、照射の時間を24時間／日として、炭酸ガス5 ％を含む空気を7 l ／min の割合で通気しつつ、第１工程として、水槽中の無機液体培地に、パブロバを重量乾燥藻体濃度40mg／l の割合で接種し、水槽の前面片面に光強度100 μE ／ｍ・ｓ2 の白色蛍光灯を照射し、培養して、100mg ／l となった後、第２工程として、水槽の前後の両面に、光強度100 μE ／ｍ・ｓ2 の白色蛍光灯を照射し、培養して、220mg ／l となった後、第３工程として、培養液のpHを7 ．0 に調整して培養して、550mg ／l となった後、培養処理を止めて、得られた培養液をシャープレス遠心分離機（15000 回／分）で分別して得た藻体を蒸留水で洗浄してから凍結乾燥して、43g の乾燥藻体を得た。 Example 6
For 50 liters of medium, NaCl is 1500 g, MgSO 4 · 7H 2 O is 350 g, KNO 3 is 20 g, CaCl 2 · 2H 2 O is 9 g, KH 2 PO 4 is 4.5 g, NaHCO 3 is 2.5 g, Tris- HCl is 130 g, vitamin B 12 is 0.35 mg, thiamine -HCl is 30mg, biotin 0. 5 mg, FeCl 2 .6H 2 O 150 mg, Na 2 EDTA 1250 mg, ZnCl 2 1 mg, H 3 BO 3 15 mg, CoCl 2 .6H 2 O 0. 50 ml of an inorganic liquid medium (pH 8.0) containing 375 mg, 1 mg of CuCl 2 .2H 2 O, 10 mg of MnCl 2 .4H 2 O, and 9.25 mg of (NH 4 ) 6 MoO 24 Put in an acrylic vertical flat water tank with a length of 2.5m, width of 2m at the front and back, and width of 16cm at the left and right sides, culture temperature of 30 ° C, irradiation time of 24 hours / day, carbon dioxide gas 5% As the first step, aerating the air containing 7 l / min, inoculate the inorganic liquid medium in the aquarium with pavlova at a weight dry alga concentration of 40 mg / l, and light intensity on the front side of the aquarium After irradiating with 100 μE / m · s 2 white fluorescent lamp and cultivating to 100 mg / l, the second step is white on both sides of the water tank with light intensity of 100 μE / m · s 2 After irradiating with a fluorescent lamp and culturing to 220 mg / l, as a third step, the pH of the culture solution is set to 7. After adjusting to 0 and culturing to 550 mg / l, the culture treatment was stopped, and the obtained culture broth was fractionated with a sharp press centrifuge (15000 times / min) to obtain the algal cells obtained in distilled water. After washing with lyophilization, 43 g of dry alga bodies were obtained.

得られた乾燥藻体中のＥＰＡ量及びＤＨＡ量を実施例１と同様にして定量した結果、ＥＰＡは1 ．2g、ＤＨＡは0 ．5gであった。   As a result of quantifying the amount of EPA and the amount of DHA in the obtained dry algal cells in the same manner as in Example 1, EPA was 1. 2 g, DHA is 0. It was 5g.

〔実施例７〕
培地500 リットルに対して、ＮａＣｌが13. 5kg 、ＭｇＳＯ4 ・７Ｈ2 Ｏが3.3kg 、ＫＮＯ3 が180g、ＣａＣｌ2 ・２Ｈ2 Ｏが70g 、ＫＨ2 ＰＯ4 が35g 、ＮａＨＣＯ3 が20g 、Tris−ＨＣｌが1.21kg、ビタミンＢ12が2.5mg 、チアミン−ＨＣｌが300mg 、ビオチンが0 ．5mg 、ＦｅＣｌ2 ・６Ｈ2 Ｏが1385mg、Ｎａ2 ＥＤＴＡが9.3g、ＺｎＣｌ2 が2mg 、Ｈ3 ＢＯ3 が30mg、ＣｏＣｌ2 ・６Ｈ2 Ｏが0 ．75mg、ＣｕＣｌ2 ・２Ｈ2 Ｏが2mg 、ＭｎＣｌ2 ・４Ｈ2 Ｏが20mg、（ＮＨ4 ）6 ＭｏＯ24が18.5mgの割合で含まれている無機液体培地（pH8 ．0 ）500 リットルを、直径１．１ｍ、高さ０．８ｍの５００リットル容積の円形ポリタンクに入れ、培養温度を26℃とし、照射の時間を18時間／日として、炭酸ガス1 ％を含む空気を5 l ／min の割合で通気しつつ、第１工程として、円形ポリタンク中の無機液体培地に、パブロバを重量乾燥藻体濃度13mg／l の割合で接種し、円形ポリタンクの上方から光強度75μE ／ｍ・ｓ2 の白色蛍光灯を照射し、培養して、50mg／l となった後、第２工程として、円形ポリタンクの上方から光強度138 μE ／ｍ・ｓ2 の白色蛍光灯を照射し、培養して、220mg ／l となった後、第３工程として、培養液のpHを7 ．0 に調整して培養して、510mg ／l となった後、培養処理を止めて、得られた培養液をシャープレス遠心分離機（15000 回／分）で分別して得た藻体を蒸留水で洗浄してから凍結乾燥して、260gの乾燥藻体を得た。 Example 7
For 500 liters of medium, NaCl is 13.5 kg, MgSO 4 .7H 2 O is 3.3 kg, KNO 3 is 180 g, CaCl 2 .2H 2 O is 70 g, KH 2 PO 4 is 35 g, NaHCO 3 is 20 g, Tris -HCl is 1.21kg, vitamin B 12 is 2.5mg, thiamine -HCl is 300mg, biotin is 0. 5 mg, FeCl 2 .6H 2 O 1385 mg, Na 2 EDTA 9.3 g, ZnCl 2 2 mg, H 3 BO 3 30 mg, CoCl 2 .6H 2 O 0. A diameter of 500 liters of an inorganic liquid medium (pH 8.0) containing 75 mg, 2 mg of CuCl 2 · 2H 2 O, 20 mg of MnCl 2 · 4H 2 O, and 18.5 mg of (NH 4 ) 6 MoO 24 Place in a 500-liter round plastic tank with a height of 1.1 m and a height of 0.8 m, culture temperature is 26 ° C, irradiation time is 18 hours / day, and air containing 1% carbon dioxide is 5 l / min As a first step, pavlova is inoculated into the inorganic liquid medium in the circular polytank at a rate of 13 mg / l by weight dry algal body concentration, and white with a light intensity of 75 μE / m · s 2 from above the circular polytank. After irradiating with fluorescent light and culturing to 50 mg / l, as a second step, irradiate with white fluorescent light with a light intensity of 138 μE / m · s 2 from above the circular plastic tank and incubate to 220 mg. / L After the third step, the pH of the culture solution is set to 7. After adjusting to 0 and culturing to 510 mg / l, the culture treatment was stopped, and the obtained culture broth was fractionated with a sharp press centrifuge (15000 times / min) to obtain the algal cells obtained in distilled water. After washing with lyophilization, 260 g of dried alga bodies were obtained.

得られた乾燥藻体中のＥＰＡ量及びＤＨＡ量を実施例１と同様にして定量した結果、ＥＰＡは1 1g、ＤＨＡは5gであった。   As a result of quantifying the amount of EPA and the amount of DHA in the obtained dry algal cells in the same manner as in Example 1, 11 g of EPA and 5 g of DHA were found.

〔実施例８〕
培地2000リットルに対して、ＮａＣｌが54Kg、ＭｇＳＯ4 ・７Ｈ2 Ｏが13.2Kg、ＫＮＯ3 が720g、ＣａＣｌ2 ・２Ｈ2 Ｏが300g、ＫＨ2 ＰＯ4 が140g、ＮａＨＣＯ3 が80g 、Tris−ＨＣｌが4.82Kg、ビタミンＢ12が10mg、チアミン−ＨＣｌが1.2g、ビオチンが2mg 、ＦｅＣｌ2 ・６Ｈ2 Ｏが5.538g、Ｎａ2 ＥＤＴＡが37.3g 、ＺｎＣｌ2 が8mg 、Ｈ3 ＢＯ3 が120mg 、ＣｏＣｌ2 ・６Ｈ2 Ｏが3mg 、ＣｕＣｌ2 ・２Ｈ2 Ｏが8mg 、ＭｎＣｌ2 ・４Ｈ2 Ｏが80mg、（ＮＨ4 ）6 ＭｏＯ24が74mgの割合で含まれている無機液体培地（pH8 ．0 ）2000リットルを、縦１０ｍ、横３ｍの２トン容の強化プラスチック製レースウェイ培養タンクに入れ、培養温度を23℃とし、照射の時間を18時間／日として、炭酸ガス1 ％を含む空気を7 l ／min の割合で通気しつつ、第１工程として、培養タンク中の無機液体培地に、パブロバを重量乾燥藻体濃度10mg／l の割合で接種し、培養タンクの前面片面に光強度75μE ／ｍ・ｓ2 の白色蛍光灯を照射し、培養して、70mg／l となった後、第２工程として、培養タンクの前後の両面に、光強度150 μE ／ｍ・ｓ2 の白色蛍光灯を照射し、培養して、220mg ／l となった後、第３工程として、培養液のpHを6 ．8 に調整して培養して、520mg ／l となった後、培養処理を止めて、得られた培養液をシャープレス遠心分離機（15000 回／分）で分別して得た藻体を蒸留水で洗浄してから凍結乾燥して、1010g の乾燥藻体を得た。 Example 8
For 2000 liters of medium, NaCl 54 kg, MgSO 4 · 7H 2 O 13.2 kg, KNO 3 720 g, CaCl 2 · 2H 2 O 300 g, KH 2 PO 4 140 g, NaHCO 3 80 g, Tris-HCl 4.82 Kg, vitamin B 12 10 mg, thiamine-HCl 1.2 g, biotin 2 mg, FeCl 2 .6H 2 O 5.538 g, Na 2 EDTA 37.3 g, ZnCl 2 8 mg, H 3 BO 3 120 mg, Inorganic liquid medium (pH 8.0) containing 3 mg of CoCl 2 .6H 2 O, 8 mg of CuCl 2 .2H 2 O, 80 mg of MnCl 2 .4H 2 O, and 74 mg of (NH 4 ) 6 MoO 24 ) Put 2000 liters into a 2 ton reinforced plastic raceway culture tank 10m long and 3m wide, culturing temperature 23 ° C, irradiation time 18 hours / day, and air containing 1% carbon dioxide. As the first step, aerating at a rate of 7 l / min, Pavlova was inoculated into the liquid medium at a rate of 10 mg / l of dry dry alga body concentration, irradiated with a white fluorescent lamp with a light intensity of 75 μE / m · s 2 on the front side of the culture tank, and cultured to 70 mg / l. After that, as the second step, the both sides of the culture tank were irradiated with a white fluorescent lamp with a light intensity of 150 μE / m · s 2 and cultured to reach 220 mg / l, then the third step The pH of the culture solution is 6. After adjusting to 8 and cultivating to 520 mg / l, the culture treatment was stopped, and the obtained culture solution was fractionated with a sharp press centrifuge (15000 times / min) to obtain the algal cells obtained in distilled water. After washing with lyophilization, 1010 g of dried algae were obtained.

得られた乾燥藻体中のＥＰＡ量及びＤＨＡ量を実施例１と同様にして定量した結果、ＥＰＡは44g 、ＤＨＡは18g であった。   As a result of quantifying the amount of EPA and the amount of DHA in the obtained dry algal cells in the same manner as in Example 1, 44 g of EPA and 18 g of DHA were found.

第１〜３工程の各工程までそれぞれパブロア藻体を３０日間培養した培養曲線を示す図である。It is a figure which shows the culture curve which culture | cultivated the Pabloa alga body for 30 days to each process of a 1st-3rd process, respectively.

Claims (2)

単細胞微細藻類を培養槽中の無機液体培地に接種して、光照射しつつ、かつ、炭酸ガス含有空気を通気しつつ培養する方法において、パブロバ藻体であるハプト植物部門(Haptophyta)に属するハプト藻網(Haptophyceae)のパブロバ目(Pavlovales)のパブロバ（Pavlova)を重量乾燥藻体濃度10〜40mg／l に接種して、培養槽に光強度75〜100 μE ／m ・s ２ の光を照射しつつ、50〜100mg ／l まで培養する第１工程と、この50〜100mg ／l 濃度の培養液に、培養槽に光強度125 〜150 μE ／m ・s ２ の光を照射しつつ、200 〜230mg ／l まで培養する第２工程と、この200 〜230mg ／l 濃度の培養液に、培養液のpHを6 ．8 〜7 ．2 の範囲に調整して、500 〜550mg ／l まで培養する第３工程とから成ることを特徴とするイコサペンタエン酸及びドコサヘキサエン酸の生産方法。 Haptophyta belonging to the haptophyta section, which is a Pavlova alga body, in a method of inoculating single-cell microalgae into an inorganic liquid medium in a culture tank, cultivating while irradiating light and aeration of carbon dioxide-containing air pavlova Pavlova th algae network (Haptophyceae) (Pavlovales) a (Pavlova) was inoculated into weight dry algal cell concentration 10 to 40 mg / l, the irradiation light of the light intensity 75~100 μE / m · s 2 to the culture tank However, the first step of culturing up to 50 to 100 mg / l, and irradiating the culture solution with a light intensity of 125 to 150 μE / m · s 2 to the culture medium at a concentration of 50 to 100 mg / l , In the second step of culturing up to ˜230 mg / l, the pH of the culture solution is adjusted to 6. 8-7. 2. A method for producing icosapentaenoic acid and docosahexaenoic acid, comprising a third step of culturing to 500 to 550 mg / l after adjusting to the range of 2 . 単細胞微細藻類を培養槽中の無機液体培地に接種して、光照射しつつ、かつ、炭酸ガス含有空気を通気しつつ培養する方法において、パブロバ藻体であるハプト植物部門(Haptophyta)に属するハプト藻網(Haptophyceae)のパブロバ目(Pavlovales)のパブロバ（Pavlova)を重量乾燥藻体濃度10〜40mg／l に接種して、培養槽の片側から光強度75〜100 μE ／m ・s ２ の光を照射しつつ、50〜100mg ／l まで培養する第１工程と、この50〜100mg ／l 濃度の培養液に、培養槽の両側から光強度125 〜150 μE ／m ・s ２ の光を照射しつつ、200 〜230mg ／l まで培養する第２工程と、この200 〜230mg ／l 濃度の培養液に、培養液のpHを6 ．8 〜7 ．2 の範囲に調整して、500 〜550mg ／l まで培養する第３工程とから成ることを特徴とするイコサペンタエン酸及びドコサヘキサエン酸の生産方法。 Haptophyta belonging to the haptophyta section, which is a Pavlova alga body, in a method of inoculating single-cell microalgae into an inorganic liquid medium in a culture tank, cultivating while irradiating light and aeration of carbon dioxide-containing air pavlova Pavlova th algae network (Haptophyceae) (Pavlovales) a (Pavlova) was inoculated into weight dry algal cell concentration 10 to 40 mg / l, the light intensity from one side of the culture tank 75~100 μE / m of · s 2 light while irradiating the irradiation with the first step of culturing to 50 to 100 mg / l, in the culture solution of the 50 to 100 mg / l concentration, the light intensity 125 ~150 μE / m · s 2 from both sides of the culture tank However, the second step of culturing up to 200 to 230 mg / l and the culture solution at a concentration of 200 to 230 mg / l are adjusted to pH 6. 8-7. 2. A method for producing icosapentaenoic acid and docosahexaenoic acid, comprising a third step of culturing to 500 to 550 mg / l after adjusting to the range of 2 .
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JP3391494B2 (en) * 1993-02-17 2003-03-31 株式会社海洋バイオテクノロジー研究所 Novel betaine lipid and its production method
JPH07316584A (en) * 1994-05-25 1995-12-05 Shimadzu Corp Purification of highly unsaturated fatty acid
JPH09252762A (en) * 1996-03-22 1997-09-30 Yoshio Tanaka Culture of cocolithophorid alga to increase production of docosahexaenoic acid
JPH09252761A (en) * 1996-03-22 1997-09-30 Yoshio Tanaka Culture of nannochloropsis algae increasing production of eicosapentaenoic acid
JP3541124B2 (en) * 1998-04-14 2004-07-07 株式会社日健総本社 Culture method of Coleoptera algae to produce polysaccharides

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