JPS6314697A - Production of eicosapentaenoic acid - Google Patents

Abstract

PURPOSE:To obtain eicosapentaenoic acid (EPA) useful as thrombosis, etc., in expensively and in high yield, by cultivating a specific microorganisms in a medium. CONSTITUTION:Mortierella elongata SAM 0219 strain capable of producing EPA is inoculated into a medium containing 0.1-30wt% carbon source (e.g. molasses), 0.01-5wt% nitrogen source (e.g. urea), hydrocarbon, fatty acid (salt), fats and oils, etc. Then, a mold is multiplied at optimum growth temperature of 20-30 deg.C, subjected to shaking culture at 10-20 deg.C at pH 4-10 for 2-10 days and lipid containing EPA in the mold is formed and accumulated. Then the mold is separated from the medium, collected and extracted with methanol, etc., to give a lipid compound of EPA. Then the compound is esterified with methanol, prepared EPA methyl ester is hydrolyzed with an alkali, extracted with an ether, etc., and purified.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing Eif-lipencitric acid by a fermentation method.

[Prior art]

Traditionally, eicosapentaenoic acid (hereinafter referred to as EPA) has been produced by extraction from fish oil or certain types of algae,
A method of purification is being used. However, in either method, the EPA content in the fish oil is low, and incomplete purification and concentration leave a fishy odor, and the cultivation of algae requires light irradiation to achieve a certain yield. There are many remaining issues.

Furthermore, there is no known method for producing EPA using microorganisms of the genus Mortierella.

[Problems to be Solved by the Invention] The present invention uses Mortierella microorganisms that have not been known to have the ability to produce EPA.
The purpose of the present invention is to provide a method for producing EPA with high yield and simple steps using an inexpensive commonly used medium.

[Means for solving problems]

The above object is to culture a microorganism belonging to the genus Mortierella and capable of producing EPA, to produce a lipid containing F, PA or EF)'A, and to collect EPA. , P by the manufacturing method.

[Specific explanation]

In the present invention, number: 1, belongs to the genus Mortierella, EP
Any microorganism that has A-producing ability can be used. Examples of such microorganisms include, for example, Mortierella coni r:I.
1 standard ■ button) IFO8570, Mortierella exigua (Mortierella qxjI(u, a)-
IPO857], Mortierella hygrophiler (,
Modan-Lprella Power■pAtsutsujiha>rp.

5941 etc. can be mentioned. Any of these strains can be obtained without any restrictions from the Fermentation Research Institute.

In addition, the bacterial strain Mortierella elongata SAM 0219 (m.
No. 03) can also be used.

Next, the above-mentioned bacterial strain SAM 0219 (Feikoken Bacteria No. 8
703) is described.

Growth status in each medium Culture conditions: 25°C, darkness 1, malt extract agar medium Colony growth is good, colonies on the 2nd day of culture have a diameter of 2
8-31 mm, colonies on the 5th day of culture have a diameter of 65-7 mm.
2 mm, colony exhibits a ruptured shape, aerial mycelium is poorly developed, sporangium spores are well formed, sporangium stalks are produced from aerial mycelia, and has a garlic-like odor.

2. Growth of colonies on potato/glucose agar medium is good. Colonies on the second day of culture have a diameter of 2.
7-31 mm, colonies on the 5th day of culture have a diameter of 75-8 mm.
0 mm, the colony has a para-like eave shape, aerial mycelia are significantly developed in the center of the colony, the back side of the colony is yellowish-white or yellow, the formation of sporangia spores is poor, there is an odor similar to garlic, and the odor is slightly strong. .

3. The growth of colonies on Czapek agar medium is relatively good, the diameter of the colonies on the second day of culture is 22-24 mm, the diameter of the colonies on the fifth day of culture is 50-53 mm, the development of aerial mycelia is poor, and the aerial mycelia are dense. Sometimes they get tangled up in each other.

The formation of sporangium spores is very good, and the sporangium has an odor similar to garlic produced by aerial mycelia.

4. LCA agar medium (medium preparation method is provided by Hiroshi Miura,
(According to Mitsuyo Kudo, "Agar Medium for Aquatic Deuteromycetes," Journal of the Japanese Painting Society Vol. II, pp. 116-118, 1970) Colony growth was good, and the diameter of the colony on the second day of culture was 27- 241 mm, the 50th cultured colony has a diameter of 64 mm.
-(i6mm, colony exhibits a ruptured shape, development of aerial mycelium is poor except in the center of the colony, formation of sporangia spores is good. Sporangium stalks arise from aerial mycelium. There is an odor similar to garlic.

Microscope observation Sporangial stalks, branching methods of sporangial stalks, sporangia, sporangial spores, etc. were observed using microscopic specimens of each culture medium and direct microscopic examination of colonies.

The sporangial stalk is 87.5-320 μm long and 3 mm wide at the base.
-7.5μm, tapering towards the tip, 1.0-2.5μm
m. Sporangial stalks are often branched at the base. The spores are spherical, 15-30 μm in diameter, contain many sporangiospores, and leave a somewhat indistinct color after detachment. Sporangium spores oval, rarely kidney-shaped, smooth surface, 7.5-
12.5X 5-7.5 μm, chlamydospores are formed in relatively large numbers. May occur singly or in rare cases in combination. Sometimes several hyphae are exposed around it. oval or slender spherical, 12
．． 5-30X 7.5-15μm0 or diameter 12.5
-15μm0 zygospores are not observed.

3. Physiological properties Optimal growth conditions pH: 6-9 Temperature: 20-30°C Growth range pH: 4-10 Temperature: 5-40°C According to the above mycological properties, the strain of the present invention (SAM-02
19) Search for taxonomic position in 1.1. Eight, von A
rx.

“The Genera of Fungi 5por
ulaLir+gin I”ureCulture,”
3rd ed, J. Cramer, 1ml, and H.

Domsch, W., Gam5. &T, 11. 8ndcrson%'Compt+ndium ofS
oil Fungi,”＾cademic Prass
According to 19FIO, the sporangium forms a spherical spore r-sac at the tip of the sporangium stalk, it does not have a columnar axis, the sporangium spore has no accessory threads, and the cultured hyphae grow on garlic [4 or more]. This strain is known as L9Lij because it emits a strong odor.
-It is thought to be a fungus belonging to the genus Crellia.

Therefore, JGams, “A Key to the 5
pecies of Mortierella,” Pe
rsoonia 9, 381-391, 1977
Comparing the mycological properties with known species of the genus Mortierella according to At 5-320 p.m., branching occurs only at the bottom, does not branch into clusters of grapes, and contains a large number of sporangiospores inside the spore clusters, so Mortierel la genus Mortierel Ia.
Subgenus (Sugen, Mortiere)
Sec.

It is considered to be included in "IW 王奸hυ" □).

There are 22 types in the h-rev. 1 category section. Comparing the various mycological properties of this strain with these 22 species, this strain is considered to be similar to the three species: Hatoumundai and Hong α 薊憑, (One Ritan Ha, and L Kennai Uta). So, K, Il, Doms
ch, W, Gam5+ & T, -H, eighterso
n.

“Compendium of 5oll Fungi
,” Academic Press.

1980, W, Gam5, “Some new o
r noteworthy 5pec, 1esof M
ortierella,” Persoonia9,
111-140.1977), and G. Linnem.
ann+”Mortierella Coemans
1863. H, Zycha & R, Siepmann
, ”Mucorales Eine Besch-r
eibung A11er Gattungen
and Arten Dieser Pilz
-gruppe,”pp, 155-140. J, Cr
Amer, 1969, the present strain was compared with these three species in terms of mycological properties. This strain is Fj, Hc h
a and e clearly differ in the length of the sporangium stalk, the width of the base, and the size of the sporangium.

It is clearly different from the L1 Ushichikken in the form and size of the sporangiospores. -M, e-I-onJli-a,,t4+
Ia refers to the fact that the sporangium stalk is rather short, the chlamydospores are oval or spherical in shape and are sometimes linked together, and the chlamydospores sometimes produce several hyphae around them. However, the present inventors believe that these differences make this strain
It was judged that this was not sufficient to classify it as a different species from Ortierella elog-ata. Therefore, the present inventors identified this strain as Mortierella el p-ta.
It was identified as SAM 0219. SAM 0219 strain was released on March 19, 1986 by the Agency of Industrial Science and Technology of the Ministry of International Trade and Industry.
Accession number FERM P-87 to Industrial Research Institute (FRI)
It has been deposited as 03'ζ.

In order to culture the bacterial strain used in the present invention, spores, hyphae, or a preculture solution obtained by culturing the strain in advance are inoculated into a liquid medium or a solid medium and cultured. In the case of a liquid medium, the carbon sources include glucose, fructose, +sylose, strawberry tucarose, maltose, soluble starch,
Any commonly used substances can be used, including but not limited to, tJ, honey, glycerol, and mannitol. Peptone is a nitrogen source,
In addition to natural nitrogen sources such as yeast extract, malt extract, meat extract, casamino acids, and corn stable liquor, organic nitrogen sources such as urea, as well as sodium nitrate, ammonium nitrate,
Inorganic nitrogen sources such as ammonium sulfate can be used. In addition, inorganic salts such as phosphates, magnesium sulfate, iron sulfate, copper sulfate, and vitamins can also be used as trace nutrient sources, if necessary. There are no particular limitations on the concentration of these medium components as long as they do not impair the growth of microorganisms. In practice, the carbon source generally ranges from 0.1 to 30% by weight, preferably from 1 to 10% by weight,
The concentration of the nitrogen source is 0.01 to 5% by weight, preferably 0.1 to 2% by weight, and hydrocarbons, fatty acids, fatty acid salts, or fats and oils may also be added. In addition, the culture temperature is 5-40℃
, preferably at 10 to 20°C from the start of culture, or at 10 to 20°C after culturing at 20 to 30°C to proliferate the bacterial cells.
Cultivation is continued at 20°C to produce EPA. Such temperature control makes it possible to increase the ratio of EPA in the produced fatty acids. The pH of the medium is set to 4 to 10, preferably 6 to 9 (10), and aerated agitation culture, shaking culture, or static culture is performed.

Cultivation is usually carried out for 2 to 10 days.

When culturing on a solid medium, 50~
Using bran, rice husks, rice bran, etc. to which 100% by weight of water has been added, culture is carried out at 5 to 40°C, preferably at the temperature mentioned above, for 3 to 14 days. In this case, a nitrogen source, inorganic salts, trace nutrients, and additives such as hydrocarbons, fatty acids, fatty acid salts, or fats and oils can be added to the medium as necessary.

After culturing in this manner, EPA-containing lipids are produced and accumulated within the bacterial cells. When a liquid medium is used, EPA is collected from the cultured bacterial cells as follows.

After completion of the culture, cultured bacterial cells are obtained by using the culture solution, centrifuging for 1 minute, and using conventional solid-liquid separation means such as filtration. The bacterial cells are thoroughly washed with water and preferably dried. Drying can be performed by freeze drying, air drying, etc. The dried bacterial cells are preferably extracted with an organic solvent under a nitrogen stream. Ether, hexane, methanol, ethanol, chloroform, dichloromethane, petroleum ether, etc. can be used as the organic solvent, and extraction can be carried out by alternate extraction with methanol and petroleum ether, or extraction using a single layer system of chloroform-methanol-water. good results can also be obtained. By distilling off the organic solvent from the extract under reduced pressure, a lipid containing a high concentration of EPA can be obtained.

Furthermore, instead of the above method, extraction can be performed using wet bacterial cells. In this case, a water-compatible solvent such as methanol or ethanol, or a water-compatible mixed solvent consisting of these and water and/or another solvent is used. Other steps are the same as above.

The lipid obtained as described above contains EPA as a component of a lipid compound, for example, fat. Although they can be separated directly, they are preferably separated as esters with lower alcohols, such as methyl eicosapentaenoate. By forming such an ester, it can be easily separated from other lipid components,
In addition, it can be easily separated from other fatty acids produced during culture, such as valmitic acid, oleic acid, linoleic acid, etc. (these are also esterified during esterification of EPA). For example, to obtain methyl ester of EPA, the above-mentioned extracted lipid is mixed with anhydrous methanol-hydrochloric acid 5% to
10%, BF3-methanol 10% to 50%, etc.
Preferably, the treatment is carried out at room temperature for 1 to 24 hours.

In order to recover eicosapentaenoic acid methyl ester from the above-mentioned treated solution, it is preferable to extract it with an aqueous solvent such as hexane, ether, or ethyl acetate.

Next, this extract is dried with anhydrous sodium acetate, etc., and the organic solvent is distilled off, preferably under reduced pressure, to obtain a mixture mainly consisting of fatty acid esters. In addition to eicosapentaenoic acid methyl ester, it contains palmitic acid methyl ester, stearic acid methyl ester, oleic acid methyl ester, etc. To isolate eicosapentaenoic acid methyl ester from a mixture of these fatty acid methyl esters, Column chromatography, low temperature crystallization method, urea inclusion method, etc. can be used alone or in combination.

From the thus isolated methyl eicosapentaenoate, E
PA can be obtained by hydrolyzing with an alkali and then extracting with an organic solvent such as ether or ethyl acetate.

In addition, in order to collect EPA without passing through its methyl ester, the extracted lipids are subjected to alkaline decomposition (for example, with 5% sodium hydroxide at room temperature for 2 to 3 hours), and then fatty acids are extracted from this decomposition solution. -Can be extracted and purified using methods commonly used for purification.

Next, the present invention will be explained in more detail with reference to Examples.

Net Equilibrium Kaito Glucose 5%, Peptone 0.5%, Yeast Extract 0.3
% and malt extract 0.3% (pH 6,0) 5
0m7! 500mj! Put it in Yosakaro flask, 120
Sterilized at ℃ for 20 minutes. Mortierella elongata S
AM 0219 (FBI Group 1' 1t7(13) l
Inoculate the platinum loop, and use a reciprocating shaker (IIOrp
M) was cultured with shaking at 12°C for 7 days. After culturing, the bacterial cells were collected by filtration, thoroughly washed with water, and then freeze-dried. As a result, 0.7 g of dried bacterial cells were extracted from 20 microbial cells per person using the Bligh & Dyer extraction method using a monolayer solvent of chloroform-methanol and water.
When the fat was extracted, 150 μ of fat rt was obtained. This lipid was methyl esterified by treatment with anhydrous methanol-hydrochloric acid (!15:5) at 20°C for 3 hours and extracted with ether to obtain 95 μm of fatty acid methyl. The k11 composition of methyl fatty acid was determined by gas chromatography as 4J1, and methyl palmitate 12
%, methyl stearate 12%, methyl oleate 24%
%, methyl linoleate 5%, γ-methyl linoleate 8%,
Methyl bishomo-gamma-lyrinnate 5%, methyl arachidonic acid 10%, methyl eicosapentaenoate 13%, others 1
It was found that the percentage was 1%. The fatty acid methyl mixture was separated by column chromatography, a methyl eicosapentaenoate fraction was collected, and the solvent was distilled off using a rotary evaporator.As a result, 6.5 μm of purified methyl eicosapentaenoate was obtained. When this specimen and a commercially available methyl eicosapentaenoate standard sample were compared by gas chromatography analysis, high performance liquid chromatography analysis, mass spectrometry analysis, and NMR analysis, they were in agreement in all analyses.Before purification And the amount of methyl eicosapentaenoate after purification is 0.25 m1r/mβ and 0.1 per medium, respectively.
3■/mI! , 18 mg/g and 9 μ/g per dry bacterial cell, respectively.

Example I A medium 51 having the same composition as in Example 1 was placed in a 15Il jar fermenter, and after sterilization at 120°C for 40 minutes, Mortierella elongata SAM O219 (FERMP-
8703') was inoculated with 200 m 12 of preculture.

Aerated agitation culture was carried out at 18°C for 5 days at an aeration rate of 0.5 ν, v, m, and the obtained wet bacterial cells were 150 g (dry weight 50 g).
) was subjected to extraction, hydrolysis, and methyl esterification in the same manner as in Example 1, yielding 13 g of total lipids and 8 g of methyl mixed fatty acids. The composition of this product is 13% methyl palmitate, 14% methyl stearate, 28% methyl oleate, 8% methyl linoleate, 8% methyl T-linoleate, 3% bishomo-T-methyl linoleate,
It was found that 13% of methyl arachidonate, 6% of methyl eicosapentaenoate, and 7% of others. The production amount of methyl eicosapentaenoate is 0.10g/per culture medium.
i, 9.6 μ/g per dry bacterial cell.

In addition, after the completion of the culture, the culture filtrate 4,2 obtained by filtration
After drying 70 mff, extraction, hydrolysis, and methyl esterification were performed in the same manner as in Example 1. As a result, 82 μl of mixed fatty acid methyl containing 7% methyl eicosapentaenoate was obtained.
It was.

Mort-jerel
Ia■ Hakawa, IFO8571), and Mortierella hygrophila (Mμ) country r-e-1jq hy-B
-rol, 14jjq, IFO5941) was carried out in the same manner as in Example 1, and 47 and 72 cubic centimeters of fatty acid methyl were obtained, respectively.

When methyl eicosapentaenoate contained in these methyl fatty acids was isolated and purified, it was found that each methyl eicosapentaenoate contained 4.8 w
x, and 7.4■ were obtained.

Jiha [4- 2% glucose, 1% yeast extract, Tween 200
．． A medium (pH 6.0) containing 2% and 0.5% of various hydrocarbons, sodium fatty acids, or fats and oils (pH 6.0) was placed in a 100 ml Mayer and sterilized at 120° C. for 20 minutes. Mortierella elongata SAM 0219 (
FERM P-8703) 1 platinum loop was inoculated and heated at 28℃ using a rotary shaker (20Orpm).
．． It was cultured for a period of time.

The obtained bacterial cells were extracted, hydrolyzed, and methyl esterified in the same manner as in Example 1. For each of the various hydrocarbons, fatty acid sodium, and fats and oils added to the medium, the obtained dry bacterial weight, total lipid amount, total fatty acid methyl amount, eicosapentaenoic acid methyl content, and eicosapentaenoic acid methyl production per medium The amounts were as shown in the table below.

When linseed oil or sodium linoleate is added to standard medium, the amount of eicosapentaenoic acid produced is 1 ml of medium.
(1.44mR, 10.32cm), which was an extremely high concentration.Also, when hydrocarbons, fatty acids, fatty acid salts, or fats and oils were added, a significant amount of eicosapentaenoic acid was produced, but when no additives were added. Almost no formation was observed.

20 mA' of a medium having the same composition as in the practical construction example was placed in a 100 m/volume Mayer and sterilized at 120°C for 20 minutes. Mortierella elongata SAM 0219 (FERM P-8
703) Inoculate a loopful of platinum, and use a rotary shaker (2
After culturing at 28° C. for 5 days at 0 Orpm), the culture temperature was changed to 12° C., and the cells were similarly cultured for 3 days. The obtained bacterial cells were subjected to extraction, hydrolysis and methyl esterification in the same manner as in Example 1, to obtain 75 ml of mixed fatty acid methyl containing 9% of methyl eicosapentaenoate.

Notification of change in accession number January - 8, 1985 Director General Kuro 1) Mr. Akihiro 1. Indication of the incident 1986 Patent Application No. 158651 2. Name of the invention Process for producing eicosapentaenoic acid 3. Procedures were carried out. Relationship with the case Patent applicant name (190) Suntory Ltd. 4, agent address 2-8-10-5 Toranomon, Minato-ku, Tokyo 105
Name of the old depository institution: Institute of Industrial Science and Technology, Microbial Technology Research Institute 7; name of the new depositary institution: Agency of Industrial Science and Technology, Microbial Technology Research Institute 8; new deposit number: FERM BP-1239 (FERM BP-1239) 9 , 1 copy of the funds certifying the new deposit number of the catalog of attached documents 1 copy of the procedural amendment (voluntary) January 1985 Commissioner of the Japanese Patent Office Kuro 1) Akio Tono 1, Indication of the case Patent Application No. 158651 of 1988 2. Name of the invention Process for producing eicosapentaenoic acid 3. Relationship with the case of the person making the amendment Patent applicant name (190) Suntory Ltd. 4. Agent address 8-10 Toranomon-chome, Minato-ku, Tokyo 105 (
5. Subject of amendment (1) "Claims" column of the specification (2) "Detailed description of the invention" column of the specification (3) Copy of the certificate of acceptance 6. Contents of the amendment (1) Amend the claims in a separate circular.

(2) ■ Specification No. 4 Jlj line 1 and lines 2 to 3, r8703)” is followed by “(Feikoken Joyori No. 1
No. 239).

■ On page 9, line 12, “It has been deposited.”
9) was transferred to an international deposit. ”.

■ 15th member, line 1, page 16, line 16 to 1
Line 7, 18Ji [Line 122 and page 20, line 6 r (FIRM P-8703) J then r (FER
M BP-1239) Join J.

■ The 19th member, line 18 rlo, 32 da” is rO, 3
Correct to 2WvJ.

■ Add the following statement after the 13th line of the 20th Sada.

“Example 6 Medium containing 2 doses of glucose and 1 portion of yeast extract (PH6,
0) Put 2011/ into a 100-volume Mayer and heat to 120°C.
sterilized for 20 minutes. Mortierella Elongata SA
M 0219 (FIRM P-870 Pier (FERM B)
P-1239) 1 platinum loop was inoculated and cultured in a rotary shaker (200 rpm) at 28°C for 4 days.
After sterilization for 5 minutes, the cells were added and cultured in the same manner for 2 days. The obtained bacterial cells were extracted, hydrolyzed, and methyl esterified in the same manner as in Example 1. The amounts of methyl eicosapentaenoate produced in the dried bacterial cells and the culture medium for each of the various sodium fatty acids and fats and oils added to the culture medium were as shown in the table below.

As mentioned above, by adding fatty acids, oils, etc. during culture (4 days after culture), eicosapentaenoic acid, which was not observed in the control no-addition area, was reduced to 0.0% per 1 d of culture medium.
06Mfl ~0.25.9 produced. (3) Leave a copy of the trust deed to the orphan.

7. List of attached documents (1) Amended patent claims 1 copy (2) Copy of depository note 1 copy 2. 4IIF-Claims 1 Belongs to the genus Mortierella (Mort1ier@l1m) and contains eicosine citric acid 1. A method for producing eicosapentaenoic acid, which comprises culturing a microorganism capable of producing eicosapentaenoic acid to produce eicosapentaenoic acid or a lipid containing all of eicosapentaenoic acid, and then collecting eicosapentaenoic acid.

2. The method according to claim 1, characterized in that the culture is carried out at a temperature below the optimum growth temperature from the start of culture or after culturing at the optimum growth temperature.

3. The method according to claim 1, which comprises adding a hydrocarbon, a fatty acid, a fatty acid salt, or an oil or fat to the medium.

4. The method according to claim 1, characterized in that fatty acids, fatty acid salts, or fats and oils are added to the culture solution during culturing.

Procedural amendment (voluntary) June 1986/? Director of the Japan Patent Office Kuro 1) Mr. Yu Akira 1. Indication of the case Patent Application No. 158651 of 1988 2. Name of the invention Process for producing eicosapentaenoic acid and lipid containing the same (new name) 3. Person making the amendment Relationship to the case Patent applicant name (190) Suntory Ltd. 4, agent address 8-10-6, Toranomon-chome, Minato-ku, Tokyo 105
Subject of amendment ill “Title of the invention” column (2) of the description
The content of the amendment shall be made in column (3) of [Special and IT Claims] of the book, and in column 7 of "Detailed Description of the Invention" of the specification.

(2. Amend the claims as per the attached sheet.

(3) ■ Specification box 1t' [Line 19, 11 "eicosapentaenoic acid" is corrected to r:l sabentaenoic acid and lipid 1 containing it.

(2) Correct rEPAJ on page 2, line 17 to "rEPA and lipids containing it."

■ Page 3, line 5, ``1 [Production method] r Production method; and a lipid containing eicosapentaenoic acid by culturing a microorganism belonging to the genus Mortierella and having the ability to produce eicosapentaenoic acid.'' Norimaki Tsumadera The method for producing a lipid containing eicosapentaenoic acid is corrected to j.

8. List of attached documents Claims 1 copy 2. Claims ■. Eicosapentaenoic acid or eicosapentaenoic acid produced by culturing a microorganism that belongs to the genus HorLierella and has the ability to produce eicosapentaenoic acid. 1. A method for producing eicosapentaenoic acid, which comprises producing a lipid containing the following and collecting eicosapentaenoic acid.

2. The method according to claim 1, characterized in that the culture is carried out at a temperature below the optimum growth temperature from the start of culture or after culturing at the optimum growth temperature.

3. The method according to claim 1, which comprises adding a hydrocarbon, a fatty acid, a fatty acid salt, or an oil or fat to the medium.

4. The method according to claim 1, characterized in that fatty acids, fatty acid salts, or fats and oils are added to the culture solution during culturing.

5. Mortietra (MorLit!rel Ia)
A microorganism that belongs to the genus niocosapentaenoic acid and has the ability to produce niocosapentaenoic acid is cultivated to produce lipids containing eicosapentaenoic acid.
1-4 A method for producing a lipid containing eicosapentaenoic acid.

6. The method according to claim 5, characterized in that the culture is carried out at a temperature below the optimum growth temperature from the start of the culture or after culturing at the optimum growth temperature.

7. The method according to claim 5, characterized in that a hydrocarbon, a fatty acid, a fatty acid salt, or an oil or fat is added to the medium.

8. The method according to claim 5, characterized in that fatty acids, fatty acid salts, or fats and oils are added to the culture solution during culturing.

Claims (1)

[Scope of Claims] 1. A microorganism belonging to the genus Mortierella and capable of producing eicosapentaenoic acid is cultured to produce eicosapentaenoic acid or a lipid containing eicosapentaenoic acid; A method for producing eicosapentaenoic acid, which comprises collecting eicosapentaenoic acid. 2. The method according to claim 1, characterized in that the culture is carried out at a temperature below the optimum growth temperature from the start of culture or after culturing at the optimum growth temperature. 3. The method according to claim 1, which comprises adding a hydrocarbon, a fatty acid, a fatty acid salt, or an oil or fat to the medium.