JPH05276963A - Production of docosahexaenoic acid - Google Patents

Abstract

PURPOSE:To produce inexpensive docosahexaenoic acid free from fish smell, having noteworthy physiological actions such as lowering action on cholesterol, anticoagulant action, carcinostatic action, effect to improve memorizing and studying ability related to cerebral metabolism system, and use as a preventive for senile dementia, a therapeutic agent for Alzheimer's disease or as a fatty acid essential for growth of young fish. CONSTITUTION:Fine algae to be produced and supplied industrially and stably in fixed qualities, is used as a raw material and subjected to liquid shaking culture or liquid submerged aerated spinner culture to extremely raise a content of docosahexaenoic acid in the algae and productivity of algae. Docosahexaenoic acid is extracted from the prepared algae to stably and inexpensively produce docosahexaenoic acid. An alga belonging to marine algae, capable of producing used docosahexaenoic acid, may be used and Crypthecodinium cohnii may be cited as the alga.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a cholesterol lowering action, an anticoagulant action, an anticancer action, and further improvement of memory and learning ability in relation to the brain metabolic system, prevention of senile dementia, and treatment of Alzheimer's disease. The present invention relates to a method for producing docosahexaenoic acid, which is one of the highly unsaturated fatty acids whose physiological effects are drawing attention, such as the use of medicines and fry for growth essential fatty acids.

[0002]

BACKGROUND ART Docosahexaenoic acid is known to have many physiologically active functions as one of fish oil components,
In recent years, many research institutes have been actively promoting the elucidation of the physiologically active function of docosahexaenoic acid by discovering a raw material (tuna orbital fat) containing docosahexaenoic acid at a high concentration and developing a new advanced purification technology. .. Regarding the purification method and use of docosahexaenoic acid, a prophylactic and therapeutic agent for thrombosis (Japanese Patent Laid-Open No. 183716/1982), a method for separating and purifying highly unsaturated fatty acid or its ester (Japanese Laid-Open Patent Publication No.
1-37752), an anticancer composition containing a polyunsaturated fatty acid (JP-A-63-258816),
Method for separating and purifying highly unsaturated fatty acid alkyl ester (JP-A-63-8356), anti-cancer agent containing docosahexaenoyl monoglyceride as an active ingredient (JP-A 1-2)
No. 03322), a cerebral function improving agent containing docosahexaenoic acid and its ester as an active ingredient (JP-A-1-15).
3629), a brain function improving composition, a learning ability enhancer, a memory enhancing agent, a dementia preventive agent, a dementia treating agent or a functional food having a brain function improving effect (JP-A-2-49723). Has been done.

On the other hand, regarding the method for producing docosahexaenoic acid, it was difficult to separate and extract docosahexaenoic acid from fish oils such as tuna, sardines and skipjacks.
Practical application was delayed. As a method other than extraction and purification from fish oil, a method of producing highly unsaturated fatty acid by a conversion reaction using Mortierella spp.
3-185389), a process for producing highly unsaturated fatty acid-enriched fats and oils by microorganisms capable of producing arachidonic acid (JP-A-1-304892), and a process for producing highly unsaturated fatty acid-containing lipids from marine microorganisms (JP-A-2-142486). Japanese Patent Laid-Open No. 23878/1990, a method for producing highly unsaturated fatty acids using a genus Echinosporangium.
Gazette), Echinosporangium Transversalie ATCC 16960, 1803
A method for producing highly unsaturated fatty acids from the culture of 6 (European Publication No. 355972) is disclosed. In addition, bacteria (DeLong, EF & Vayanos, AA: A
ppl. Environ. Microbiol., 51 (4) , 730 (1986), Fungal Thraustochytrium (Haskias, R.
H. et al; Can. J. Microbiol. 10 , 187 (1964)), Entotophthora (Tyrrell; Can. J. Microbio
l., 13 , 755 (1967)), Japonochyt
rium) sp. ATCC 28207 (JP-A-1-199588), and algae include dinoflagellates and haptoalgae.
(Joseph. JD; Lipids, 10 , 395 (1975), Nichols,
PD et al; Phytochemistry, 23 , 1043 (1984))
There are no natural growth methods or static culture methods that simply increase docosahexaenoic acid in algal cells, and many technical methods are required to put these methods into practical use. Need to overcome challenges.

[0004]

The target substance of the present invention, docosahexaenoic acid, is a substance expected to have many physiological activities such as anti-cancer effect, but its supply is unstable because it depends on fish oil as a raw material. Quality is inconsistent. Furthermore, many steps and costs are required for refining to remove the odor peculiar to fish oil, but nonetheless it is difficult to use because the odor peculiar to fish oil cannot be sufficiently removed. However, there is a problem that the product is expensive because it requires a technical problem. In order to solve these problems, the present invention provides a method for stably and inexpensively producing docosahexaenoic acid using a raw material other than fish.

[0005]

Means for Solving the Problems As a result of diligent efforts to solve these problems, the present inventor uses microalgae as a raw material, which is industrially stable and can be produced and supplied with a constant quality,
By liquid shaking culture or liquid submerged aeration stirring culture, the content of docosahexaenoic acid in the algal cells is dramatically increased, and the algal cell productivity is also increased, and docosahexaenoic acid is extracted from the obtained algal cells by extracting docosahexaenoic acid. We have found a stable and inexpensive manufacturing method. More specifically, the present invention succeeded in producing a high concentration of algal cells by liquid shaking culture or liquid submerged culture of marine microalgae, in which the algal cells were conventionally grown only by the static culture method. Furthermore, it was found that the content of docosahexaenoic acid in alga can be dramatically increased by this method,
A process for obtaining docosahexaenoic acid by completing extraction or purification of docosahexaenoic acid from the alga body after recovering the alga body as it is or after drying is completed.

[0006]

The alga used in the present invention belongs to marine alga and may be any alga that produces docosahexaenoic acid.
For example, Crypthecodinium cornii. These are known algae, such as ATCC (American
It is available from storage organizations such as the Type Culture Collection), and (Crypthecodinium cohnii ATCC 30021,30543,3
0556,30571,30572,30775,50051,50053,50055,50056,500
58,50060) can be exemplified.

As a medium for culturing algae used in the present invention, a medium of any composition can be used as long as the medium can grow well. Suitable carbon sources, nitrogen sources, inorganic salts and the like can be contained as medium components. As such a carbon source, any carbon source that can be used by the alga of the present invention can be used. Examples of organic substances that can be used as the carbon source include organic compounds such as glycerin, carbohydrates such as glucose and fructose, and organic acids such as malonic acid and citric acid. Examples of the nitrogen source include commonly used inorganic nitrogen compounds such as ammonium sulfate and ammonium nitrate, and organic nitrogen sources such as bepton, yeast extract and casein hydrolyzate.

Natural seawater is the best inorganic salt, but various kinds of known artificial seawater artificially prepared can be widely used. In addition, various sodium salts, phosphates, magnesium salts, potassium salts, and borate salts can be used. , Carbonate, etc. can be used. Furthermore, trace amounts of heavy metal salts such as iron salts, manganese salts, cobalt salts, zinc salts, chlorine compounds and bromine compounds can be used.

As a culturing method, a static culturing method can be used, but the present inventor has diligently made efforts toward more efficient culturing, which has been conventionally performed only by static culturing. As a result, by shaking culture or liquid submerged aeration and agitation culture, the amount of alga bodies was improved by about 1.5 times in 7 days of culture as compared with ordinary static culture, and the content ratio of docosahexaenoic acid in alga bodies was improved. It is 1.3 to 6 times, and it is discovered that it increases dramatically.

Shake culture is also called suspension culture, and algae that produce docosahexaenoic acid are planted in a liquid medium and cultivated on a shaker with continuous shaking. The device used in this method includes a device that shakes the culture flask reciprocally to the left and right, a device that horizontally swivels, a device that keeps the elongated culture tube horizontal and vertically reciprocates both ends. The rotary tube culture method is also shaking culture.

The liquid submerged aeration and agitation culture is also called tank culture, and preferably the culture solution is put in a stainless steel closed tank and sterilized, and then algae for the purpose of culture are inoculated, and agitator is aerated while aerating sterile air. Rotate and culture. The ventilation volume is
Usually, it is 0.2 to 1.5 l / l / min.

The culture temperature can be 15 to 34 ° C., but 25 to 32 ° C. is optimal. The pH should be around neutral, and the number of days of culture should be determined depending on the remaining amount of carbon source and the amount of secreted substances into the medium, but it is usually about 2 to 12 days. In addition, a defoaming agent such as a fatty acid ester-based or silicon-based defoaming agent may be added in the jar fermenter aeration stirring culture. Examples thereof include glycerin monooleate, glycerin monostearate, glycerin monopalmitate, glycerin monolinoleate and sorbitan fatty acid ester.

After the completion of the culturing, a general centrifugation method is used for recovering the algal cells from the culture broth. Usually, the culturing temperature is preferably below, for example, by centrifugation at 10 ° C. and 12,000 × g for 10 minutes. To do. Docosahexaenoic acid can be extracted from the recovered alga body as raw alga or as a dried alga body by freeze-drying, heating, and ventilation drying.

Extraction and purification of docosahexaenoic acid from algal cells can be carried out by adding an antioxidant of fatty acid such as α-tocopherol or by an extraction method using an organic solvent such as methanol / chloroform in a nitrogen stream. How to extract lipids, Folch, Lee (L
ees) or Stanley et al. The crude product thus obtained can be purified by various methods such as column chromatography or recrystallization.

The docosahexaenoic acid produced by the method of the present invention has no odor peculiar to fish, is easy to purify, and is useful as a health food or a physiologically active substance.

[0016]

The present invention will be described in more detail with reference to the following examples. (Example 1) The medium used had a different composition.
Crypthecodinium cornii is added to 100 ml of the medium described below.
One (1) platinum loop of (Crypthecodinium cohnii) ATCC 30021 was inoculated and statically cultured at 28 ° C for 7 days. 1 of the obtained culture solution
0 ml was inoculated into a 300 ml flask charged with 100 ml of the same medium, and shake culture was carried out for 5 days at a temperature of 28 ° C, pH 6.8 and a rotation speed of 180 rpm with a rotary shaker. The algal cells were separated by centrifugation for 15 minutes. Then, it is washed with water and centrifuged in the same manner to separate the algal bodies, and the algal bodies are dried at 105 ° C. for 5 hours.
8 g / l of dried algal cells were obtained.

The extraction and purification of docosahexaenoic acid from dried algal cells is carried out by a conventional extraction method Blign-Dyer method using a warning blender containing 20 times the amount of algal cells in methanol / chlorohelm (1/2). Chemistry experiment course 4 Lipid II p9- (1991) Tokyo Kagaku Dojin Co., Ltd.) was carried out to obtain a crude product. This was subjected to methyl esterification (ibid., P54) to identify by analysis by gas chromatograph using authentiic docosahexaenoic acid as a standard substance. Moreover, 242 mg of docosahexaenoic acid having a purity of 99.2% by weight was obtained by silica gel thin layer chromatography according to a conventional method (ibid., P12, p30).

The analysis values of the fatty acid composition in the algal cells by gas chromatography were as follows. Fatty acids Static culture algal cells Shaking culture algal cells ─── ────── ────── C 12: 0 33.4% 5.7% C 14: 0 40.2% 27.5% C 16: 0 10.9% 22.7% C 18: 0 0.9% 1.6% C 18: 1 3.3% 8.0% C 22: 6 11.3% 34.5% (docohexaenoic acid) ─── ────── ────── 100.0% 100.0%

(Example 2) Using a 300 ml volume flask charged with 100 ml of the medium having the same composition as that used in Example 1, 10 ml of the statically cultivated culture solution of Kryptecogeni cornii ATCC30021 was inoculated, and 28
The culture was carried out by shaking on a rotary shaker at 180 ° C. and 180 rpm. 500 ml of the obtained culture solution was inoculated into a 10 L jar fermenter containing 7 L of the same medium,
Culture was performed for 5 days. Culture conditions are temperature 28 ℃, pH
7.0, stirring 300 rpm, aeration 3.5 l / min (0.
5 V.VM culture solution volume volume / aeration volume volume / min). 0.75 g of sorbitan fatty acid ester was used as an antifoaming agent. After completion of the culture, the algal cells were collected by centrifugation at 12,000 × g for 15 minutes. The cell yield was 43.4 g after drying at 105 ° C. for 5 hours. From the obtained dried cells, Example 1
Extraction and treatment were carried out in the same manner as above to obtain 5.5 g of docosahexaenoic acid.

(Example 3) Culturing was carried out in the same manner as in Example 2, and in 10 L jar fermenter culture, glucose as a carbon source and a nitrogen source as a carbon source were calculated for 60 hours, 80 hours and 100 hours as the culture progressed. Yeast extract and minerals were aseptically added, and a cumulative total of 10% carbon source and the same ratio of yeast extract and minerals were added. Also,
The temperature was kept constant at 28 ° C, but the stirring was gradually increased from 300 rpm to 350 rpm according to the addition of the medium, and the aeration rate was also increased from 0.3 V.VM to 1.0 V.VM. ,
2.25 g of defoamer was used. After 120 hours 184.
1 g of dried algal cells was obtained. Example 1 from the obtained dried algal cells
Extraction and treatment were carried out in the same manner as above to obtain 23.8 g of docosahexaenoic acid.

(Example 4) Medium components Glucose as a carbon source, yeast extract as a nitrogen source / vitamin source, and commercially available artificial seawater aquamarine as a mineral source were used. 182.0 g of a body and 21.7 g of docosahexaenoic acid were obtained.

(Example 5) Medium components Dried algal cells were cultured in the same manner as in Example 3 except that glucose was used as a carbon source, yeast extract was used as a nitrogen source / vitamin source, mineral source and seawater was used as distilled water. 180.6 g and docosahexaenoic acid 21.0 g were obtained.

Example 6 The used microalgae was ATCC3
Crypthec other than 0021
odinium cohnii (C. cohnii) and other conditions were the same as in Example 1 to obtain docosahexaenoic acid. Regarding the obtained results, the amount of algal cells and docosahexaenoic acid (DHA)
The amounts are summarized in Table 1.

[0024]

Comparative Example 1 Static culture was carried out under the same conditions as in Example 1 except that the shaking culture of the culture method was static culture. The results are shown in Table 2.

[0026]

Composition of medium NaCl 23.48 g FeCl ₃ · 6H ₂ O 0.01 g MgCl ₂ · 6H ₂ O 10.63 g Na ₂ glycerophosphate 0.15 g Na ₂ SO ₄ 3.92 g (NH ₄ ) ₂ SO ₄ 0.05 g CaCl ₂ (anhydrous 1.11 g Tris buffer 3.0 g KCl 0.66 g Vitamin mixture 1.0 ml NaHCO ₃ 0.19 g K ₂ HPO ₄ 0.01 g KBr 0.1 g Glucose 20.0 g H ₃ BO ₃ 0.03 g Glutamic acid 1.5 g SrCl ₃・ 6H ₂ O 0.04 g Casein hydrolyzate 20.0 g trace mineral mixture 3.0 ml distilled water 1.0 L trace mineral mixture vitamin mixture a ₂ EDTA 1.0 g biotin _{0.003g FeCl 3 · 6H 2 O 0.05} g thiamine 1.0 g H ₃ BO ₃ 1.0 g distilled water 1.0 L MnCl ₂・ 4H ₂ O 0.15 g ZnCl ₂ 0.01 g CoCl ₂・ 6H ₂ O 0.005 g Distilled water 100.0 ml Aquamarine composition (Yasusu Co., Ltd.) MgSO ₄・ 6H ₂ O 11.11 g KBr 0.10 g CaCl ₃・ 2H ₂ O 1.54 g H ₃ BO ₃ 0.03 g SrCl ₃・ 6H ₂ O 0.04 g NaF 0.003 g KCl 0.69 g NaCl 24.53 g NaHCO ₃ 0.20 g Na ₂ SO ₄ 4.09 g Distilled water 1.0 L pH 7.0 (adjusted with 1-N HCl) Seawater Seawater off Kawasaki Town, Chiba City Collected and used.

[0028]

Industrial Applicability As described above, according to the method of the present invention, since natural products such as tuna and sardine oil are conventionally extracted from fish oil of which production amount and quality are unstable depending on the season and region,
Docosahexaenoic acid, which was produced, can be industrially stably produced at a high concentration, and docosahexaenoic acid can be extracted from the algal cells produced by the liquid shaking culture method and liquid deep aeration stirring culture of marine microalga, resulting in inexpensive fish. Odorless docosahexaenoic acid can be stably supplied.

[Procedure amendment]

[Submission date] April 1, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0006

[Correction method] Change

[Correction content]

[0006]

The alga used in the present invention belongs to marine alga and may be any alga that produces docosahexaenoic acid.
For example, there is such Kuriputeko di chloride-Koni over.
These are known algae, such as ATCC (America
n Type Culture Collection), Crypthecodinium cohnii ATCC 30021,30543,
30556,30571,30572,30775,50051,50053,50055,50056,50
058,50060 can be exemplified.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0007

[Correction method] Change

[Correction content]

As a medium for culturing algae used in the present invention, a medium of any composition can be used as long as the medium can grow well. Suitable carbon sources, nitrogen sources, inorganic salts and the like can be contained as medium components. As such a carbon source, any carbon source that can be used by the alga of the present invention can be used. The organic materials can be used as such a carbon source, an organic compound such as glycerin, glucose, carbohydrates such as fructose, there are organic acids such as acetic acid, using any substrate that can be cultured.
Further, ammonium sulfate, which is usually used as a nitrogen source,
Inorganic nitrogen compounds such as ammonium nitrate, and bepton, yeast extract, casein hydrolyzate , glutamic acid,
An organic nitrogen source such as corn steep liquor can be exemplified.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0011

[Correction method] Change

[Correction content]

[0011] Liquid deep aeration spinner culture, also known as tank culture, preferably culture medium placed in a sealed vessel stainless, after sterilization, inoculated with algae culture purposes, a stirrer while passing sterile air Rotate and culture. The ventilation volume is
Usually, it is 0.2 to 1.5 l / l / min.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0016

[Correction method] Change

[Correction content]

[0016]

The present invention will be described in more detail with reference to the following examples. (Example 1) The medium used had a different composition.
Kuriputeko di chloride-Koni below in the medium 100ml
One (1) platinum loop of (Crypthecodinium cohnii) ATCC 30021 was inoculated and statically cultured at 28 ° C. for 7 days. 300 ml prepared by adding 10 ml of the obtained culture solution to 100 ml of the same medium
Inoculate the flask for 5 days, temperature 28 ° C, pH 6.8,
Shaking culture was performed with a rotary shaker at a rotation speed of 180 rpm, and after the culture was completed, the alga was separated by centrifugation at 5 ° C. and 12,000 × g for 15 minutes. Then, it was washed with water and centrifuged in the same manner to separate algal cells, and the algal cells were dried at 105 ° C. for 5 hours to obtain 1.8 g / l of dried algal cells.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0017

[Correction method] Change

[Correction content]

Extraction and purification of docosahexaenoic acid from 1.8 g of dried algal cells is carried out by a conventional method using a warning blender containing 20 times the amount of algal cells in methanol / chlorohelm (1/2). Law (new chemistry experiment course
4 lipid II p9- (1991) Tokyo Kagaku Dojin)
Was carried out according to the procedure described above to obtain a crude product. This was subjected to methyl esterification (ibid., P54) to identify by analysis by gas chromatograph using authentiic docosahexaenoic acid as a standard substance. Moreover, 242 mg of docosahexaenoic acid having a purity of 99.2% by weight was obtained by silica gel thin layer chromatography according to a conventional method (ibid., P12, p30).

[Procedure Amendment 6]

[Document name to be amended] Statement

[Correction target item name] 0018

[Correction method] Change

[Correction content]

The analysis values of the fatty acid composition in the algal cells by gas chromatography were as follows. Fatty acids Static culture algal cells Shaking culture algal cells ─── ────── ────── C 12: 0 33.4% 5.7% C 14: 0 40.2% 27.5% C 16: 0 10.9% 22.7% C 18: 0 0.9% 1.6% C 18: 1 3.3% 8.0% C 22: 6 11.3% 34.5% ( DoCoMo service hexaenoic acid) ─── ────── ────── 100.0% 100.0%

[Procedure Amendment 7]

[Document name to be amended] Statement

[Name of item to be corrected] 0019

[Correction method] Change

[Correction content]

[0019] (Example 2) using a 300ml flask charged with medium 100ml of the same composition as that used in Example 1, static culture was Kuriputeko di Niu culture-time code <br/> two over ATCC30021 Inoculate 10 ml of liquid,
The culture was carried out at 28 ° C. and 180 rpm with shaking on a rotary shaker. 500 ml of the obtained culture solution was inoculated into a 10 L jar fermenter charged with 7 L of the same medium, and cultured for 5 days. The culture conditions are a temperature of 28 ° C., p
H7.0, stirring 300 rpm, aeration 3.5 l / min
(0.5 V.VM culture solution volume volume / aeration volume volume / minute). 0.75 sorbitan fatty acid ester as an antifoaming agent
g used. After completion of the culture, the algal cells were collected by centrifugation at 12,000 × g for 15 minutes. The cell yield was 43.4 g after drying at 105 ° C. for 5 hours. The dried cells were extracted and treated in the same manner as in Example 1 to obtain 5.5 g of docosahexaenoic acid.

[Procedure Amendment 8]

[Document name to be amended] Statement

[Name of item to be corrected] 0023

[Correction method] Change

[Correction content]

Example 6 The used microalgae was ATCC3
0021 other than the crypto Te co-di chloride Koh knee Crypth
Docosahexaenoic acid was obtained by culturing under the same conditions as in Example 1 except that ecodinium cohnii (C. cohnii) was used. Regarding the obtained results, the amount of algal cells and docosahexaenoic acid (DH
A) The amounts are summarized in Table 1.

[Procedure Amendment 9]

[Document name to be amended] Statement

[Name of item to be corrected] 0027

[Correction method] Change

[Correction content]

Composition of medium NaCl 23.48 g FeCl ₃ · 6H ₂ O 0.01 g MgCl ₂ · 6H ₂ O 10.63 g Na ₂ glycerophosphate 0.15 g Na ₂ SO ₄ 3.92 g (NH ₄ ) ₂ SO ₄ 0.05 g CaCl ₂ (anhydrous 1.11 g Tris buffer 3.0 g KCl 0.66 g Vitamin mixture 1.0 ml NaHCO ₃ 0.19 g K ₂ HPO ₄ 0.01 g KBr 0.1 g Glucose 20.0 g H ₃ BO ₃ 0.03 g Glutamic acid 1.5 g SrCl ₃・ 6H ₂ O 0.04 g Casein hydrolyzate 20.0 g trace mineral mixture 3.0 ml distilled water 1.0 L trace mineral mixture vitamin cocktail Na ₂ EDTA 1.0 g biotin _{0.003g FeCl 3 · 6H 2 O 0.05} g thiamine 1.0 g H ₃ BO ₃ 1.0 g distilled water 1.0 L MnCl ₂・ 4H ₂ O 0.15 g ZnCl ₂ 0.01 g CoCl ₂・ 6H ₂ O 0.005 g Distilled water 100.0 ml Aquamarine composition (Yasusu Co., Ltd.) MgSO ₄・ 6H ₂ O 11.11 g KBr 0.10 g CaCl ₃・ 2H ₂ O 1.54 g H ₃ BO ₃ 0.03 g SrCl ₃・ 6H ₂ O 0.04 g NaF 0.003 g KCl 0.69 g NaCl 24.53 g NaHCO ₃ 0.20 g Na ₂ SO ₄ 4.09 g Distilled water 1.0 L pH 7.0 (adjusted with 1-N HCl) Seawater Seawater off Kawasaki Town, Chiba City Collected and used.

[Procedure Amendment 10]

[Document name to be amended] Statement

[Correction target item name] 0028

[Correction method] Change

[Correction content]

[0028]

Industrial Applicability As described above, according to the method of the present invention, since natural products such as tuna and sardine oil are conventionally extracted from fish oil of which production amount and quality are unstable depending on the season and region,
Docosahexaenoic acid that had been produced can be industrially stably produced at a high concentration, and docosahexaenoic acid can be stably extracted by extracting it from the alga cells produced by the liquid shaking culture method or liquid deep aeration stirring culture of marine microalgae. It is possible to stably supply docosahexaenoic acid that is inexpensive and has no fishy odor.

Claims (2)

[Claims] 1. A method for producing docosahexaenoic acid, which comprises obtaining docosahexaenoic acid from an alga obtained by liquid shaking culture of a marine microalgae that produces docosahexaenoic acid. 2. A method for producing docosahexaenoic acid, which comprises obtaining docosahexaenoic acid from an alga obtained by culturing a marine microalgae that produces docosahexaenoic acid in a deep liquid (tank) with aeration.