JPH09252764A - Cultivation of monodus algae producing eicosapentaenoic acid - Google Patents
Cultivation of monodus algae producing eicosapentaenoic acidInfo
- Publication number
- JPH09252764A JPH09252764A JP8091770A JP9177096A JPH09252764A JP H09252764 A JPH09252764 A JP H09252764A JP 8091770 A JP8091770 A JP 8091770A JP 9177096 A JP9177096 A JP 9177096A JP H09252764 A JPH09252764 A JP H09252764A
- Authority
- JP
- Japan
- Prior art keywords
- culturing
- concentration
- monodus
- pcv
- water tank
- 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.)
- Pending
Links
- 241000180113 Monodus Species 0.000 title claims abstract description 16
- 241000195493 Cryptophyta Species 0.000 title claims abstract description 14
- 235000020673 eicosapentaenoic acid Nutrition 0.000 title abstract description 26
- JAZBEHYOTPTENJ-JLNKQSITSA-N all-cis-5,8,11,14,17-icosapentaenoic acid Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O JAZBEHYOTPTENJ-JLNKQSITSA-N 0.000 title abstract description 5
- 229960005135 eicosapentaenoic acid Drugs 0.000 title abstract description 5
- JAZBEHYOTPTENJ-UHFFFAOYSA-N eicosapentaenoic acid Natural products CCC=CCC=CCC=CCC=CCC=CCCCC(O)=O JAZBEHYOTPTENJ-UHFFFAOYSA-N 0.000 title abstract description 5
- 238000012258 culturing Methods 0.000 claims abstract description 30
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000007788 liquid Substances 0.000 claims abstract description 19
- 229910002092 carbon dioxide Inorganic materials 0.000 claims abstract description 14
- 239000001569 carbon dioxide Substances 0.000 claims abstract description 13
- 238000005273 aeration Methods 0.000 claims abstract description 5
- 239000013505 freshwater Substances 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 48
- 230000001678 irradiating effect Effects 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 7
- 238000012136 culture method Methods 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 abstract 3
- 238000009630 liquid culture Methods 0.000 abstract 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 16
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 12
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 12
- 239000004317 sodium nitrate Substances 0.000 description 8
- 235000010344 sodium nitrate Nutrition 0.000 description 8
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 7
- 239000012153 distilled water Substances 0.000 description 7
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 6
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 6
- 239000004327 boric acid Substances 0.000 description 6
- 239000001110 calcium chloride Substances 0.000 description 6
- 229910001628 calcium chloride Inorganic materials 0.000 description 6
- 229960002413 ferric citrate Drugs 0.000 description 6
- 239000007789 gas Substances 0.000 description 6
- NPFOYSMITVOQOS-UHFFFAOYSA-K iron(III) citrate Chemical compound [Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NPFOYSMITVOQOS-UHFFFAOYSA-K 0.000 description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 6
- 235000019341 magnesium sulphate Nutrition 0.000 description 6
- VLAPMBHFAWRUQP-UHFFFAOYSA-L molybdic acid Chemical compound O[Mo](O)(=O)=O VLAPMBHFAWRUQP-UHFFFAOYSA-L 0.000 description 6
- 229910000160 potassium phosphate Inorganic materials 0.000 description 6
- 235000011009 potassium phosphates Nutrition 0.000 description 6
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 6
- 229910000368 zinc sulfate Inorganic materials 0.000 description 6
- 229960001763 zinc sulfate Drugs 0.000 description 6
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 5
- 229910000365 copper sulfate Inorganic materials 0.000 description 5
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 238000009423 ventilation Methods 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- CUXYLFPMQMFGPL-BGDVVUGTSA-N (9Z,11E,13Z)-octadecatrienoic acid Chemical compound CCCC\C=C/C=C/C=C\CCCCCCCC(O)=O CUXYLFPMQMFGPL-BGDVVUGTSA-N 0.000 description 1
- 239000004925 Acrylic resin Substances 0.000 description 1
- 229920000178 Acrylic resin Polymers 0.000 description 1
- 241000206761 Bacillariophyta Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000206731 Phaeodactylum Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000004523 agglutinating effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 229940036886 copper sulfate 25 mg Drugs 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 235000019387 fatty acid methyl ester Nutrition 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- PDKHNCYLMVRIFV-UHFFFAOYSA-H molybdenum;hexachloride Chemical compound [Cl-].[Cl-].[Cl-].[Cl-].[Cl-].[Cl-].[Mo] PDKHNCYLMVRIFV-UHFFFAOYSA-H 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 1
- 239000004926 polymethyl methacrylate Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 102220201851 rs143406017 Human genes 0.000 description 1
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【0001】[0001]
【発明の属する技術分野】本発明は、エイコサペンタエ
ン酸(以下EPAという)を生産する淡水産単細胞藻類
のモノダス(Monodus)藻体などの培養法に関す
る。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for culturing a freshwater unicellular alga, Monodus algal cells, which produces eicosapentaenoic acid (hereinafter referred to as EPA).
【0002】[0002]
【従来の技術】従来、窒素源(硝酸ナトリウム)を添加
した、ガラス製偏平フラスコ中の無機液体培地中に、モ
ノダス・サズテラネウスATCC30593 ・CTCC848
を入れて、4〜10キロルックスの光照射と、炭酸ガス
5%を含む空気の通気下で、約6〜10日間の1次培養
した後、得られた培養物から藻体を分取し、これを、実
質的に窒素源(硝酸ナトリウム)を含まない培地で、約
10〜15日間の2次培養して藻体内に多量に蓄積させ
るエイコサペンタエン酸の生産方法は特公平4−383
97号公報に開示されており公知である。2. Description of the Related Art Conventionally, in an inorganic liquid medium in a glass flat flask to which a nitrogen source (sodium nitrate) has been added, Monodas Sazterraneus ATCC30593.CTCC848
After irradiating with 4 to 10 kilolux of light and aeration of air containing 5% of carbon dioxide gas, primary culture was carried out for about 6 to 10 days, and then algae were collected from the obtained culture. The production method of eicosapentaenoic acid, which is a secondary culture of about 10 to 15 days in a medium substantially containing no nitrogen source (sodium nitrate) to accumulate a large amount in the alga body, is disclosed in JP-B-4-383.
It is disclosed in Japanese Patent Publication No. 97 and is publicly known.
【0003】[0003]
【発明が解決しようとする課題】本発明者は、EPAを
生産する淡水産単細胞微細類のモノダス属に属する藻体
を大量に培養する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。The present inventors have accomplished the present invention as a result of researching a culture method for culturing a large amount of algae belonging to the genus Monodus, which is a freshwater single-cell microgenus that produces EPA.
【0004】[0004]
【課題を解決するための手段】本発明は、アクリル樹脂
製またはガラス製の偏平縦型水槽水槽中の無機液体培地
に、モノダス属に属する藻体を接種して、10〜30℃
の温度下で、炭酸ガスを0.3〜3.0%含む空気を、
通気量5〜35l/minの割合で通気しながら培養す
る培養法において、培養開始濃度を0.9〜1.7ml
/lとなし、照度6000〜21000lxの光を水槽
の前後面のいずれかの片面に照射して、PCV濃度1.
8〜4.9ml/lまで培養する第1工程と、該PCV
濃度1.8〜4.9ml/lの培養液に、水槽の前後の
両面に照度11000〜21000lx光を照射して、
PCV濃度5.0〜5.5ml/lまで培養する第2工
程と、該PCV濃度5.0〜5.5ml/lの培養液
に、水槽の前後の両面に照度10000〜35000l
xの光を照射して、PCV濃度10.0〜11.0まで
培養する第3工程とから成るEPAを産生するモノダス
藻体属藻体の培養法である。According to the present invention, an inorganic liquid medium in an acrylic resin or glass flat vertical aquarium is inoculated with algae belonging to the genus Monodus, and the temperature is 10 to 30 ° C.
At the temperature of, the air containing 0.3 to 3.0% of carbon dioxide gas,
In the culture method of culturing while aerating at an aeration rate of 5 to 35 l / min, the culture start concentration is 0.9 to 1.7 ml.
/ L, and irradiate light with an illuminance of 6000 to 21000 lx to either one of the front and rear surfaces of the water tank to obtain a PCV concentration of 1.
First step of culturing up to 8 to 4.9 ml / l and the PCV
A culture solution having a concentration of 1.8 to 4.9 ml / l was irradiated with illuminance of 11000 to 21000 lx on both surfaces before and after the water tank,
The second step of culturing to a PCV concentration of 5.0 to 5.5 ml / l, and using a culture solution having a PCV concentration of 5.0 to 5.5 ml / l, illuminance of 10000 to 35000 l on both sides before and after the water tank.
and a third step of culturing to a PCV concentration of 10.0 to 11.0 by irradiating x light, and a method for culturing an EPA-producing Monodaceous alga.
【0005】本発明で使用する、上端開口が開放型の縦
型偏平水槽とは、厚さ3cm程度のガラス製またはアク
リル製で、高さが1.5〜2.5mで、前後面の各面の
幅が0.5〜2.0程度で、左右側面の各面の幅が7〜
17cmの縦型で偏平の水槽で、上端に形成された長方
形の開口部には、周壁に適数個の通気孔が設けられ、先
端が閉鎖されている通気管が、その先端部から挿入さ
れ、また、船底形状に形成された底部には培養液が排出
管が着脱自在に設けられて成る縦型の偏平な水槽を意味
する。当該水槽の側面の幅は、17cmを超えると光の
照射が充分にならず好ましくない。そして、該水槽の前
後面の受光面積が広く好ましく、また、挿入された通気
管から炭酸ガス含有空気が送入されると、水槽中の培地
が攪拌されて、炭酸ガスの吸収効率が高く、かつ、上端
開口部が細長く狭いので雑菌の混入が少なくて、微細藻
類の大量培養に好適なのである。また、本発明での光照
射とは、白色蛍光灯、昼色蛍光灯または白熱灯の照射を
意味する。The vertical flat water tank having an open upper end used in the present invention is made of glass or acrylic having a thickness of about 3 cm, has a height of 1.5 to 2.5 m, and has front and rear surfaces. The width of the surface is about 0.5 to 2.0, and the width of each of the left and right side surfaces is 7 to
A 17 cm vertical, flat water tank with a rectangular opening formed at the upper end and a suitable number of ventilation holes in the peripheral wall, and a ventilation pipe with a closed tip inserted from the tip. Further, it means a vertical flat water tank in which a culture solution discharge pipe is detachably provided at a bottom portion formed in a ship bottom shape. If the width of the side surface of the water tank exceeds 17 cm, light irradiation is not sufficient, which is not preferable. And, the light receiving area of the front and rear surfaces of the aquarium is wide and preferable, and when the carbon dioxide-containing air is fed from the inserted ventilation pipe, the medium in the aquarium is agitated and the absorption efficiency of carbon dioxide is high, Moreover, since the upper end opening is long and narrow, it is suitable for large-scale culturing of microalgae with less contamination of various bacteria. Further, the light irradiation in the present invention means irradiation of a white fluorescent lamp, a daylight fluorescent lamp or an incandescent lamp.
【0006】本発明での無機液体培地とは、培地100
リットルに対して,硝酸ナトリウムが150〜180
g、燐酸カリウムが20〜30g、硫酸マグネシウムが
20〜30g、塩化カルシウムが10〜20 g、クエン
酸第二鉄が0.45〜2.45g、硼酸が0.11〜
0.55g、モリブデン酸が30〜150mg、塩化コ
バルトが10〜50mg、硫酸銅が10〜50mg、硫
酸亜鉛が10〜5mgを含む無機液体培地を意味する。Inorganic liquid medium in the present invention means medium 100
Sodium nitrate is 150 to 180 per liter
g, potassium phosphate 20 to 30 g, magnesium sulfate 20 to 30 g, calcium chloride 10 to 20 g, ferric citrate 0.45 to 2.45 g, and boric acid 0.11 to
It means an inorganic liquid medium containing 0.55 g, molybdic acid 30 to 150 mg, cobalt chloride 10 to 50 mg, copper sulfate 10 to 50 mg, and zinc sulfate 10 to 5 mg.
【0007】本発明で使用するモノダス藻類としては、
真正眼点藻に属するモノダス・サブテラネウス(mon
odus・subterraneus)や、珪藻に属す
るフェオダクチラム(Phaeodactylum s
p.)などが挙げられる。The monodus algae used in the present invention include:
Monodas subterraneus (mon) belonging to true eye spot algae
odus ・ subterraneus) and Phaeodactylums belonging to diatoms
p. ).
【0008】本発明において、モノダス藻類をPCV濃
度0.9〜1.7ml/lの割合で縦型偏平水槽の無機
液体培地に接種して、10〜30℃下で、炭酸ガス0.
5〜5.0%含有空気を通気量5〜35l/minの割
合で通気しつつ、第1工程にで、照度8000〜180
00lxの光を水槽の前後の何れかの片面に照射して培
養するのは、藻体を凝集させず順調に培養することがで
き、そして、PCV濃度1.8〜4.9ml/lで培養
を止めるのは、PCV濃度4.9ml/lを超えると、
照度不足を来してEPA生産速度が低下し、かつ、藻体
を凝集させて、順調に培養を第2工程に移行することが
できないからである。In the present invention, monodus algae are inoculated into an inorganic liquid medium in a vertical flat water tank at a PCV concentration of 0.9 to 1.7 ml / l, and carbon dioxide gas of 0.
Illuminance of 8000 to 180 in the first step while aerating 5 to 5.0% contained air at a rate of 5 to 35 l / min.
By irradiating either side of the front or rear of the water tank with 001x of light for culturing, it is possible to cultivate smoothly without agglutinating algal cells, and to cultivate at a PCV concentration of 1.8 to 4.9 ml / l. Is stopped when the PCV concentration exceeds 4.9 ml / l,
This is because illuminance becomes insufficient and the EPA production rate decreases, and the alga bodies cannot be aggregated to smoothly transfer the culture to the second step.
【0009】第1工程で培養したPCV濃度1.8〜
4.9ml/lの水槽中の培養液を、第2工程におい
て、水槽の前後の両面に照度11000〜21000l
x光を照射して培養するのは、順調に培養させてEPA
の生産をさせることがてきるからであり、そして、PC
V濃度5.0〜5.5ml/lで培養を止めるのは、P
CV濃度5.5ml/lで培養を止めるのは、PCV濃
度5.5ml/lを超えるまで培養を続けると、照度不
足のためEPAの生産速度を低下させ、かつ、藻体を凝
集させて、第3工程に順調に移行することができないか
らである。The concentration of PCV cultured in the first step is 1.8-
In the second step, the culture solution in a water tank of 4.9 ml / l was applied to both front and rear surfaces of the water tank with an illuminance of 11000 to 21000 l.
When culturing by irradiating x-ray, culturing is performed smoothly and EPA is performed.
Because it will be possible to produce
Stopping the culture at a V concentration of 5.0 to 5.5 ml / l is due to P
Stopping the culture at a CV concentration of 5.5 ml / l is because if the culture is continued until the PCV concentration exceeds 5.5 ml / l, the production rate of EPA is decreased due to insufficient illuminance and algae are aggregated, This is because it is not possible to smoothly shift to the third step.
【0010】第2工程で培養した水槽中のPCV濃度
5.0〜5.5ml/lの培養液を、第3工程におい
て、照度10000〜35000lxの光を、水槽の前
後の両面に照射して培養するのは、順調にEPAの生産
をさせるためであり、そして、培養をPCV濃度10.
0〜11.0で止めるのは、PCV濃度の増加は殆ど認
められず、従って、EPAの生産もなくなるので培養を
止めるのである。The culture solution having a PCV concentration of 5.0 to 5.5 ml / l in the water tank cultivated in the second step was irradiated with light having an illuminance of 10,000 to 35000 lx in both the front and rear sides of the water tank in the third step. The culturing was carried out in order to allow the EPA production to proceed smoothly, and the culturing was performed at a PCV concentration of 10.
The reason for stopping at 0 to 11.0 is that the increase in PCV concentration is scarcely recognized, and therefore the production of EPA is also stopped, so that the culture is stopped.
【0011】[0011]
【実施例1】培地100リットルに対して、硝酸ナトリ
ウムが150g、燐酸カリウムが20g、硫酸マグネシ
ウムが20g、塩化カルシウムが10g、クエン酸第二
鉄が0.49g、硼酸が0.11g、モリブデン酸が3
0mg、塩化モリブデンが10mg、塩化コバルトが1
0mg、硫酸銅が10mg、硫酸亜鉛10mgを含む無
機液体培地(pH7.5)100リットルを、高さ2
m、前後面の各幅1m、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25℃とし、照
射時間を18時間/日として、炭酸ガス1%を含む空気
を20l/minの割合で通気しつつ、第1工程とし
て、モノダス・サブテラネウスを、PCV濃度1.2m
l/lの割合で接種し、水槽の前後面の何れかの片面
に、12000lxの白色蛍光灯を照射し、培養して、
PCV濃度2.2ml/lとなった後、第2工程とし
て、水槽の前・後面の両面に、15000lxの白色蛍
光灯を照射し、培養して、PCV濃度5.5ml/lと
なった後、第3工程として、水槽の前・後面の両面に、
20000lxの白色蛍光灯を照射し、培養して、PC
V濃度が10.6ml/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(150
00回転/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、203gの乾燥藻体を得た。Example 1 150 g of sodium nitrate, 20 g of potassium phosphate, 20 g of magnesium sulfate, 10 g of calcium chloride, 0.49 g of ferric citrate, 0.11 g of boric acid and 100 g of molybdic acid per 100 liters of a medium. Is 3
0 mg, molybdenum chloride 10 mg, cobalt chloride 1
100 liters of an inorganic liquid medium (pH 7.5) containing 0 mg, 10 mg of copper sulfate and 10 mg of zinc sulfate, and a height of 2
m, the width of each of the front and rear surfaces is 1 m, and the width of each of the left and right sides is 13 cm. The container is placed in a vertical flat water tank made of acrylic, the culture temperature is 25 ° C., the irradiation time is 18 hours / day, and the air containing 1% carbon dioxide gas is 20 l. As the first step, while ventilating at a rate of / min, use Monodus subterraneus with a PCV concentration of 1.2 m.
Inoculate at a ratio of 1 / l, irradiate 12,000 lx of white fluorescent lamp on either one of the front and back surfaces of the water tank, and culture
After the PCV concentration reached 2.2 ml / l, in the second step, both front and rear surfaces of the water tank were irradiated with 15000 lx white fluorescent lamps and cultured to reach a PCV concentration of 5.5 ml / l. , As the third step, on both front and rear surfaces of the aquarium,
Irradiate with 20000 lx white fluorescent lamp, incubate, and
After the V concentration reached 10.6 ml / l, the culture treatment was stopped, and the obtained culture solution was subjected to a Sharpless centrifuge (150
The algal cells obtained by fractionation at 00 rpm were washed with distilled water and freeze-dried to obtain 203 g of dried algal cells.
【0012】得られた乾燥藻体を、塩酸5%を含むメタ
ノール溶媒に入れ、95℃で3時間還流した後、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クロマトグラフィー分析法で定量
した結果、18.3gのEPAを得た。The obtained dried algal cells were placed in a methanol solvent containing 5% hydrochloric acid and refluxed at 95 ° C. for 3 hours, and then fatty acid methyl ester was extracted with a hexane solvent, and trichosanoic acid was analyzed by gas chromatography using an internal standard. As a result of quantification by the method, 18.3 g of EPA was obtained.
【0013】[0013]
【実施例2】実施例1と同様に、縦型偏平水槽中に10
0リットルの無機液体培地を入れ、培養温度を23℃と
し、照射時間を18時間/日として、炭酸ガス1%含有
空気を20l/minの割合で通気しつつ、第1工程と
して、モノダス・サブテラネウスCTCC848を、培
養開始濃度5ml/lとして接種し、水槽の前後面の何
れかの片面から12500lxの白色蛍光灯を照射し、
培養して、PCV濃度が2.1ml/lとなったとき、
第2工程として、水槽の前後面の両面から16000l
xの白色蛍光灯を照射し、培養してPCV濃度5.4m
l/lとなったとき、第3工程として、水槽の前後面の
両面の両面から20000lxの白色蛍光灯を照射し、
培養してPCV濃度が10.3ml/lとなったとき、
培養処理を止めて、得られた100リットルの培養液か
ら分別した70リットルの培養液をシャープレス遠心分
離処理(15000回転/分)して得た藻体を蒸留水で
洗浄し、凍結乾燥して乾燥藻体を128gを得た。そし
て、得られた乾燥藻体中のEPAを実施例1記載と同様
に定量した結果、12.3gのEPAを得た。[Embodiment 2] As in the case of Embodiment 1, 10
0 liters of an inorganic liquid medium was added, the culture temperature was 23 ° C., the irradiation time was 18 hours / day, and the air containing 1% carbon dioxide was aerated at a rate of 20 l / min, and the first step was Monodus subterraneus. CTCC848 was inoculated at a culture starting concentration of 5 ml / l, and 12500 lx of white fluorescent lamp was irradiated from either one of the front and rear surfaces of the water tank,
When the culture reached a PCV concentration of 2.1 ml / l,
As the second step, 16000l from both front and back of the water tank
x white fluorescent lamp, cultivated and PCV concentration 5.4m
When it becomes 1 / l, as a third step, irradiate a white fluorescent lamp of 20,000 lx from both front and rear surfaces of the water tank,
When the culture reached a PCV concentration of 10.3 ml / l,
After stopping the culturing process, 70 liters of the culture broth separated from the obtained 100 liters of culture broth were subjected to Sharpless centrifugation (15,000 rpm) to wash the alga bodies with distilled water and freeze-drying. 128 g of dried algal cells was obtained. Then, as a result of quantifying the EPA in the obtained dried algal cells in the same manner as described in Example 1, 12.3 g of EPA was obtained.
【0014】得られた乾燥藻体中のEPA量を、実施例
1と同様にガスクロマトグラフィー分析法で定量した結
果、乾燥藻体中のEPA量は14.3gであった。The amount of EPA in the obtained dried algal cells was quantified by the gas chromatography analysis method as in Example 1, and as a result, the amount of EPA in the dried algal cells was 14.3 g.
【0015】また、上記70リットルを分別した残余
の、PCV濃度10.3ml/lの30リットル培養液
に、上記実施例1の無機液体培地70リットルを加え
て、20000lxの白色蛍光灯を照射し、培養するこ
とを6回繰り返し培養して得た培養液をシャープレス遠
心分離機(15000回転/分)で得た藻体を蒸留水で
洗浄し、凍結乾燥して得た乾燥藻体中のEPAを定量し
た結果、78gのEPAを得た。70 liters of the inorganic liquid medium of Example 1 was added to the remaining 30 liters of the PCV concentration of 10.3 ml / l, which was obtained by separating the above 70 liters, and a 20000 lx white fluorescent lamp was irradiated. , The culture solution obtained by repeatedly culturing 6 times was washed by distilled water with a Sharpless centrifuge (15000 rpm), and the freeze-dried dried alga bodies As a result of quantifying EPA, 78 g of EPA was obtained.
【0016】[0016]
【実施例3】培地200リットルに対して、硝酸ナトリ
ウムが300g、燐酸カリウムが40g、硫酸マグネシ
ウム40g、塩化カルシウムが20g、クエン酸第二鉄
が0.98g、硼酸が0.22g、モリブデン酸が60
mg、塩化コバルト20mg、硫酸銅が20mg、硫酸
亜鉛が20mgを含む無機液体培地(pH7.5)20
0リットルを、高さ2m、前・後面の幅2m、左・右側
面の幅13cmのアクリル製の縦型偏平水槽に入れ、培
養温度23℃とし、照射時間を18時間/日として、炭
酸ガス1%含有空気を35l/mlの割合で通気しつ
つ、第1工程として、上記無機液体培地にフェオダクチ
ラムを1.3ml/l接種し、水槽の前後の何れかの片
面に12000lxの白色蛍光灯を照射し、培養して、
PCV濃度が2.3となょた後、第2工程として、水槽
の前・後面の両面に照度15000lxの白色蛍光灯を
照射し、培養して、PCV濃度が5.0ml/lとなっ
た後、第3工程として、水槽の前・後面の両面に照度2
0000lxの白色蛍光灯を照射し、培養して、PCV
濃度が10.1ml/lとなった後、培養処理を止め
て、得られた培養液をシヤープレス遠心分離機(150
00回転/分)で分別して得た藻体を蒸留水で洗浄し、
凍結乾燥して395gの乾燥藻体を得た。得られた乾燥
藻体中のEPA量を、実施例1と同様にして定量した結
果、36.3gであった。Example 3 For 200 liters of medium, 300 g of sodium nitrate, 40 g of potassium phosphate, 40 g of magnesium sulfate, 20 g of calcium chloride, 0.98 g of ferric citrate, 0.22 g of boric acid and molybdic acid were used. 60
Inorganic liquid medium (pH 7.5) containing 20 mg, 20 mg of cobalt chloride, 20 mg of copper sulfate and 20 mg of zinc sulfate.
0 liters were placed in a vertical flat water tank made of acrylic with a height of 2 m, a front / rear surface width of 2 m, and left / right side surfaces of 13 cm width, the culture temperature was 23 ° C., the irradiation time was 18 hours / day, and carbon dioxide gas was added. As a first step, 1.3 ml / l of pheoductilam was inoculated into the above-mentioned inorganic liquid medium while aerating 1% -containing air at a rate of 35 l / ml, and a 12,000 lx white fluorescent lamp was provided on either side of the front and rear of the water tank. Irradiate, incubate,
After the PCV concentration was 2.3, in the second step, both front and rear surfaces of the water tank were irradiated with a white fluorescent lamp with an illuminance of 15000 lx and cultured to give a PCV concentration of 5.0 ml / l. After that, as the third step, the illuminance of 2 on both the front and rear surfaces of the water tank.
Irradiate with 0000 lx white fluorescent lamp, incubate, and
After the concentration reached 10.1 ml / l, the culturing process was stopped, and the obtained culture broth was transferred to a shear press centrifuge (150
(00 revolutions / minute), the algal cells obtained by separation are washed with distilled water,
It was freeze-dried to obtain 395 g of dried algal cells. The amount of EPA in the obtained dried algal cells was quantified in the same manner as in Example 1 and the result was 36.3 g.
【0017】[0017]
【実施例4】培地50リットルに対して、硝酸ナトリウ
ムが75g、燐酸カリウムが10g、硫酸マグネシウム
が10g、塩化カルシウムが5g、クエン酸第二鉄が
0.25g、硼酸が0.055g、モリブデン酸が15
mg、塩化コバルトが5mg、硫酸銅が5mg、硫酸亜
鉛が5mgの割合で含む無機液体培地(pH7.5)5
0リットルを、高さ2m、前・後面の幅0.5m、左・
右側面の幅13cmのアクリル製の縦型偏平水槽に入れ
て、培養温度を23℃とし、照射時間を18時間/日と
して、炭酸ガス1%を含有する空気を20l/minの
割合で通気しつつ、第1工程として、該培地に、モノダ
ス・サブテラネウスCTCC848を培養開始濃度1.
1ml/l接種し、水槽の前面片面に照度12000ル
ックスの白色蛍光灯を照射し、培養して、PCV濃度が
2.0ml/lとなった後、第2工程として、照度15
000lxの白色蛍光灯を、水槽の前後の両面に照射
し、培養して藻体濃度が5.4ml/lとなった後、第
3工程として、照度20000lx白色蛍光灯を、水槽
の前後の両面に照射し、培養してPCV濃度が10.8
ml/lとなったとき培養を止めで、得られた培養液を
シャープレス遠心分離機(15000回転/分)で分別
して得た藻体を、蒸留水で洗浄し、凍結乾燥して97g
の乾燥藻体を得た。 得られた乾燥藻体中のEPA量
を、実施例1と同様に、カスクロマトグラフィー分析法
で定量した結果、EPA量は8.9gであった。Example 4 75 g of sodium nitrate, 10 g of potassium phosphate, 10 g of magnesium sulfate, 5 g of calcium chloride, 0.25 g of ferric citrate, 0.055 g of boric acid and 50% of molybdic acid per 50 liters of medium. Is 15
Inorganic liquid medium (pH 7.5) containing 5 mg of cobalt chloride, 5 mg of cobalt chloride, 5 mg of copper sulfate and 5 mg of zinc sulfate 5
0 liter, height 2m, front / rear width 0.5m, left
It was placed in an acrylic vertical flat water tank with a width of 13 cm on the right side, the culture temperature was 23 ° C., the irradiation time was 18 hours / day, and air containing 1% carbon dioxide was aerated at a rate of 20 l / min. Meanwhile, in the first step, the culture starting concentration of Monodus subterraneus CTCC848 was 1.
Inoculate 1 ml / l, irradiate one side of the front surface of the water tank with a white fluorescent lamp with an illuminance of 12000 lux, and culture it until the PCV concentration became 2.0 ml / l.
After irradiating 000 lx white fluorescent lamps on both sides of the front and rear of the water tank, and culturing to reach an algal concentration of 5.4 ml / l, as a third step, illuminance 20000 lx white fluorescent lamps on both sides of the front and rear of the water tank. To a PCV concentration of 10.8
When the amount reached to ml / l, the culture was stopped, and the obtained culture solution was fractionated with a Sharpless centrifuge (15,000 revolutions / minute) to obtain algal cells, which were washed with distilled water and freeze-dried to give 97 g.
The dried alga bodies of The amount of EPA in the obtained dried algal cells was quantified by the Cass chromatography analysis method as in Example 1, and as a result, the amount of EPA was 8.9 g.
【0018】[0018]
【実施例5】培地50リットルに対して、硝酸ナトリウ
ムが75g、燐酸カリウムが10g、硫酸マグネシウム
が10g、塩化カルシウム5g、クエン酸第二鉄が0.
25g、硼酸が0.055g、モリブデン酸が15g、
塩化コバルトが5mg、硫酸銅が5mg、硫酸亜鉛が5
mgの割合で含む無機液体培地(pH5.5)50リッ
トルを高さ1.5m、前・後面の幅0.5m、左・右側
面の幅7cmのアクリル製の縦型偏平水槽に入れ、培養
温度を10℃とし、照射時間を24時間/日として、炭
酸ガス5%含有空気を35l/minの割合で通気しつ
つ、第1工程として、水槽中の50リットルの無機液体
培地に、モノダス・サブテラネウスを、PCV濃度0.
9ml/の割合で接種し、水槽の前面片面に6000l
xの昼色蛍光灯を照射し、培養して、PCV濃度が1.
8ml/lとなったとき、第2工程として、水槽の前・
後の両面に、11000lxの昼色蛍光灯を照射し、培
養して、PCV濃度が5.0ml/lとなったとき、第
3工程として、水槽の前・後の両面に10000lxの
昼色蛍光灯を照射し、培養して、PCV濃度10.0m
l/lとなったとき、培養を止めて、得られた培養液を
シャープレス遠心分離機(15000回転/分)で分別
した藻体を蒸留水で洗浄し、凍結乾燥して、95gの乾
燥藻体を得た。得られた乾燥藻体中のEPA量を、実施
例1と同様にして定量した結果、8.1gであった。Example 5 To 50 liters of a medium, 75 g of sodium nitrate, 10 g of potassium phosphate, 10 g of magnesium sulfate, 5 g of calcium chloride, and ferric citrate of 0.
25g, boric acid 0.055g, molybdic acid 15g,
Cobalt chloride 5 mg, copper sulfate 5 mg, zinc sulfate 5
50 liters of inorganic liquid medium (pH 5.5) containing 50 mg in a height of 1.5 m, width of front / rear surface of 0.5 m, width of left / right side of 7 cm in an acrylic vertical flat water tank, and cultured The temperature was set to 10 ° C., the irradiation time was set to 24 hours / day, and 5% carbon dioxide gas-containing air was aerated at a rate of 35 l / min, and the first step was to add 50 mL of an inorganic liquid medium in a water tank to Monodasu. Subterraneus was treated with a PCV concentration of 0.
Inoculate at a rate of 9 ml / 6000l on one side of the front of the aquarium
PCV concentration is 1.
When it reaches 8 ml / l, as the second step, in front of the water tank
When both sides of the rear are irradiated with 11,000 lx of daylight fluorescent light and cultured, and the PCV concentration becomes 5.0 ml / l, the third step is 10,000 lx of daylight fluorescent on both the front and rear of the water tank. Irradiate with light and culture, PCV concentration 10.0m
When it reached 1 / l, the culture was stopped, and the obtained culture solution was separated by a Sharpless centrifuge (15000 rpm), the alga was washed with distilled water, freeze-dried, and dried to 95 g. I got algae. The amount of EPA in the obtained dried algal cells was quantified in the same manner as in Example 1, and the result was 8.1 g.
【0019】[0019]
【実施例6】培地50リットルに対して、硝酸ナトリウ
ムが90g、燐酸カリウムが15g、硫酸マグネシウム
が15g、塩化カルシウムが10g、クエン酸第二鉄が
1.23g、硼酸が0.28g、モリブデン酸が75m
g、塩化コバルトが23mg、硫酸銅が25mg、硫酸
亜鉛が25mgの割合で含む無機液体培地(pH8.
0)50リットルを、高さが2.5m、前・後面の幅が
2m、左・右側面の幅が17cmのガラス製の縦型偏平
水槽に入れ、培養温度を30℃とし、照射時間を24時
間/ひとして、炭酸ガス5%含有空気を35l/min
の割合で通気しつつ、第1工程として、水槽中の50リ
ットルの無機液体培地に、モノダス・サブテラネウス
を、PCV濃度1.7ml/lの割合で接種し、水槽の
前面片面に、21000lxの白熱灯を照射し、培養し
て、PCV濃度が4.9ml/lとなったとき、第2工
程として、水槽の前・後の両面に、21000lxの白
熱灯を照射し、培養して、PCV濃度が5.0ml/l
となったとき、第3工程として、水槽の前・後の両面
に、35000lxの白熱灯を照射し、培養して,PC
V濃度が11.0ml/lとなったとき、培養を止め
て、得られた培養液をシャーブレス遠心分離機(150
00回転/分)で分別して得た藻体を蒸留水で洗浄し、
凍結乾燥して、97gの乾燥藻体を得た。得られた乾燥
藻体中のEPA量を、実施例1と同様にして定量した結
果、8.9gであった。Example 6 90 g of sodium nitrate, 15 g of potassium phosphate, 15 g of magnesium sulfate, 10 g of calcium chloride, 1.23 g of ferric citrate, 0.28 g of boric acid and 50 g of molybdic acid per 50 liters of a medium. Is 75m
g, cobalt chloride 23 mg, copper sulfate 25 mg, and zinc sulfate 25 mg in an inorganic liquid medium (pH 8.
0) 50 liters were placed in a vertical glass flat water tank with a height of 2.5 m, a width of front and rear sides of 2 m, and widths of left and right sides of 17 cm, the culture temperature was 30 ° C., and the irradiation time was 24 hours / hour, 35 l / min of air containing 5% carbon dioxide
As a first step, 50 L of an inorganic liquid medium in a water tank was inoculated with Monodas subterraneus at a PCV concentration of 1.7 ml / l as a first step while aeration of 21,000 lx of incandescent water was applied to the front surface of the water tank. When the PCV concentration reached 4.9 ml / l by illuminating with a lamp and culturing, the second step was irradiating with 21000 lx incandescent lamps on both the front and rear sides of the water tank, culturing, and culturing the PCV concentration. Is 5.0 ml / l
Then, as a third step, both the front and the rear of the water tank are irradiated with an incandescent lamp of 35000 lx, cultured, and
When the V concentration reached 11.0 ml / l, the culturing was stopped, and the obtained culture broth was collected using a sherbless centrifuge (150
(00 revolutions / minute), the algal cells obtained by separation are washed with distilled water,
It was freeze-dried to obtain 97 g of dried algal cells. The amount of EPA in the obtained dried algal cells was quantified in the same manner as in Example 1, and the result was 8.9 g.
【0020】[0020]
【発明の効果】本発明によれば、モノダス藻類の大量培
養が容易にでき、かつ、EPAを高能率で生産できる有
用な培養法が得られたのである。EFFECTS OF THE INVENTION According to the present invention, a useful culture method has been obtained in which large-scale culture of Monodus algae can be easily carried out and EPA can be produced with high efficiency.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 FI 技術表示箇所 (C12P 7/64 C12R 1:89) ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 6 Identification number Office reference number FI technical display location (C12P 7/64 C12R 1:89)
Claims (1)
機液体培地に接種して、光照射と、炭酸ガスを含む空気
を通気しつつ、培養する培養法において、モノダス藻類
を無機液体培地にPCV濃度0.9〜1.7ml/l接
種し、水槽の前後面の何れかの片面から照度6000〜
12500lxの光を照射して、PCV濃度1.8〜
4.9ml/lになるまで培養する第1工程と、当該P
CV濃度1.8〜4.9ml/lの培養液に、水槽の前
後の両面から照度11000〜21000lxの光を照
射して、PCV濃度5.0〜5.5ml/lになるまで
培養する第2工程と、当該PCV濃度5.0〜5.5m
l/lの培養液に、水槽の前後の両面から照度1000
0〜35000lxの光を照射して、PCV濃度10.
9〜11.0ml/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産を生産さ
せるモノダス藻類の培養法。1. A culture method in which freshwater unicellular algae are inoculated into an inorganic liquid medium in a vertical flat water tank and cultured while irradiating with light and aeration containing carbon dioxide, monodus algae are cultured in the inorganic liquid medium. PCV concentration of 0.9 to 1.7 ml / l was inoculated into the water, and illuminance of 6000 to 6000 from either one of the front and back surfaces of the water tank.
By irradiating 12,500 lx of light, the PCV concentration is 1.8-
The first step of culturing to 4.9 ml / l and the P
A culture solution having a CV concentration of 1.8 to 4.9 ml / l is irradiated with light having an illuminance of 11000 to 21000 lx from both front and rear surfaces of a water tank to culture until a PCV concentration of 5.0 to 5.5 ml / l. 2 steps and the PCV concentration 5.0 to 5.5 m
In 1 / l culture solution, illuminance 1000
A PCV concentration of 10.
And a third step of culturing to 9 to 11.0 ml / l, which is a culturing method of monodus algae for producing eicosapentaene.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP8091770A JPH09252764A (en) | 1996-03-22 | 1996-03-22 | Cultivation of monodus algae producing eicosapentaenoic acid |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP8091770A JPH09252764A (en) | 1996-03-22 | 1996-03-22 | Cultivation of monodus algae producing eicosapentaenoic acid |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH09252764A true JPH09252764A (en) | 1997-09-30 |
Family
ID=14035817
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP8091770A Pending JPH09252764A (en) | 1996-03-22 | 1996-03-22 | Cultivation of monodus algae producing eicosapentaenoic acid |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH09252764A (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012168662A1 (en) | 2011-06-08 | 2012-12-13 | Fermentalg | Method for the epa enrichment of microalgae of the monodus genus cultivated in mixotrophic mode |
WO2013136023A1 (en) | 2012-03-16 | 2013-09-19 | Fermentalg | Production of eicosapentaenoic acid and/or arachidonic acid in mixotrophic mode using euglena |
JP2017060478A (en) * | 2011-01-28 | 2017-03-30 | アルガサイツ リミテッドAlgaeCytes Limited | Process for production of microalgae, cyanobacteria and metabolites thereof |
-
1996
- 1996-03-22 JP JP8091770A patent/JPH09252764A/en active Pending
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2017060478A (en) * | 2011-01-28 | 2017-03-30 | アルガサイツ リミテッドAlgaeCytes Limited | Process for production of microalgae, cyanobacteria and metabolites thereof |
WO2012168662A1 (en) | 2011-06-08 | 2012-12-13 | Fermentalg | Method for the epa enrichment of microalgae of the monodus genus cultivated in mixotrophic mode |
WO2013136023A1 (en) | 2012-03-16 | 2013-09-19 | Fermentalg | Production of eicosapentaenoic acid and/or arachidonic acid in mixotrophic mode using euglena |
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