JP2001245688A - Lipid containing 5,11,14-eicosatrienoic acid and/or 5,11,14,17- eicosatetraenoic acid and method for producing the lipid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for the microbial production of 5,11,14- eicosatrienoic acid and/or 5,11,14,17-eicosatetraenoic acid. SOLUTION: The objective method for the production of 5,11,14-eicosatrienoic acid and/or 5,11,14,17-eicosatetraenoic acid or a triglyceride or lipid containing the fatty acids comprises the culture of a microorganism capable of producing arachidonic acid in a medium added with a Δ6 unsaturation reaction inhibitor and the collection of the produced lipid from the cultured product. The method enables the hitherto impossible industrial production of these fatty acids by microbial means.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing 5,11,14-eicosatrienoic acid and / or arachidonic acid by culturing a microorganism capable of producing arachidonic acid in a medium supplemented with a Δ6 desaturation inhibitor. 5, 11, 14, 17-eicosatrienoic acid or 5, 11, 14, 17-eicosatrienoic acid and / or 5, 11, 14, 17-eicosatrienoic acid and / or 5, 11, 14, 17-eicosatrienoic acid. -A process for producing eicosatetraenoic acid or a lipid containing these as a component, and containing 5,11,14-eicosatrienoic acid and / or 5,11,14,17-eicosatetraenoic acid. The present invention relates to lipids or triglycerides, particularly lipids or triglycerides containing at least 12% by weight of 5,11,14-eicosatrienoic acid, and compositions containing these.

[0002]

2. Description of the Related Art 5,11,14-Eicosatrienoic acid and 5,11,14,17-eicosatetraenoic acid are polyunsaturated fatty acids in which the arrangement of double bonds is non-methylene mediated. Arachidonic acid or eicosapentaenoic acid (EP
It is thought to antagonize the action of A), and its physiological action is expected. At present, the elucidation of these physiological actions is progressing, but 5, 11, 14-eicosatrienoic acid and 5, 1
1,14,17-Eicosatetraenoic acid is thought to have different actions (Ikeda et al., L. et al.
ipids, 27, 500-504, 1992. Sugano Food and Development, 34, 4-
8, 1999).

[0003] 5,11,14-eicosatrienoic acid and 5,11,14,17-eicosatetraenoic acid are naturally present in nagi seeds, odoriferous (biota) seeds, yew seeds, clams and starfishes. However, they are poor and unstable, and expensive and impractical. In addition, the content of 5,11,14-eicosatrienoic acid and 5,11,14,17-eicosatetraenoic acid is low, and both are always contained and their composition cannot be controlled. There's a problem.

As a method for concentrating these fatty acids, 5,11,14-eicosatrienoic acid and 5,11,1 obtained by decomposing oils and fats of scented odor (biota) seeds are used.
A method is known in which free fatty acids containing 4,17-eicosatetraenoic acid are concentrated using a lipase derived from Candida (Jie MSFLK et al., J. Am. Oil Chem. Soc.,
72, 245-249 (1995)), as well as the above-mentioned problems, but also problems in that only free fatty acids can be obtained and in terms of cost. As a production method using a microorganism, a production method using a microorganism having an arachidonic acid-producing ability and having a reduced Δ6 desaturation activity is known (Japanese Unexamined Patent Application Publication No. Hei 5-
276964), since the degree of decrease in Δ6 desaturation activity is not so remarkable, the production amount and content are low,
It was not always practical.

[0005] Δ6 For compounds that inhibit desaturation reaction, p animals - iso Pentoxyresorufin aniline (p-isopento
xyaniline) has an inhibitory effect (Obukowicz MG et al., Bioc
hem. Pharmacol., 55, 1045-1058 (1998)) and SC-26
196 and nifedipine have an inhibitory effect in in vitro experiments using rat liver microsomal fractions (Obukowicz MG et al., J. Pharmacol. Exp. Ther., 287, 1
57-166 (1998); Kawashima et al., Biosci. Biotech. Biochem.,
60, 1672-1676 (1996)), all of these compounds
Only effects on the animal or on the microsomal fraction of the animal were known.

As for microorganisms, the addition of propyl gallate during the cultivation of Mortierella filamentous fungi can simultaneously inhibit the Δ5 desaturation reaction and the Δ6 desaturation reaction (Kawashima et al., Biochim. Biophy
s. Acta, 1299, 34-38 (1996)). However, in this case, no accumulation of 5,11,14-eicosatrienoic acid or 5,11,14,17-eicosatetraenoic acid was observed. In order for 5,11,14-eicosatrienoic acid to be formed, linoleic acid must have a longer chain length and ΔΔ
It is necessary to undergo two reactions of 5 desaturation. However, when propylgallate is added, 5,11,14-eicosatrienoic acid is produced because the Δ5 desaturation reaction is strongly inhibited. Probably not.

That is, when a microorganism is added to a culture medium during its culture, the Δ6 desaturation reaction is specifically inhibited, and as a result, 5, 11, 14-eicosatrienoic acid or 5, 11 No compound having an action of accumulating 14,17-eicosatetraenoic acid has been known. Thus, a method for producing 5,11,14-eicosatrienoic acid, 5,11,14,17-eicosatetraenoic acid, or a lipid containing these as a component with high production efficiency, and 5,11 There is a strong demand for the development of lipids or triglycerides having a high content of, 14-eicosatrienoic acid and compositions containing them.

[0008]

Therefore, the present invention relates to
11,14-eicosatrienoic acid and / or 5,11,
A method for producing 14,17-eicosatetraenoic acid or a lipid containing the same as a component, and 5,11,
An object of the present invention is to provide a lipid or triglyceride containing 14-eicosatrienoic acid as a constituent, and a composition containing these.

[0009]

Means for Solving the Problems The present inventors have conducted various studies in order to achieve the above-mentioned object.
It has been found that there are compounds that specifically inhibit only the desaturation reaction. Such compounds include 3 ′,
4 '-(methylenedioxy) acetophenone, 3,4-
Compounds having a methylenedioxyphenyl group, such as (methylenedioxy) aniline, 1,2- (methylenedioxy) -4-nitrobenzene, piperonyl alcohol, piperonylamine, piperonilonitrile, safrole, and paracoumarates; Para - anisidine
Is mentioned.

By culturing a microorganism having an arachidonic acid-producing ability in a medium to which these substances have been added, linoleic acid, which is a substrate of the Δ6 desaturation reaction, is accumulated, and the linoleic acid is grown by the chain lengthening reaction to 11,14. -Eicosadienoic acid, and the fatty acid is further converted to 5,11,14-eicosatrienoic acid by a Δ5 desaturation reaction,
The lipid containing this fatty acid is produced and accumulated in the cells,

At this time, α-linolenic acid or a derivative thereof and / or 11,14,17-eicosatrienoic acid or a derivative thereof, or an oil or fat containing these as a component is added to the medium to obtain α-linolenic acid. The acid is converted to 11,14,17-eicosatrienoic acid by a chain elongation reaction, and the fatty acid is further converted to 5,11,14,17-eicosatetraenoic acid by a Δ5 desaturation reaction and Lipids containing, 11,14-eicosatrienoic acid and 5,11,14,17-eicosatetraenoic acid are produced and accumulated in the cells, or

By culturing a microorganism having arachidonic acid-producing ability at a temperature lower than 20 ° C. in a medium to which a compound that specifically inhibits only the Δ6 desaturation reaction is added, 5, 11 , 14-Eicosatrienoic acid is further converted to 5,11,1 by ω3 desaturation reaction.
Converted to 4,17-eicosatetraenoic acid and converted to 5,1
1,14-eicosatrienoic acid and 5, 11, 14, 17
The present inventors have found that lipids containing eicosatetraenoic acid are produced and accumulated in cells, and have completed the present invention.

Further, the present inventors have maintained that the ratio of linoleic acid / γ-linolenic acid in the cells is maintained at 30 or more so that the conversion of linoleic acid to 11,14-eicosadienoic acid can be reduced from γ-linolenic acid. It has been found that the reaction proceeds sufficiently prior to the conversion to dihomo-γ-linolenic acid, and as a result, a lipid containing a remarkable amount of 5,11,14-eicosatrienoic acid is formed, thereby completing the present invention. .

That is, the present invention is characterized in that a microorganism capable of producing arachidonic acid is cultured in a medium to which a Δ6 desaturation inhibitor has been added, and lipids are collected from the culture. -A process for producing eicosatrienoic acid-containing lipids;
(6) containing 5,11,14-eicosatrienoic acid, which is characterized by culturing in a medium supplemented with a desaturation inhibitor, collecting lipid from the culture, and separating and purifying triglyceride from the lipid. A process for producing triglycerides; and

The microorganism having arachidonic acid-producing ability is identified as Δ
The cells are cultured in a medium supplemented with 6 desaturation inhibitors, lipids are collected from the culture, and 5, 11, 14-
A method for producing 5,11,14-eicosatrienoic acid, which comprises separating and purifying eicosatrienoic acid; and a method for producing a microorganism having an arachidonic acid-producing ability in the above-mentioned production method, by using saturated fatty acids, oleic acid, and linoleic acid. Acid, 1
Provided is a production method characterized by culturing in a medium to which any one of 1,14-eicosadienoic acid or a derivative thereof, or an oil or fat containing these as a component is further added.

The present invention also relates to a method for producing a microorganism having an arachidonic acid-producing ability by using α-linolenic acid or a derivative thereof and / or
Alternatively, 11,14,17-eicosatrienoic acid or a derivative thereof or an oil or fat containing these as a component is added, and the mixture is further cultured in a medium to which a Δ6 desaturation inhibitor is added, and lipids are separated from the culture. The present invention provides a method for producing 5, 11, 14, 17-eicosatetraenoic acid-containing lipid, which is characterized by being collected. The present invention also comprises culturing a microorganism having arachidonic acid-producing ability at a temperature lower than 20 ° C. in a medium to which a Δ6 desaturation reaction inhibitor has been added,
And collecting lipids from the culture 5,
Provided is a method for producing a lipid containing 11,14,17-eicosatetraenoic acid.

The present invention further relates to a method of culturing a microorganism having an arachidonic acid-producing ability while maintaining the ratio of linoleic acid / γ-linolenic acid in the cells at 30 or more, or obtaining the microorganism having the arachidonic acid-producing ability. Linoleic acid /
The method comprises culturing a microorganism having a reduced Δ6 desaturation reaction having a ratio of γ-linolenic acid of 30 or more, and collecting lipids from the culture, wherein 12% by weight of 5, 11, 14-eicosatrienoic acid is obtained. % Is provided.

The present invention further relates to 3 ', 4'-(methylenedioxy) acetophenone, 3,4- (methylenedioxy) aniline, 1,2- (methylenedioxy) -4-nitrobenzene, piperonyl alcohol Compounds with methylenedioxyphenyl groups, such as thiamine, piperonylamine, piperonilonitrile, and safrole, and inhibitors of the Δ6 desaturation reaction of microorganisms containing arachidonic acid that contain paracoumarates and p- anisidine provide.

The present invention further provides a lipid containing 5,11,14-eicosatrienoic acid, wherein the content of 5,11,14-eicosatrienoic acid is at least 15% by weight based on the total fatty acids in the lipid, And 5.1 for total fatty acids in lipids
5,11,14-eicosatriene having a content of 1,14-eicosatrienoic acid of 15% by weight or more and a content of 5,11,14,17-eicosatetraenoic acid of 0.1% by weight or less Lipids containing acids,

5,5 based on the total fatty acids in the triglyceride
Triglycerides containing 5,11,14-eicosatrienoic acid having a content of 11,14-eicosatrienoic acid of 12% by weight or more, and 5,11,14-eicosatrienoic acid based on the total fatty acids in the triglyceride. 5, 11, 14, and 17, the content of 5,11,14,17-eicosatetraenoic acid is 0.1% by weight or less.
-A triglyceride containing eicosatrienoic acid, and a composition containing the above lipid or triglyceride.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, any microorganism capable of producing arachidonic acid can be used. As microorganisms having arachidonic acid-producing ability,
Mortierella (Mortierella) genus, Koni audio bolus (Conidiobolus) genus, Fichiumu (Pythium) genus Phytophthora (Phytophthora) genus, Penishiryumu (Penici
llium ), Cladosporium ,
Genus Mucor, genus Fusarium ,
Aspergillus (Aspergillus) genus, Rhodotorula (Rh
odotorula ), Entomophthora
And microorganisms belonging to the genus, Echinosporangium , and Saprolegnia .

Microorganisms belonging to the subgenus Mortierella of the genus Mortierella include, for example, Mortierella elonga
ta ), Mortierella exig
ua ), Mortierella figurofila ( Mortierella)
hygrophila ), Mortierell alpina ( Mortierell)
a alpina ) and the like.

More specifically, Mortierella elongata IFO8570, Mortierella elongata
Exigua ( Mortierella exigua ) IFO8571, Mortierella hygrophila ( Mortierella hygrophila ) IF
O5941, Mortierella alpi
na ) IFO8568, ATCC16266, ATCC32221, ATCC42430,
CBS219.35, CBS224.37, CBS250.53, CBS343.66, CB
S527.72, CBS529.72, CBS608.70, CBS754.68 and the like can be mentioned.

All of these strains were obtained from the Institute of Fermentation (IFO) in Osaka and the American Type Culture Collection in the United States.
Collection, ATCC) and Centralbureau voor Schimm
It can be obtained without any restrictions from elcultures (CBS). In addition, the strain Mortierella elongata SAM0219 (Microtechnical Research Laboratories No. 8703) (Microtechnical Research Laboratories No. 1239) isolated by the research group of the present invention from the soil can be used, but is limited to these strains. I am not doing it. Strains belonging to these type cultures or strains isolated from the natural world can be used as they are, but natural mutants having different properties from the original strain obtained by performing growth and / or isolation one or more times can be used. It can also be used.

In the present invention, a microorganism having a reduced Δ6 desaturation activity obtained from a microorganism having an arachidonic acid-producing ability can be used. That is, by culturing a microorganism having a decreased Δ6 desaturation activity obtained from a microorganism having arachidonic acid-producing ability in a medium to which a Δ6 desaturation reaction inhibitor has been added, 5,11,14 in lipids or triglycerides -Eicosatrienoic acid and / or 5,11,14,17
-It is possible to increase the proportion of eicosatetraenoic acid.

In addition, among the microorganisms having a decreased Δ6 desaturation activity, a microorganism having a ratio of linoleic acid / γ-linolenic acid in the cells of 30 or more is used, so that the Δ6 desaturation inhibitor can be used in a medium. 5, 11,
Lipids containing significant amounts of 14-eicosatrienoic acid can be obtained. Such a microorganism having a decreased Δ6 desaturation activity can be obtained by, for example, the following mutation treatment using the microorganism having the arachidonic acid-producing ability. In addition, Δ Δ of microorganisms having arachidonic acid-producing ability
6 It can also be obtained by genetic manipulation to reduce or delete the activity of an enzyme involved in the desaturation reaction. For example, knockout of the gene encoding the enzyme,
Although inactivation by antisense is mentioned, the method is not limited to these methods.

The present invention further relates to the aforementioned arachidonic acid in order to increase the proportion of 5,11,14-eicosatrienoic acid and / or 5,11,14,17-eicosatetraenoic acid in lipids or triglycerides. A microorganism having enhanced chain length elongation activity or Δ5 desaturation activity obtained by subjecting a microorganism having a production ability or a microorganism having a decreased Δ6 desaturation activity obtained from this microorganism to a mutation treatment or a genetic manipulation. The use of is also included.

In the present invention, the mutation treatment is performed using radiation (X
Radiation, gamma rays, neutron rays) irradiation, ultraviolet irradiation, high heat treatment, etc., or suspending the microorganisms in an appropriate buffer, adding a mutagen, incubating for a certain time,
General mutagenesis such as appropriate dilution and inoculation on an agar medium to obtain mutant colonies can also be performed.

The mutagens include nitrogen mustard, methyl methanesulfonate and N-methyl-N'-nitro-N
-Nitrosoguanidine (NTG) and other alkylating agents, base analogs such as 5-bromouracil, antibiotics such as mitomycin C, base synthesis inhibitors such as 6-mercaptopurine, pigments such as proflavine, 4-nitro Certain carcinogens such as quinoline-N-oxide, and compounds such as manganese chloride and formaldehyde can be mentioned. The colonies obtained by the mutation operation are analyzed for the fatty acid composition in the cells according to a general method, and bacteria having a reduced Δ6 desaturation reaction are selected.

For example, as the mutant strain of the present invention, a strain Mortierella alpina SAM1969 having reduced Δ6 desaturation activity, which was obtained by the research group of the present invention by performing a mutagenesis treatment.
(Microtechnical Laboratory No. 12901) can also be used,
It is not limited to these strains. In order to culture the strain used in the present invention, spores, hyphae of the strain, or a preculture obtained by pre-culturing is inoculated into a liquid medium or solid medium and cultured. In the case of a liquid medium, as a carbon source, glucose, fructose, xylose, saccharose, maltose, soluble starch, molasses, glycerol, mannitol, and other commonly used ones can be used, but are not limited thereto. Not something.

Examples of the nitrogen source include natural nitrogen sources such as peptone, yeast extract, malt extract, meat extract, casamino acid, corn steep liquor, soy protein, defatted soybean, cottonseed scum, and organic nitrogen sources such as urea. In addition, inorganic nitrogen sources such as sodium nitrate, ammonium nitrate, and ammonium sulfate can be used. In addition, inorganic salts such as phosphates, magnesium sulfate, iron sulfate, and copper sulfate, vitamins, and the like can be used as trace nutrients if necessary.

[0032] These medium components are not particularly limited as long as they do not impair the growth of microorganisms. In general, the carbon source is preferably used at a concentration of 0.1 to 40% by weight, preferably 1 to 25% by weight. The carbon source may be fed sequentially during the culture. Also, the initial nitrogen source addition amount is 0.1 to 10% by weight,
The content is preferably 0.1 to 6% by weight, and a nitrogen source may be fed during the culturing.

The Δ6 desaturation inhibitor used in the present invention specifically inhibits the Δ6 desaturation reaction of the microorganism to be used, and enables 5,11,14-eicosatrienoic acid and 5,11,1.
All compounds that accumulate 4,17-eicosatetraenoic acid can be used. Preferably, a Δ5 desaturation reaction for converting 11,14-eicosadienoic acid to 5,11,14-eicosatrienoic acid, and converting 11,14,17-eicosatrienoic acid to 5,11,14,17-eicosatrienoic acid In order for the Δ5 desaturation reaction to be converted to satetraenoic acid to proceed, it is preferable that the Δ5 desaturation reaction inhibitory action is small. Examples of such compounds include the following compounds.

[0034]

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[0035]

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[0036]

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Compounds having a methylenedioxyphenyl group include 3 ′, 4 ′-(methylenedioxy) acetophenone, 3,4- (methylenedioxy) aniline,
2- (methylenedioxy) -4-nitrobenzene, piperonyl alcohol, piperonylamine, piperonilonitrile and safrole are preferred, and the paracoumarate ester is preferably methyl paracoumarate. Δ
The total amount of the 6-unsaturation reaction inhibitor is not particularly limited as long as it does not impair the growth of microorganisms. In practice, generally 0.0001 to 10% by weight, preferably 0.01 to 10% by weight of the medium
%.

These inhibitors of Δ6 desaturation reaction may be added before or immediately after inoculation of the microorganism, or may be added after the start of the culture, or may be added at both times. The addition may be performed once after the start of the culture, or may be performed intermittently in multiple portions. Alternatively, it can be added continuously. The Δ6 desaturation inhibitor may be used alone, or a plurality of compounds may be used in combination.

The cultivation temperature of the microorganism of the present invention varies depending on the microorganism used, but is 5 to 40 ° C., preferably 10 to 3 ° C.
0 ° C. Further, when producing a lipid containing 5,11,14-eicosatrienoic acid substantially without containing 5,11,14,17-eicosatetraenoic acid, the culture temperature is 20 ° C. or higher. Preferably it is 20-40 degreeC, More preferably, it is 20-30 degreeC. The term "substantially not contained" means a case where 5, 11, 14, 17-eicosatetraenoic acid is 0.1% by weight or less based on all fatty acids in the lipid.

However, even when culturing at this temperature, α-linolenic acid, a precursor of 5,11,14,17-eicosatetraenoic acid, or a derivative thereof and / or 1
When cultivation is performed by adding 1,14,17-eicosatrienoic acid or a derivative thereof, or an oil or fat containing these as a component, 5,11,14,17-eicosatetraenoic acid is also produced. .

Furthermore, α-linolenic acid or a derivative thereof and / or 11,14,17-eicosatrienoic acid or a derivative thereof, or an oil or fat containing these as a component is added to the medium without adding 5,11,14. , 17-
The culture temperature for producing eicosatetraenoic acid is
The temperature is lower than 20 ° C, preferably 5 ° C or higher and lower than 20 ° C, more preferably 10 ° C or higher and lower than 20 ° C. The medium is adjusted to a pH of 4 to 10, preferably 5 to 9, for aeration, stirring, shaking, or stationary culture. The culture is usually performed for 2 to 30 days, preferably for 5 to 20 days, more preferably for 5 to 15 days.

In the case of culturing by solid culture, bran, rice husk, and water containing 50 to 100% by weight of water based on the weight of the solid material,
Cultivation is performed for 3 to 14 days using rice bran or the like at 5 to 40 ° C, preferably at the above-mentioned temperature. In this case, a nitrogen source, an inorganic salt, and a trace nutrient can be added to the medium as needed.

In the present invention, any of saturated fatty acid, oleic acid, linoleic acid, 11,14-eicosadienoic acid, a derivative thereof, or a derivative thereof is used as a precursor of 5,11,14-eicosatrienoic acid. The production of 5,11,14-eicosatrienoic acid can be increased by adding a fat or oil contained as a component to the medium. In addition, α-linolenic acid or a derivative thereof and / or 11,14,17-eicosatrienoic acid or a derivative thereof as a precursor of 5,11,14,17-eicosatetraenoic acid, or containing these as a constituent component 5, 11, 14, 17-eicosatetraenoic acid can be produced by adding the resulting fat or oil to the medium.

In each case, the total amount of the precursor added is 0.001 to 10% by weight, preferably 0.5 to 10% by weight, based on the medium.
% By weight. These precursors may be added before or immediately after inoculation of the producing microorganism, or may be added after the start of the culture, or may be added at both time points. The addition may be performed once after the start of the culture, or may be performed intermittently in multiple portions. Alternatively, it can be added continuously.

In addition, irrespective of the production of 5, 11, 14, 17-eicosatetraenoic acid, 5, 11, 14
-In order to increase the production of eicosatrienoic acid, its substrate can be added. Examples of the substrate include hydrocarbons such as tetradecane, hexadecane, and octadecane; fatty acids such as tetradecanoic acid, hexadecanoic acid, and octadecanoic acid; and salts (eg, sodium salts and potassium salts) and esters thereof; and fats and oils containing fatty acids as constituents ( Examples thereof include olive oil, safflower oil and palm oil), but are not limited thereto.

The total amount of substrate added is 0.001 to 10
%, Preferably 0.5 to 10% by weight. These substrates may be added before or immediately after inoculation of the producing microorganism, or may be added after the start of the culture, or may be added at both time points. The addition may be performed once after the start of the culture, or may be performed intermittently in multiple portions. Alternatively, it can be added continuously.

Further, 5,11,14-eicosatrienoic acid and / or
Or to obtain a lipid or triglyceride containing 5,11,14,17-eicosatetraenoic acid in a commercially viable yield,
Using a liquid medium, aeration and agitation culture is preferable, and a usual agitation type fermenter or bubble column type culture apparatus can also be used. Desirably, the aeration amount is 0.1 to 3 vvm, and the stirring speed is 10 to 500 rpm.

By culturing in this manner, lipids containing 5,11,14-eicosatrienoic acid and / or 5,11,14,17-eicosatetraenoic acid are produced and accumulated in the cells. When a liquid medium is used, a culture solution in the course of producing lipids by cell culture or a sterilized culture solution thereof, or a culture solution at the end of culture or a sterilized culture solution thereof, or a culture bacterium collected from each of them 5,1 from body or its dry matter
A lipid containing 1,14-eicosatrienoic acid and / or 5,11,14,17-eicosatetraenoic acid is collected. For example,
Collection of lipids containing 5,11,14-eicosatrienoic acid and / or 5,11,14,17-eicosatetraenoic acid from the cultured cells, and 5,11,14- Triglycerides containing eicosatrienoic acid and / or 5,11,14,17-eicosatetraenoic acid, or 5,11,14-eicosatrienoic acid and / or 5,11,14,17-eicosatetraic acid Enoic acid is isolated.

After completion of the culturing, the cultured cells are obtained from the culture solution by conventional solid-liquid separation means such as centrifugation or filtration. The cells are thoroughly washed with water and preferably dried. Drying can be performed by freeze drying, air drying, or the like. The dried cells are crushed by, for example, a dynomill or ultrasonic waves, and then subjected to extraction treatment with an organic solvent, preferably under a nitrogen stream. Organic solvents include ether, hexane, methanol, ethanol,
Chloroform, dichloromethane, petroleum ether and the like can be used, and good results can also be obtained by alternate extraction of methanol and petroleum ether or extraction using a single-layer solvent of chloroform-methanol-water. By distilling off the organic solvent from the extract under reduced pressure, 5.1
1,14-eicosatrienoic acid and / or 5,11,1
A lipid containing 4,17-eicosatetraenoic acid is obtained.

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

In the lipid obtained as described above,
5, 11, 14-eicosatrienoic acid and / or 5, 1
1,14,17-eicosatetraenoic acid is used for neutral lipids such as triglyceride, diglyceride, monoglyceride, and sterol ester, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, and lysophosphatidylethanol. Amine, phosphatidylinositol, lysophosphatidylinositol, phosphatidylserine, lysophosphatidylserine, phosphatidic acid, as a component of polar lipids such as lysophosphatidic acid, or as a free fatty acid I have.

For example, lipids and fats containing 5,11,14-eicosatrienoic acid and / or 5,11,14,17-eicosatetraenoic acid produced using a microorganism belonging to the subgenus Mortierella. As a composition, the neutral lipid is 70 to 100% by weight, the polar lipid is 0 to 30% by weight, and triglyceride which is a main component of the neutral lipid is 70 to 100% by weight.
99% by weight.

In the case of lipids substantially free of 5,11,14,17-eicosatetraenoic acid, the content of 5,11,14-eicosatrienoic acid in the lipid is 0.2% based on the total fatty acids. ~ 80% by weight, preferably 1.3-80% by weight,
More preferably, it is 2.5 to 80% by weight. Also, 5, 1
In the case of a lipid containing 1,14,17-eicosatetraenoic acid, the content of 5,11,14,17-eicosatetraenoic acid in the lipid is 0.1 to 70% by weight based on the total fatty acids,
The content is preferably from 0.2 to 70% by weight, more preferably from 0.5 to 70% by weight. The content of 5,11,14,17-eicosatetraenoic acid varies depending on the type and amount of the precursor or the culture temperature. I do.

The ratio of triglycerides to total fatty acids in lipids is 5, 1 in the case of lipids substantially free of 5, 11, 14, 17-eicosatetraenoic acid.
3.0 to 80% by weight of 1,14-eicosatrienoic acid, preferably 12.1 to 80% by weight, more preferably 13.4 to 80% by weight
In the case of a lipid containing 5,11,14,17-eicosatetraenoic acid, 5,11,1
The content of 4,17-eicosatetraenoic acid is 0.1 to 70% by weight, preferably 0.4 to 70% by weight, more preferably 0.5 to 70% by weight based on the total fatty acids.
The 4,17-eicosatetraenoic acid content can be freely changed by adjusting the type and amount of the precursor or the culture temperature.

In the present invention, a lipid containing 5,11,14-eicosatrienoic acid having a content of 5,11,14-eicosatrienoic acid of 15% by weight or more based on the total fatty acids in the lipid is used. Can be obtained. Further, the content of 5,11,14-eicosatrienoic acid is 15% by weight or more and 5,11,14,17-
A lipid containing 5,11,14-eicosatrienoic acid having an eicosatetraenoic acid content of 0.1% by weight or less can be obtained.

The above-mentioned lipid means that a higher fatty acid forms some chemical bond, typically an ester bond, in the molecule, and is insoluble in water and contains alcohol, chloroform,
A substance soluble in organic solvents such as benzene. 5, 1
1,14-eicosatrienoic acid and / or 5,11,
Examples of lipids containing 14,17-eicosatetraenoic acid include 5,11,14-eicosatrienoic acid and / or
Or a lower alkyl ester of 5,11,14,17-eicosatetraenoic acid, and / or 5,11,14-eicosatrienoic acid and / or 5,11,14,17-eicosatetraenoic acid. And sterol esters, and mixtures of any two or more of these.

The above-mentioned lower alkyl ester of 5,11,14-eicosatrienoic acid and / or 5,11,14,17-eicosatetraenoic acid means 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms. And more preferably 1 to 3 lower alcohols and esters of 5,11,14-eicosatrienoic acid and / or 5,11,14,17-eicosatetraenoic acid. A glycerin ester containing 5,11,14-eicosatrienoic acid and / or 5,11,14,17-eicosatetraenoic acid as a component is defined as at least one molecule per glycerin molecule. 5, 11, 14-eicosatrienoic acid and / or 5,
It refers to a substance in which 11, 14, 17-eicosatetraenoic acid is ester-bonded.

Examples thereof include triglyceride, diglyceride, monoglyceride, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylinositol, lysophosphatidylinositol, phosphatidylcholine. Examples thereof include gillserin, lysophosphatidylserine, phosphatidic acid, lysophosphatidic acid, and glyceroglycolipid.

The sterol ester containing 5,11,14-eicosatrienoic acid and / or 5,11,14,17-eicosatetraenoic acid as a constituent is
A substance in which sterol and 5,11,14-eicosatrienoic acid and / or 5,11,14,17-eicosatetraenoic acid are ester-linked. Examples thereof include cholesterol esters and desmosterol esters. 5, 11, 14-eicosatrienoic acid and / or
Alternatively, the lipid containing 5, 11, 14, 17-eicosatetraenoic acid is not limited to the above examples, and the above-described lipids such as sphingolipids, other phospholipids, ceramides, glycosphingolipids, and other glycolipids may be used. And any lipids included in the definition of

Further, in the present invention, the content of 5,11,14-eicosatrienoic acid is 12% by weight or more, preferably 1% by weight, based on the total fatty acids in the triglyceride in the lipid.
It is possible to obtain a triglyceride containing 5,11,14-eicosatrienoic acid of 3% by weight or more, more preferably 14% by weight or more. Further, the content of 5,11,14-eicosatrienoic acid is 12% by weight or more and 5,11,1 based on the total fatty acids in the triglyceride in the lipid.
The content of 4,17-eicosatetraenoic acid is 0.1% by weight.
Below, preferably 13% by weight or more and 5, 11, 14,
The content of 17-eicosatetraenoic acid is 0.1% by weight or less, more preferably 14% by weight or more and 5, 11, 1
The content of 4,17-eicosatetraenoic acid is 0.1% by weight.
The following triglyceride containing 5, 11, 14-eicosatrienoic acid can be obtained.

From the lipids containing 5,11,14-eicosatrienoic acid and / or 5,11,14,17-eicosatetraenoic acid collected from the culture, 5,11,14
-Eicosatrienoic acid and / or 5, 11, 14, 17
-To separate and purify triglyceride containing eicosatetraenoic acid, according to a conventional method, for example, deacidification method, deodorization method, degumming method, dehydration method, steam distillation method, molecular distillation method, cooling separation method, column This is performed by a chromatography method or the like. For example, a lipid containing 5,11,14-eicosatrienoic acid and / or 5,11,14,17-eicosatetraenoic acid is extracted from the culture using hexane according to the above-mentioned operation, and the extracted oil is extracted from the extracted oil. For example, the triglyceride of the present invention can be obtained by purification treatment such as deacidification, deodorization, and degumming.

From a lipid containing 5,11,14-eicosatrienoic acid and / or 5,11,14,17-eicosatetraenoic acid, 5,11,14-eicosatrienoic acid and / or 5,11 To separate, 14,17-eicosatetraenoic acid, a concentrated fatty acid or a mixed fatty acid ester is concentrated and separated by a conventional method, for example, a urea addition method, a cooling separation method, a column chromatography method or the like. Performed by

More specifically, it is also possible to directly separate 5,11,14-eicosatrienoic acid and / or 5,11,14,17-eicosatetraenoic acid. It is preferably separated as the ethyl ester of 5,11,14-eicosatrienoic acid and / or 5,11,14,17-eicosatetraenoic acid. By making such an ester, it can be easily separated from other lipid components, and other fatty acids produced during culture, such as palmitic acid (also these
5, 11, 14-eicosatrienoic acid and / or 5, 1
1,14,17-Eicosatetraenoic acid).

For example, to obtain an ethyl ester of a polyunsaturated fatty acid, the above extracted lipid is treated with anhydrous ethanol-hydrochloric acid.
5 ~10%, BF ₃ - ethanol 10-50%, etc., at room temperature
Preferably, the treatment is for 1 to 24 hours. 5, 11, 14-eicosatrienoic acid and / or 5, 1
In order to recover the ethyl ester of 1,14,17-eicosatetraenoic acid, it is preferable to extract with an organic solvent such as hexane, ether or ethyl acetate. Next, this extract is dried with anhydrous sodium sulfate or the like, and the organic solvent is distilled off, preferably under reduced pressure, to obtain a mixture containing a fatty acid ester as a main component. This mixture can be used in the composition of the present invention as it is or further to increase the concentration of the fatty acid of the present invention.

This mixture contains the desired 5, 11, 1
4-eicosatrienoic acid and / or 5, 11, 14, 1
In addition to the ethyl ester of 7-eicosatetraenoic acid, fatty acid ethyl esters such as palmitic acid ethyl ester are included. From these fatty acid ethyl ester mixtures, 5, 11, 14-eicosatrienoic acid and / or 5,
In order to isolate the ethyl ester of 11,14,17-eicosatetraenoic acid, column chromatography, low-temperature crystallization method, urea clathration method, liquid-liquid AC distribution chromatography, etc. are used alone or in combination. can do.

The thus isolated 5,11,14-eicosatrienoic acid and / or the free 5,1 from the ethyl ester of 5,11,14,17-eicosatetraenoic acid
1,14-eicosatrienoic acid and / or 5,11,1
In order to obtain 4,17-eicosatetraenoic acid, it may be hydrolyzed with an alkali and then extracted with an organic solvent such as ether or ethyl acetate. In order to ingest 5,11,14-eicosatrienoic acid and / or 5,11,14,17-eicosatetraenoic acid without passing through the ethyl ester, the extracted lipid is subjected to alkaline degradation (for example, After the reaction with 5% sodium hydroxide at room temperature for 2 to 3 hours), the decomposed solution can be extracted and purified by a method commonly used for extracting and purifying fatty acids. The resulting free fatty acids of the invention and salts thereof can also be used for the compositions of the invention.

The lipid or triglyceride containing 5,11,14-eicosatrienoic acid and / or 5,11,14,17-eicosatetraenoic acid according to the present invention is a lipid or triglyceride rich in the fatty acid. And there are endless possibilities for its uses: food, beverages, cosmetics,
It can be used as raw materials and additives for pharmaceuticals and animal feed. For example, general foods, beverages, functional foods, dietary supplements, milk preparations, lotions, emulsions, enteral nutrition, powders, granules, troches, syrups, tablets, capsules, infusions, injections, gels for application, Examples thereof include a poultice, a powder feed, a solid feed, a liquid feed, and the like, but are not limited thereto, and there are no restrictions on the purpose of use, the amount of use, and the form of processing.

[0068]

Next, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the embodiments. Embodiment 1 FIG . 5, 11, using Δ6 desaturation inhibitor
14-eicosatrienoic acid, 5, 11, 14, 17-e
A method for producing icosatetraenoic acid One platinum loop of Mortierella alpina IFO8568, a microorganism having the ability to produce arachidonic acid, was placed in a 10 mL Erlenmeyer flask in 2 mL of a liquid medium (4% glucose, 1% yeast extract, And the following Δ
6 Inoculation of desaturation inhibitor (concentration shown in Table 1)
Shaking culture was performed at 28 ° C. and 120 rpm for 10 days.

After the culture, the cells were collected by filtration and dried. According to a conventional method, fatty acids in the cells were methyl-esterified with methanolic hydrochloric acid, extracted with hexane, and the fatty acid methyl ester obtained by distilling off hexane was analyzed by gas chromatography. Table 1 shows the results.

(1) 3 ', 4'-(methylenedioxy)
Acetophenone (2) 3,4- (methylenedioxy) aniline (3) 1,2- (methylenedioxy) -4-nitrobenzene (4) piperonyl alcohol (5) piperonylamine (6) piperonilonitrile (7 ) Safflor (8) Methyl paracoumarate (9) Paraanisidine

[0071]

[Table 1]

All the Δ6 desaturation inhibitors were found to accumulate linoleic acid and 5,11,14-eicosatrienoic acid. All 5, 11, 14, 17-
Eicosatetraenoic acid was not detected.

Embodiment 2 FIG. Fine with arachidonic acid producing ability
Production of 5, 11, 14-eicosatrienoic acid by living organisms
Method One platinum loop of a microorganism having the following arachidonic acid-producing ability was placed in a 10 mL Erlenmeyer flask in a liquid medium 2.
The cells were inoculated into mL (glucose 4%, yeast extract 1%, piperonylamine 0.05%), and cultured with shaking at 28 ° C and 120 rpm for 10 days. After the culturing, methyl esterification was performed in the same manner as in Example 1, and the obtained fatty acid methyl ester was analyzed by gas chromatography. Table 2 shows the results.

[0074] (1) Mortierella alpina (Mortie
rella alpina ) IFO8568 (2) Mortierella alpin
a ) SAM1969 (Microtechnical Laboratories No. 12901) (3) Mortierella figlophila
hygrophila ) IFO5941 (4) Conidiobolus thromoboides
us thromoboides ) CBS183.60 (5) Pythium irregulare
CBS494.86 (6) Phytophthora
infestans ) IFO4872 (7) Echinosporandium transversale ( Echi
nosporangium transversale ) NRRL3116 (8) Saprolegnia lappon
ica ) CBS313.81

[0075]

[Table 2]

In all the strains, the production of 5,11,14-eicosatrienoic acid was confirmed by adding piperonylamine. Mortierella alpina SAM 1969 which produces 5,11,14-eicosatrienoic acid without the addition of piperonylamine
Also, in the case of (Microtechnical Laboratories No. 12901), the amount of production and the ratio to the total fatty acid amount were significantly increased by the addition. In any case, 5, 11, 14, 17-eicosatetraenoic acid was not detected.

Embodiment 3 FIG. 5, 11, 1 by low temperature culture
Method for producing 4,17 -eicosatetraenoic acid Mortierella alpina IFO8
568 or Mortierella alpi
na ) One platinum loop of SAM1969 (Microtechnical Laboratory No. 12901)
2mL of liquid medium in 10mL Erlenmeyer flask
(Glucose 4%, yeast extract 1%, piperonylamine
0.05%), and cultured at 12 to 28 ° C. with shaking at 120 rpm for 10 days. After the culturing, methyl esterification was performed in the same manner as in Example 1, and the obtained fatty acid methyl ester was analyzed by gas chromatography. Table 3 shows the results.

[0078]

[Table 3] All strains are only available when cultured below 20 ° C.
1,14,17-Eicosatetraenoic acid was produced.

Embodiment 4 FIG. α-linolenic acid, 11, 14,
17-eicosatrienoic acid, or a compound thereof
5, 11, 14, 17-eicosatetraene by addition
Method for producing acid Mortierella alpina IFO8
One loop of 568 was placed in a 10 mL Erlenmeyer flask containing 2 mL of liquid medium (4% glucose, 1% yeast extract,
The compound shown in Table 4 was added to a medium consisting of 0.05% piperonylamine (0.5%), and cultured with shaking at 28 ° C and 120 rpm for 10 days.

After the culture, a part of the cells was methylesterified in the same manner as in Example 1, and the obtained fatty acid methylester was analyzed by gas chromatography. The remaining cells were dried and extracted with chloroform-methanol. Further, according to a conventional method, the extracted oil is subjected to thin-layer chromatography,
Fractionation by silica column chromatography gave triglycerides. The obtained triglyceride was also analyzed by gas chromatography according to the method described above. Table 4 shows the results.

[0081]

[Table 4]

The medium containing piperonylamine was further added with α
-Linolenic acid or a derivative thereof, 11, 14, 17-
By adding eicosatrienoic acid or a derivative thereof, or an oil or fat containing these as a component,
Lipids containing 5, 11, 14, 17-eicosatetraenoic acid were produced. The lipids produced include 5,11,
Triglycerides containing 14,17-eicosatetraenoic acid were included.

Embodiment 5 FIG. 5, 11, 14-eikosatori
Effect of substrate addition on enic acid production Mortierella alpina IFO8
One loop of 568 was placed in a 10 mL Erlenmeyer flask containing 2 mL of liquid medium (4% glucose, 1% yeast extract,
The compound shown in Table 5 was added to a medium consisting of 0.05% piperonylamine (0.5%), and cultured with shaking at 28 ° C and 120 rpm for 10 days. After the culturing, methyl esterification was performed in the same manner as in Example 1, and the obtained fatty acid methyl ester was analyzed by gas chromatography. Table 5 shows the results.

[0084]

[Table 5] Each of the substrates had an effect of increasing the production amount of 5, 11, 14-eicosatrienoic acid.

Embodiment 6 FIG. Using 10L aeration tank culture
Mass culture method of 11,14-eicosatrienoic acid Mortierella alpina SAM1
Inoculate one loop of 969 (Microtechnical Laboratories No. 12901) into 100 mL of liquid medium (2% glucose, 1% yeast extract) in a 500 mL Erlenmeyer flask, and incubate at 28 ° C for 120 r.
Preculture for 7 days at pm. 5L medium (2% glucose, 1% yeast extract, piperonylamine
0.05%), inoculate the above pre-cultured liquid, 28 ℃, 300r
Culture was performed at 1 pm and aeration of 1 vvm. The cells were cultured for 10 days while maintaining the glucose concentration at 0.5 to 1.5% by a fed-batch method.

The cells were sampled with time, methylesterified in the same manner as in Example 1, and the resulting fatty acid methylester was analyzed by gas chromatography. Also,
After completion of the culture, the cells were collected, dried, and subjected to chloroform-methanol extraction. Further, according to a conventional method, the extracted oil was fractionated by thin-layer chromatography and silica column chromatography. Of the extracted oil, 92% by weight of neutral lipid (triglyceride was 91% by weight of the extracted oil), The polar lipid was 8% by weight. The obtained triglyceride was methylesterified by the above-mentioned method, and analyzed by gas chromatography. The changes over time are shown in Tables 6 and 7.

[0087]

[Table 6]

[0088]

[Table 7]

It has been found that a remarkable amount of 5,11,14-eicosatrienoic acid can be produced by a large-scale culture method using a 10-L aeration culture tank. In addition, a remarkable amount of 5, 11, 14-eicosatrienoic acid was contained in the obtained triglyceride.

Embodiment 7 FIG. Preparation of capsules Water was added to 100 parts by weight of gelatin and 35 parts by weight of glycerin for food addition and dissolved at 50 to 60 ° C to prepare a gelatin film having a viscosity of 20,000 cps. Next, the dried cells obtained by the method described in Example 6 were crushed and extracted with hexane. The hexane-extracted oil or the triglyceride obtained by deoxidizing, deodorizing, and degumming the hexane-extracted oil contained 3% by weight. The contents were prepared by mixing vitamin E oil. Using these, capsule molding and drying were performed according to a conventional method to produce soft capsules containing 180 mg of content per grain.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C11B 3/00 C11B 3/00 4C083 C11C 1/08 C11C 1/08 4H059 3/00 3/00 C12N 9 / 99 C12N 9/99 // C12N 1/20 C12N 1/20 A (C12P 7/64 (C12P 7/64 C12R 1: 645) C12R 1: 645) (C12P 7/64 (C12P 7/64 C12R 1:80 ) C12R 1:80) (C12P 7/64 (C12P 7/64 C12R 1: 785) C12R 1: 785) (C12P 7/64 (C12P 7/64 C12R 1:77) C12R 1:77) (C12P 7 / 64 (C12P 7/64 C12R 1:66) C12R 1:66) F term (reference) 4B018 LB07 LB08 MD14 ME02 4B026 DC05 DG20 DX01 4B064 AD85 AD88 CB24 CC03 CD07 CD30 DA01 DA10 DA11 DA16 4B065 AA57X AA60X AA6 6X AA67X AA78X AC14 BA22 BB10 BB40 BC03 BD14 CA13 CA41 CA44 4C076 AA16 AA22 AA29 AA36 AA53 DD41 DD46 4C083 AC251 AC421 CC01 CC04 DD14 DD15 DD17 DD31 4H059 BA26 BA30 BA33 BA35 BA43 BB05 CA48 BB07 CA48 BB07 CA48

Claims (35)

[Claims] 1. A microorganism capable of producing arachidonic acid,
5. A method comprising culturing in a medium supplemented with a Δ6 desaturation inhibitor, and collecting lipid from the culture.
A method for producing a 1,14-eicosatrienoic acid-containing lipid. 2. A microorganism capable of producing arachidonic acid,
A culture medium containing a Δ6 desaturation inhibitor is added, lipids are collected from the culture, and triglycerides are separated and purified from the lipids, which contains 5,11,14-eicosatrienoic acid. A method for producing triglycerides. 3. A microorganism capable of producing arachidonic acid,
The cells are cultured in a medium supplemented with a Δ6 desaturation inhibitor, lipids are collected from the culture, and 5,11, 14
-A process for producing 5,11,14-eicosatrienoic acid, comprising separating and purifying eicosatrienoic acid. 4. A microorganism capable of producing arachidonic acid,
Saturated fatty acid, oleic acid, linoleic acid or 11,1
The production according to any one of claims 1 to 3, wherein the culture is carried out in a medium to which any one of 4-eicosadienoic acid, a derivative thereof, or an oil or fat containing these as a component is further added. Method. 5. A microorganism capable of producing arachidonic acid,
The method according to any one of claims 1 to 3, wherein the culturing is performed while maintaining the ratio of linoleic acid / γ-linolenic acid in the cells to 30 or more. 6. A microorganism capable of producing arachidonic acid,
The cultivation at a temperature of 0 ° C. or more, characterized in that:
6. The production method according to any one of items 5 to 5. 7. A microorganism having a reduced ratio of Δ6 desaturation, wherein the ratio of linoleic acid / γ-linolenic acid obtained from a microorganism capable of producing arachidonic acid is 30 or more, is cultured, and lipids are collected from the culture. 5, 1 characterized by the above-mentioned
A method for producing a lipid containing at least 12% by weight of 1,14-eicosatrienoic acid. 8. A microorganism having a reduced ratio of Δ6 desaturation, wherein the ratio of linoleic acid / γ-linolenic acid obtained from a microorganism having an arachidonic acid-producing ability is 30 or more, is cultured, and 5, 11, 14 5, 11, 1 wherein a lipid containing eicosatrienoic acid of 12% by weight or more is collected, and triglyceride is separated and purified from the lipid.
A method for producing triglyceride containing 4-eicosatrienoic acid. 9. A microorganism having a reduced linoleic acid / γ-linolenic acid ratio of 30 or more, which is obtained from a microorganism having an arachidonic acid-producing ability and having a reduced Δ6 desaturation reaction, is cultured, and 5, 11, 14 Collecting a lipid containing eicosatrienoic acid in a proportion of at least 12% by weight, and extracting 5,1 from the lipid;
A method for producing 5,11,14-eicosatrienoic acid, comprising separating and purifying 1,14-eicosatrienoic acid. 10. A microorganism having an arachidonic acid-producing ability is cultured at a temperature lower than 20 ° C. in a medium to which a Δ6 desaturation inhibitor is added, and lipids are collected from the culture. , 11, 14, 17-Method for producing lipid containing eicosatetraenoic acid. 11. A microorganism capable of producing arachidonic acid is cultured at a temperature lower than 20 ° C. in a medium to which a Δ6 desaturation inhibitor is added, lipids are collected from the culture, and triglycerides are separated from the lipids. A process for producing 5, 11, 14, 17-eicosatetraenoic acid-containing triglyceride, comprising purifying. 12. A microorganism capable of producing arachidonic acid is cultured at a temperature lower than 20 ° C. in a medium to which a Δ6 desaturation inhibitor is added, lipids are collected from the culture, and 5, 11 , 14, 17-eicosatetraenoic acid, characterized in that it is separated and purified.
A method for producing 17-eicosatetraenoic acid. 13. A microorganism having an arachidonic acid-producing ability is produced by using α-linolenic acid or a derivative thereof and / or
1,14,17-Eicosatrienoic acid or a derivative thereof or an oil or fat containing these as a component is added, and the mixture is further cultured in a medium containing a Δ6 desaturation inhibitor, and lipids are collected from the culture. A method for producing 5, 11, 14, 17-eicosatetraenoic acid-containing lipid. 14. A microorganism having an arachidonic acid-producing ability is produced by using α-linolenic acid or a derivative thereof and / or
1,14,17-Eicosatrienoic acid or a derivative thereof or an oil or fat containing these as a component is added, and the mixture is further cultured in a medium to which a Δ6 desaturation inhibitor is added, and lipids are collected from the culture. 5, 11, 1, 1 characterized in that triglyceride is separated and purified from the lipid.
A method for producing 4,17-eicosatetraenoic acid-containing triglyceride. 15. A microorganism having an arachidonic acid-producing ability is produced by using α-linolenic acid or a derivative thereof and / or
1,14,17-Eicosatrienoic acid or a derivative thereof or an oil or fat containing these as a component is added, and the mixture is further cultured in a medium to which a Δ6 desaturation inhibitor is added, and lipids are collected from the culture. And a method for producing 5,11,14,17-eicosatetraenoic acid, comprising separating and purifying 5,11,14,17-eicosatetraenoic acid from the lipid. 16. microorganisms having arachidonic acid producing ability, Mortierella (Mortierella) genus, Koni audio bolus (Conidiobolus) genus Fichiumu (Pythium) genus,
Phytophthora genus, penicillium ( Pe
nicillium ), Cladosporium
Sp., Mucor (Mucor) genus, Fuzaryumu (Fusarium)
Genus Aspergillus (Aspergillus) genus, Rhodotorula (Rhodotorula) genus, Entomofutora (Entomophthora
) Genus, Echinosporangium
The production method according to any one of claims 1 to 15, wherein the microorganism is a microorganism belonging to the genus or the genus Saprolegnia . 17. The microorganism according to claim 1, wherein the microorganism capable of producing arachidonic acid is a microorganism belonging to the genus Mortierella and the subgenus Mortierella.
The production method according to the paragraph. 18. The production method according to claim 1, wherein the Δ6 desaturation reaction inhibitor is a compound having a methylenedioxyphenyl group. 19. The compound having a methylenedioxyphenyl group is 3 ', 4'-(methylenedioxy) acetophenone, 3,4- (methylenedioxy) aniline,
1,2- (methylenedioxy) -4-nitrobenzene,
19. The method according to claim 18, wherein the method is piperonyl alcohol, piperonylamine, piperonilonitrile, or safrole. 20. The method according to claim 1, wherein the Δ6 desaturation inhibitor is paracoumarate.
8. The method according to any one of items 7 to 7. 21. The method according to claim 2, wherein the paracoumarate is methyl paracoumarate.
0. The production method according to item 0. 22. The method according to claim 1, wherein the Δ6 desaturation inhibitor is para - anisidine. 23. The amount of 5, 11,
A lipid containing 5,11,14-eicosatrienoic acid having a content of 14-eicosatrienoic acid of 15% by weight or more. 24. The amount of 5, 11,
5,11,14-Eicosatrienoic acid having a content of 14-eicosatrienoic acid of 15% by weight or more and a content of 5,11,14,17-eicosatetraenoic acid of 0.1% by weight or less is used. Contains lipids. 25. A triglyceride containing 5,11,14-eicosatrienoic acid having a content of 5,11,14-eicosatrienoic acid of 12% by weight or more based on the total fatty acids in the triglyceride. 26. The content of 5,11,14-eicosatrienoic acid is 12% by weight or more and the content of 5,11,14,17-eicosatetraenoic acid is 0.1% by weight based on the total fatty acids in the triglyceride. 5, 11, 14- which is less than 1% by weight
Triglycerides containing eicosatrienoic acid. A food composition comprising the lipid or triglyceride according to any one of claims 23 to 26. A cosmetic composition comprising the lipid or triglyceride according to any one of claims 23 to 26. 29. A pharmaceutical composition comprising the lipid or triglyceride according to any one of claims 23 to 26. 30. An animal feed containing the lipid or triglyceride according to any one of claims 23 to 26. 31. A Δ6 desaturation inhibitor for a microorganism having arachidonic acid-producing ability, comprising a compound having a methylenedioxyphenyl group. 32. 3 ′, 4 ′-(methylenedioxy) acetophenone, 3,4- (methylenedioxy) aniline, 1,2- (methylenedioxy) -4-nitrobenzene, piperonyl alcohol, piperonylamine, Comprising either piperonilonitrile or safrole,
A Δ6 desaturation inhibitor for a microorganism having arachidonic acid-producing ability. 33. A composition comprising a paramalic acid ester,
A Δ6 desaturation inhibitor for a microorganism having arachidonic acid-producing ability. 34. An inhibitor of a Δ6 desaturation reaction of a microorganism having arachidonic acid-producing ability, comprising a paramalic acid methyl ester. 35. An inhibitor of Δ6 desaturation of a microorganism having arachidonic acid-producing ability, comprising para - anisidine.