JP2006340733A - Lipid containing n-4 system and/or n-7 system highly unsaturated fatty acids and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a ≥18C n-4 system and/or n-7 system highly unsaturated fatty acids or a lipid or triglyceride containing the unsaturated fatty acids, to provide the lipid or triglyceride containing the ≥18C n-4 system and/or n-7 system highly unsaturated fatty acids, to provide a composition containing them, and to provide a method for producing the same. <P>SOLUTION: This method for producing the lipid containing the n-4 system and/or n-7 system highly unsaturated fatty acids is characterized by culturing a microorganism lowered or lacked a fatty acid chain length elongation activity from palmitic acid to stearic acid and obtained from a microorganism having an arachidonic acid-producing ability, and then collecting the lipid from the culture product. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention provides a microorganism having an ability to produce arachidonic acid, by culturing a microorganism obtained by reducing or lacking the fatty acid chain length-extending activity from palmitic acid to stearic acid obtained from a microorganism having an ability to produce arachidonic acid. , Palmitoleic acid or its derivatives, or lipids containing n-4 and / or n-7 polyunsaturated fatty acids are collected from the culture by culturing in a medium supplemented with fats and oils containing these as constituents Or triglyceride containing n-4 and / or n-7 polyunsaturated fatty acid, or n-4 and / or n-7 polyunsaturated fatty acid is separated and purified from the lipid. N-4 series or n-7 series highly unsaturated fatty acids, or a method for producing lipids containing these, and n-4 series or n-7 series highly unsaturated fats Lipids containing, and compositions containing them.

  In general, n-4 series or n-7 series polyunsaturated fatty acids have two or more double bonds and the first double bond on the methyl group side at the 4th or 7th carbon from the end of the methyl group. It has fatty acids. Double bonds of n-4 or n-7 highly unsaturated fatty acids are usually n-3 series, n-6 series highly unsaturated fatty acids such as linoleic acid, α-linolenic acid, arachidonic acid and the like present in the living body. It is considered to antagonize the action of these normal fatty acids, and its physiological action is expected, and it is expected to be applied to foods, cosmetics, pharmaceuticals, animal feeds, etc. Has been.

  In fact, n-7 polyunsaturated fatty acids having 18 or more carbon atoms are known to exist in animals lacking essential fatty acids and are considered to have various actions in animals (Wolff RL " New trends in lipid and lipoprotein analyzes "American Oil Chemists' Society, 147-180, 1995). It is known that n-4 or n-7 highly unsaturated fatty acids having 16 carbon atoms are present in microorganisms and algae, but n-4 or n-7 highly unsaturated fatty acids having 18 or more carbon atoms. The above animal is known only as the source of saturated fatty acid, and extraction from the body of the animal has a problem in that the supply stability and content are low, and its practicality is low.

  Therefore, in particular, an n-4 series or n-7 series highly unsaturated fatty acid having 18 or more carbon atoms, or a method for producing a lipid containing these, and an n-4 or n-7 series having 18 or more carbon atoms Development of lipids or triglycerides containing polyunsaturated fatty acids and compositions containing them is strongly desired.

  Therefore, the present invention provides a method for producing n-4 or n-7 polyunsaturated fatty acids having 18 or more carbon atoms, or lipids or triglycerides containing these, and n-4 or n-carbon having 18 or more carbon atoms. It is intended to provide a lipid or triglyceride containing a -7 series polyunsaturated fatty acid, and a composition containing them.

  As a result of various studies to achieve the above-mentioned object, the present inventors cultured microorganisms having arachidonic acid-producing ability in a medium to which palmitoleic acid or a derivative thereof, or an oil containing these as a constituent component was added. By this, it discovered that the lipid containing n-4 type | system | group and / or n-7 type | system | group highly unsaturated fatty acid was produced | generated. Furthermore, by obtaining a microorganism in which the activity of extending the fatty acid chain length from palmitic acid to stearic acid is reduced or deficient from a microorganism having the ability to produce arachidonic acid, and culturing this microorganism, the n-4 system and / or the n-7 system The inventors have also found that lipids containing polyunsaturated fatty acids can be produced, thereby completing the present invention.

That is, the present invention is characterized in that a microorganism having an ability to produce arachidonic acid is cultured in a medium to which palmitoleic acid or a derivative thereof, or an oil containing these as a constituent component is added, and lipids are collected from the culture. Method for producing lipids containing n-4 and / or n-7 polyunsaturated fatty acids; and reduced fatty acid chain extension activity from palmitic acid to stearic acid obtained from microorganisms capable of producing arachidonic acid Alternatively, the present invention provides a method for producing a lipid containing an n-4 series and / or an n-7 series highly unsaturated fatty acid, which comprises culturing a deficient microorganism and collecting a lipid from the culture.

The present invention further comprises 7,10,13,16-eicosatetraenoic acid having a content of 7,10,13,16-eicosatetraenoic acid of 1.5% by weight or more based on the total fatty acids in the lipid. Lipids,
A lipid containing 5,8,11,14-octadecatetraenoic acid having a content of 5,8,11,14-octadecatetraenoic acid of 0.5% by weight or more based on the total fatty acids in the lipid;
A lipid containing 8,11,14-octadecatrienoic acid having a content of 8,11,14-octadecatrienoic acid of 0.1% by weight or more based on the total fatty acids in the lipid;

A lipid containing an n-4 highly unsaturated fatty acid having 18 or more carbon atoms, wherein the content of an n-4 highly unsaturated fatty acid having 18 or more carbon atoms is 2.8% by weight or more based on the total fatty acids in the lipid;
A lipid containing 7,10,13-eicosatrienoic acid having a content of 7,10,13-eicosatrienoic acid of 0.1% by weight or more based on the total fatty acids in the lipid;
A lipid containing 5,8,11-octadecatrienoic acid having a content of 5,8,11-octadecatrienoic acid of 0.1% by weight or more based on the total fatty acids in the lipid;
A lipid containing 8,11-octadecadienoic acid having a content of 8,11-octadecadienoic acid of 0.1% by weight or more based on the total fatty acids in the lipid;

Contains n-7 polyunsaturated fatty acid having 18 or more carbon atoms and a content of n-7 polyunsaturated fatty acid having 18 or more carbon atoms with respect to the total fatty acids in the lipid. Lipids,
A triglyceride containing 7,10,13,16-eicosatetraenoic acid in which the content of 7,10,13,16-eicosatetraenoic acid is 1.5% by weight or more based on the total fatty acids in the triglyceride,
A triglyceride containing 5,8,11,14-octadecatetraenoic acid having a content of 5,8,11,14-octadecatetraenoic acid of 0.5% by weight or more based on the total fatty acids in the triglyceride,

A triglyceride containing 8,11,14-octadecatrienoic acid having a content of 8,11,14-octadecatrienoic acid of 0.1% by weight or more based on the total fatty acids in the triglyceride,
A triglyceride containing an n-4 highly unsaturated fatty acid having 18 or more carbon atoms and a content of n-4 highly unsaturated fatty acids having 18 or more carbon atoms with respect to all fatty acids in the triglyceride,
A triglyceride containing 7,10,13-eicosatrienoic acid having a content of 7,10,13-eicosatrienoic acid of 0.1% by weight or more based on the total fatty acids in the triglyceride,

A triglyceride containing 5,8,11-octadecatrienoic acid having a content of 5,8,11-octadecatrienoic acid of 0.1% by weight or more based on the total fatty acids in the triglyceride,
The triglyceride containing 8,11-octadecadienoic acid having a content of 8,11-octadecadienoic acid of 0.1% by weight or more based on the total fatty acids in the triglyceride, and the carbon number of all fatty acids in the triglyceride A triglyceride containing an n-7 polyunsaturated fatty acid having 18 or more carbon atoms and an n-7 polyunsaturated fatty acid content of 0.3% by weight or more,
As well as compositions containing these lipids or triglycerides.

  In the present invention, n-4 series or n-7 series polyunsaturated fatty acid means 18 or more carbon atoms, two or more double bonds, and methyl at the 4th or 7th carbon from the methyl group end. It is a fatty acid having the first double bond on the base side. Specifically, as an n-4 polyunsaturated fatty acid, 7,10,13,16-eicosatetraenoic acid, 5,8,11,14 -Octadecatetraenoic acid and 8,11,14-octadecatrienoic acid are 7,10,13-eicosatrienoic acid and 5,8,11-octadecateriene as n-7 polyunsaturated fatty acids Acid, and 8,11-octadecadienoic acid.

  In the present invention, any microorganism having an ability to produce arachidonic acid can be used. The microorganisms capable of producing arachidonic acid include the genus Mortierella, Conidiobolus, Pythium, Phytophthora, Penicillium, and Cladosporium. , Belonging to the genus Mucor, Fusarium, Aspergillus, Rhodotorula, Entomophthora, Echinosporangium, Saprolegnia Can do. For microorganisms belonging to the genus Mortierella (Mortierella), for example, Mortierella elongata, Mortierella exigua, Mortierella hygrophila, Mortierella alpina (Mortierella alpina).

  More specifically, Mortierella elongata IFO8570, Mortierella exigua IFO8571, Mortierella hygrophila IFO5941, Mortierella alpina ATCC30 266, ATCC16 Examples include CBS219.35, CBS224.37, CBS250.53, CBS343.66, CBS527.72, CBS529.72, CBS608.70, CBS754.68 and the like.

  All of these strains can be obtained without limitation from Osaka Institute of Fermentation (IFO), American Type Culture Collection (ATCC), or Centrralbureau voor Schimmelcultures (CBS). be able to. In addition, the strain Mortierella elongata SAM0219 (Mikken Kenyoku No. 8703) (Mikken Kenjyo No. 1239) isolated from soil by the research group of the present invention can also be used, but is limited to these strains. I'm not doing it. Although strains belonging to these type cultures or strains isolated from nature can be used as they are, natural mutant strains having different properties from the original strains obtained by performing growth and / or isolation once or more can be used. It can also be used.

  Furthermore, in the present invention, a microorganism in which the fatty acid chain length extension reaction from palmitic acid to stearic acid obtained from a microorganism having an arachidonic acid-producing ability is reduced or lost can be obtained by using, for example, the following microorganism having an arachidonic acid-producing ability. It can be obtained by the mutation treatment described in 1. It can also be obtained by genetic manipulation that reduces or eliminates the activity of the enzyme involved in the fatty acid chain lengthening reaction from palmitic acid to stearic acid in microorganisms having the ability to produce arachidonic acid. Examples thereof include knockout of a gene encoding the enzyme and inactivation by antisense, but are not limited to these methods.

  Furthermore, the present invention provides the above-mentioned arachidonic acid production in order to increase the ratio of n-4 series and / or n-7 series polyunsaturated fatty acids in the lipid or triglyceride as a whole, or to increase the ratio of specific fatty acids. The use of microorganisms obtained by performing mutation treatment or genetic manipulation on microorganisms having the ability to reduce or lack fatty acid chain extension reaction from palmitic acid to stearic acid obtained from the microorganisms is also included. . As an example, a microorganism having a reduced or deficient Δ12 desaturation reaction obtained from a microorganism capable of producing arachidonic acid can be used.

  That is, by culturing the microorganism in a medium to which palmitoleic acid or a derivative thereof, or an oil containing these as a constituent component is added, the ratio of n-4 highly polyunsaturated fatty acids is small or not contained, n-7 A highly unsaturated fatty acid-containing lipid can be obtained. In addition, by using a microorganism in which the fatty acid chain length extension reaction from palmitic acid to stearic acid obtained by further performing mutation treatment or genetic manipulation on a microorganism in which this Δ12 desaturation reaction is reduced or missing is reduced or absent, It is possible to obtain an n-7 polyunsaturated fatty acid-containing lipid in which the ratio of the n-4 polyunsaturated fatty acid is small or not contained.

Further, by using a microorganism with enhanced Δ6 desaturation reaction, it is possible to increase the ratio of n-7 highly unsaturated fatty acid, or by using a microorganism with enhanced Δ12 desaturation reaction. It is possible to increase the ratio of n-4 polyunsaturated fatty acids.
In the present invention, the mutation treatment is performed by irradiation with radiation (X-ray, gamma ray, neutron beam), ultraviolet irradiation, high heat treatment, etc., or suspending microorganisms in an appropriate buffer, etc., adding the mutagen and incubating for a certain period of time. Thereafter, a general mutation operation such as appropriately diluting and inoculating the agar medium to obtain a mutant colony can also be performed.

  Mutagens include nitrogen mustard, methyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine (NTG), alkylating agents, base analogs such as 5-bromouracil, mitomycin C, etc. Examples include antibiotics, base synthesis inhibitors such as 6-mercaptopurine, pigments such as proflavine, certain carcinogens such as 4-nitroquinoline-N-oxide, and compounds such as manganese chloride and formaldehyde. The microorganism used may be a growing cell (mycelium) or a spore.

  For example, as a mutant strain of the present invention, Mortierella alpina SAM 2268 (FERM P-17762) having a reduced fatty acid chain length-inducing activity derived from Mortierella alpina IFO8568 capable of producing arachidonic acid was used. However, the present invention is not limited to this strain, and all strains that have the ability to produce arachidonic acid and have reduced or lacked fatty acid chain length extension activity can be used.

  In order to culture the strain used in the present invention, the spore, mycelium of the strain or a preculture obtained by culturing in advance is inoculated into a liquid medium or a solid medium and cultured. In the case of a liquid medium, commonly used carbon sources such as glucose, fructose, xylose, saccharose, maltose, soluble starch, molasses, glycerol, mannitol, etc. can be used, but are not limited to these. It is not a thing.

  Nitrogen sources include natural nitrogen sources such as peptone, yeast extract, malt extract, meat extract, casamino acid, corn steep liquor, soy protein, defatted soybean, cottonseed residue, etc., organic nitrogen sources such as urea, and sodium nitrate Inorganic nitrogen sources such as ammonium nitrate and ammonium sulfate can be used. In addition, inorganic salts such as phosphate, magnesium sulfate, iron sulfate, and copper sulfate, vitamins, and the like can be used as a trace nutrient source as necessary.

  These medium components are not particularly limited as long as they do not impair the growth of microorganisms. In general, the carbon source should have 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. The initial nitrogen source addition amount is 0.1 to 10% by weight, preferably 0.1 to 6% by weight, and the nitrogen source may be fed during the cultivation.

The culture temperature of the microorganism of the present invention varies depending on the microorganism to be used, but it is 5 to 40 ° C., preferably 10 to 30 ° C., and the pH of the medium is 4 to 10, preferably 5 to 9. Alternatively, static culture is performed. The culture is usually performed for 2 to 30 days, preferably 5 to 20 days, more preferably 5 to 15 days.
When cultivating in solid culture, use bran, rice bran, rice bran, etc. to which 50 to 100% by weight of water has been added based on the weight of the solids, using a temperature of 5 to 40 ° C, preferably 10 to 30 ° C. Incubate for ~ 14 days. In this case, a nitrogen source, inorganic salts, and a trace nutrient source can be added to the medium as necessary.

  In the present invention, a hydrocarbon such as tetradecane and hexadecane, a fatty acid such as tetradecanoic acid and hexadecanoic acid, or a salt thereof (for example, sodium) in order to increase the production amount of n-4 series and / or n-7 series highly unsaturated fatty acid. Salts, potassium salts and the like) and esters, or fats and oils (for example, coconut oil and palm kernel oil) containing the fatty acid as a constituent component can be added as a substrate. When using a microorganism having an ability to produce arachidonic acid, palmitoleic acid or a derivative thereof, or an oil containing these as a constituent component is added as a precursor. The total amount of each of these substrates or precursors is 0.001 to 10% by weight, preferably 0.5 to 10% by weight, based on the medium.

These additives may be added before or immediately after inoculation with the production microorganism, or may be added after the start of culture, or may be added at both time points. The addition after the start of the culture may be performed once, or may be added intermittently divided into a plurality of times. Alternatively, it can be added continuously. Alternatively, these additives may be cultured as the sole carbon source.
In addition, in order to obtain n-4 series and / or n-7 series highly unsaturated fatty acid-containing lipids or triglycerides in a yield that can be commercialized, aeration stirring culture is preferable using a liquid medium, and normal stirring fermentation is used. A tank or a bubble column type culture apparatus can also be used. The aeration rate is preferably 0.1 to 3 vvm, and the stirring speed is preferably 10 to 500 rpm.

  By culturing in this way, lipids containing n-4 and / or n-7 polyunsaturated fatty acids are produced and accumulated in the cells. When a liquid medium is used, a culture solution in the middle of producing lipids by cell culture or a sterilized culture solution thereof, a culture solution at the end of the culture or a sterilized culture solution thereof, or a culture collected from each The n-4 series and / or n-7 series highly unsaturated fatty acid-containing lipid is collected from the body or its dried product. For example, n-4 and / or n-7 highly unsaturated fatty acid-containing lipids are collected from cultured cells as follows, and n-4 and / or n-7 highly unsaturated fatty acids are collected from the lipid. The contained triglycerides or n-4 and / or n-7 polyunsaturated fatty acids are isolated.

  After completion of the culture, cultured cells are obtained from the culture solution by conventional solid-liquid separation means such as centrifugation or filtration. The cells are washed thoroughly 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, dynomill or ultrasonic waves, and then extracted with an organic solvent, preferably under a nitrogen stream. As the organic solvent, ether, hexane, methanol, ethanol, chloroform, dichloromethane, petroleum ether, etc. can be used. Alternatively, alternate extraction of methanol and petroleum ether and extraction using a one-layer solvent of chloroform-methanol-water can also be used. Good results can be obtained. By distilling off the organic solvent from the extract under reduced pressure, a lipid containing an n-4 series and / or an n-7 series highly unsaturated fatty acid is obtained.

Moreover, it can replace with said method and can extract using a wet cell. In this case, a solvent compatible with water such as methanol and ethanol, or a mixed solvent compatible with water composed of these and water and / or other solvents is used. Other procedures are the same as described above.
Among the lipids obtained as described above, n-4 series and / or n-7 series highly unsaturated fatty acids are neutral lipids such as triglyceride, diglyceride, monoglyceride, sterol ester, phosphatidylcholine, Lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylinositol, lysophosphatidylinositol, phosphatidylserine, lysophosphatidylserine, phosphatidic acid, lysophosphatidine It exists as a constituent of polar lipids such as acids or as free fatty acids.

For example, the fat and oil composition of lipids containing n-4 series and / or n-7 series highly unsaturated fatty acids produced using microorganisms belonging to the genus Mortierella are neutral lipids of 70-100% by weight, polar The lipid is 0 to 30% by weight, and the triglyceride which is the main component of the neutral lipid is 70 to 99% by weight. The fatty acid composition of the lipid is based on the total fatty acid in the lipid,
7,10,13,16-eicosatetraenoic acid is 1.5 to 70% by weight, preferably 2.0 to 70% by weight, more preferably 5.0 to 70% by weight,

5,8,11,14-octadecatetraenoic acid is 0.5-60% by weight, preferably 0.6-60% by weight, more preferably 1.0-60% by weight,
8,11,14-octadecatrienoic acid is 0.1 to 60% by weight, preferably 0.5 to 60% by weight, more preferably 0.7 to 60% by weight,
7,10,13-eicosatrienoic acid is 0.1-60% by weight, preferably 0.4-60% by weight, more preferably 0.5-60% by weight,
5,8,11-octadecatrienoic acid is 0.1 to 60% by weight, preferably 0.2 to 60% by weight, more preferably 0.3 to 60% by weight,
8,11-octadecadienoic acid is 0.1-60 wt%, preferably 0.2-60 wt%, more preferably 0.3-60 wt%,

Also, for all fatty acids in triglycerides in lipids,
7,10,13 16-eicosatetraenoic acid is 1.5 to 70% by weight, preferably 4.8 to 70% by weight, more preferably 7.8 to 70% by weight,
5,8,11,14-octadecatetraenoic acid is 0.5-60% by weight, preferably 1.0-60% by weight, more preferably 2.1-60% by weight,

8,11,14-octadecatrienoic acid is 0.1-60% by weight, preferably 0.5-60% by weight, more preferably 0.8-60% by weight,
7, 10, 13-eicosatrienoic acid is 0.1-60 wt%, preferably 0.5-60 wt%, more preferably 1.1-60 wt%,
5,8,11-octadecatrienoic acid is 0.1 to 60% by weight, preferably 0.2 to 60% by weight,
8,11-octadecadienoic acid is 0.1 to 60% by weight, preferably 0.2 to 60% by weight.

In the present invention,
The content of 7,10,13,16-eicosatetraenoic acid is 1.5% by weight or more, preferably 2.0% by weight or more, more preferably 5.0% by weight or more based on the total fatty acids in the lipid. , 16-lipid containing eicosatetraenoic acid,
The content of 5,8,11,14-octadecatetraenoic acid is 0.5% by weight or more, preferably 0.6% by weight or more, more preferably 1.0% by weight or more based on the total fatty acids in the lipid. , 14-octadecatetraenoic acid containing lipids,

8,11,14-octadecataenoic acid having a content of 8,11,14-octadecatrienoic acid of 0.1% by weight or more, preferably 0.5% by weight or more, more preferably 0.7% by weight or more based on the total fatty acids in the lipid Lipids containing trienoic acid,
The content of n-4 highly unsaturated fatty acids having 18 or more carbon atoms with respect to all fatty acids in the lipid is 2.8% by weight or more, preferably 3.5% by weight or more, more preferably 6.8% by weight or more, A lipid containing 18 or more n-4 polyunsaturated fatty acids,
The 7,10,13-eicosatrienoic acid content with respect to the total fatty acids in the lipid is 0.1% by weight or more, preferably 0.4% by weight or more, more preferably 0.5% by weight or more. Lipids containing satrienoic acid,

5,8,11-octadecataic acid having a content of 5,8,11-octadecatrienoic acid of 0.1% by weight or more, preferably 0.2% by weight or more, more preferably 0.3% by weight or more based on the total fatty acids in the lipid Lipids containing trienoic acid,
Contains 8,11-octadecadienoic acid with a content of 8,11-octadecadienoic acid of 0.1% by weight or more, preferably 0.2% by weight or more, more preferably 0.3% by weight or more based on the total fatty acids in the lipid The content of the n-7 polyunsaturated fatty acid having 18 or more carbon atoms with respect to the total fatty acid in the lipid or the lipid is 0.3 wt% or more, preferably 0.9 wt% or more, more preferably 1.2 wt% or more A lipid containing an n-7 polyunsaturated fatty acid having 18 or more carbon atoms,
Can be obtained.

  The above-mentioned lipid means a substance in which a higher fatty acid forms some chemical bond in the molecule, typically an ester bond, and is insoluble in water and soluble in organic solvents such as alcohol, chloroform and benzene. . Examples of lipids containing n-4 and / or n-7 highly unsaturated fatty acids include n-4 and / or n-7 highly unsaturated fatty acids, n-4 and / or n-7. Lower alkyl esters of highly polyunsaturated fatty acids, glycerol esters or sterol esters containing n-4 and / or n-7 highly unsaturated fatty acids as components, and mixtures of any two or more of these Can be mentioned.

  The lower alkyl ester of the n-4 series and / or n-7 series highly unsaturated fatty acid is a C1-C6, preferably 1-4, more preferably 1-3 lower alcohol and n-. Refers to esters of 4- and / or n-7 polyunsaturated fatty acids. Moreover, the glycerol ester which contains n-4 type | system | group and / or n-7 type | system | group highly unsaturated fatty acid as a structural component is at least 1 molecule | numerator n-4 type | system | group and / or n-7 type | system | group high with respect to 1 molecule of glycerol. A substance in which unsaturated fatty acids are ester-bonded.

  Examples include triglycerides, diglycerides, monoglycerides, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylinositol, lysophosphatidylinositol, phosphatidylserine, Examples include lysophosphatidylserine, phosphatidic acid, lysophosphatidic acid, and glyceroglycolipid.

Moreover, the sterol ester which contains n-4 type and / or n-7 type | system | group highly unsaturated fatty acid as a structural component is the substance which the sterol and the n-4 type | system | group and / or n-7 type | system | group highly unsaturated fatty acid ester-bonded. To tell. Examples thereof include cholesterol esters and desmosterol esters.
Lipids containing n-4 and / or n-7 polyunsaturated fatty acids are not limited to the above examples. Sphingophospholipids, other phospholipids, ceramides, glycosphingolipids, other glycolipids, etc. Any lipid included in the definition of

Furthermore, in the present invention,
The content of 7,10,13,16-eicosatetraenoic acid is 1.5% by weight or more, preferably 4.8% by weight or more, more preferably 7.8% by weight or more based on the total fatty acids in the triglycerides in the lipids. , 10,13,16-triglycerides containing eicosatetraenoic acid,
The content of 5,8,11,14-octadecatetraenoic acid is 0.5% by weight or more, preferably 1.0% by weight or more, more preferably 2.1% by weight or more, based on the total fatty acids in the triglyceride, A triglyceride containing 11,14-octadecatetraenoic acid,

The 8,11,14-octadecatrienoic acid content of the total fatty acid in the triglyceride is 0.1% by weight or more, preferably 0.5% by weight or more, more preferably 0.8% by weight or more. Triglycerides containing decatrienoic acid,
The content of n-4 polyunsaturated fatty acids having 18 or more carbon atoms with respect to all fatty acids in triglycerides is 3.0 wt% or more, preferably 6.6 wt% or more, more preferably 10.4 wt% or more. A triglyceride containing the above n-4 polyunsaturated fatty acid,

7,10,13-eicosatrie having a content of 7,10,13-eicosatrienoic acid of 0.1% by weight or more, preferably 0.5% by weight or more, more preferably 1.1% by weight or more based on the total fatty acids in the triglyceride Triglycerides containing enoic acid,
A triglyceride containing 5,8,11-octadecatrienoic acid having a content of 5,8,11-octadecatrienoic acid of 0.1% by weight or more, preferably 0.2% by weight or more based on the total fatty acids in the triglyceride,

A triglyceride containing 8,11-octadecadienoic acid having a content of 8,11-octadecadienoic acid of 0.1% by weight or more, preferably 0.2% by weight or more, based on the total fatty acids in the triglyceride,
The content of n-7 polyunsaturated fatty acid having 18 or more carbon atoms with respect to all fatty acids in the triglyceride is 0.3 wt% or more, preferably 0.9 wt% or more, more preferably 1.5 wt% or more. A triglyceride containing the above n-7 polyunsaturated fatty acid,
Can be obtained.

  To separate and purify triglycerides containing n-4 and / or n-7 highly unsaturated fatty acids from lipids containing n-4 and / or n-7 highly unsaturated fatty acids collected from the culture According to a conventional method, for example, a deoxidation method, a deodorization method, a degumming method, a dehydration method, a steam distillation method, a molecular distillation method, a cooling separation method, a column chromatography method or the like is performed. For example, a lipid containing n-4 and / or n-7 polyunsaturated fatty acids is extracted from the culture using hexane according to the above-described operation, and purification such as deoxidation, deodorization, degumming and the like is performed from the extracted oil. By the treatment, triglycerides containing n-4 and / or n-7 polyunsaturated fatty acids can be obtained.

To separate n-4 series and / or n-7 series highly unsaturated fatty acids from lipids containing n-4 series and / or n-7 series highly unsaturated fatty acids, in the state of mixed fatty acid or mixed fatty acid ester In the usual manner, for example, it is carried out by concentration and separation by the urea addition method, the cooling separation method, the column chromatography method or the like.
More specifically, n-4 series and / or n-7 series highly unsaturated fatty acids can be directly separated, but esters with lower alcohols such as n-4 series and / or n-7 series highly unsaturated. It is preferably separated as the fatty acid ethyl ester.

  By making such an ester, it can be easily separated from other lipid components, and other fatty acids produced during cultivation, such as palmitic acid (also these are n-4 series and / or n- Can be easily separated from the 7-series highly unsaturated fatty acid. For example, in order to obtain ethyl ester of highly unsaturated fatty acid, it is preferable to treat the extracted lipid with anhydrous ethanol-hydrochloric acid 5-10%, BF3-ethanol 10-50%, etc. at room temperature for 1-24 hours. .

  In order to recover the ethyl ester of the n-4 series and / or n-7 series highly unsaturated fatty acid from the treatment liquid, it is preferable to extract with an organic solvent such as hexane, ether, ethyl acetate or the like. Next, this extract is dried with anhydrous sodium sulfate or the like, and the organic solvent is preferably distilled off under reduced pressure to obtain a mixture containing a fatty acid ester as a main component. This mixture contains fatty acid ethyl esters such as palmitic acid ethyl ester in addition to the desired n-4 and / or n-7 highly unsaturated fatty acid ethyl ester.

  This mixture can be used in the composition of the present invention as it is or after increasing the concentration of the fatty acid of the present invention. To isolate ethyl esters of n-4 and / or n-7 polyunsaturated fatty acids from these fatty acid ethyl ester mixtures, column chromatography, low temperature crystallization, urea inclusion, liquid-liquid exchange Chromatography and the like can be used alone or in combination.

  In order to obtain free n-4 and / or n-7 polyunsaturated fatty acid from the ethyl ester of n-4 and / or n-7 polyunsaturated fatty acid thus isolated, hydrolysis with alkali is required. Then, extraction with an organic solvent such as ether or ethyl acetate may be performed. In addition, in order to take n-4 series and / or n-7 series highly unsaturated fatty acid without passing through its ethyl ester, the extracted lipid is subjected to alkaline decomposition (for example, 5% sodium hydroxide at room temperature). After 3 hours), the decomposition solution can be extracted and purified by a method commonly used for extraction and purification of fatty acids. The resulting free fatty acids of the invention and their salts can also be used for the compositions of the invention.

  The lipid or triglyceride containing the n-4 series and / or n-7 series highly unsaturated fatty acid of the present invention is an n-4 series and / or an n-7 series having 18 or more carbon atoms and 2 or more double bonds. It is rich in polyunsaturated fatty acids and has unlimited possibilities for its use and can be used as a raw material and additive for foods, beverages, cosmetics, pharmaceuticals, animal feeds and the like. For example, general foods, beverages, functional foods, dietary supplements, formulas, lotions, emulsions, enteral nutrients, powders, granules, troches, syrups, tablets, capsules, infusions, injections, gels for application, Examples thereof include compresses, powdered feeds, solid feeds, liquid feeds, and the like, but are not limited thereto, and there are no restrictions on the purpose of use, the amount used, and the form of processing.

Next, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the examples.
Example 1. Mortierella alpina acquisition Mortierella alpina strains fatty-acid-chain elongase activity from palmitic acid to stearic acid was reduced by mutagenesis of IFO8568 (Mortierella alpina) IFO8568 a Czapek agar medium _{(0.2% NaNO 3, 0.1%} K 2 HPO ₄ , 0.05% MgSO ⁴ · 7H ₂ O, 0.05% KCl, 0.01% FeSO ₄ · 7H ₂ O, 3% sucrose, 2% agar, pH6.0) Inoculated into a large slant bottle containing 300mL, 28 Culturing was carried out at 2 ° C for 2 weeks.

After culturing, 50 mL of sterilized water was added, shaken, filtered through quadruple gauze, centrifuged at 8,000 xg for 10 minutes, and then suspended in 50 mM Tris buffer solution (pH 7.5) to prepare a spore suspension. . Add 0.5 mL of 0.5% NTG (N-methyl-N′-nitro-N-nitrosoguanidine) solution to 1.5 mL of 1 × 10 ⁶ / mL spore suspension and perform mutation treatment at 28 ° C. for 15 minutes. It was. After adding ₃ mL of 10% Na ₂ S ₂ O ₃ and centrifuging at 5,500 × g for 10 minutes, it was washed with sterilized water to obtain an NTG-treated spore suspension.

  Apply NTG-treated spore suspension to GY agar medium (1% glucose, 0.5% yeast extract, 0.005% Triton X-100, 1.5% agar, pH 6.0), and separate colonies grown at 28 ° C. Transferred to GY agar medium. A portion of the grown cells are dried, and in accordance with a conventional method, the fatty acid in the cells is methyl esterified with hydrochloric acid methanol, then extracted with hexane, and the hexane is distilled off to obtain the fatty acid methyl ester obtained by gas chromatography. Analyzed with As a result of examining about 1,000 colonies, a strain SAM 2268 (FERM P-17762) in which the activity of extending the fatty acid chain length from palmitic acid to stearic acid was reduced was obtained.

Example 2 Method for producing n-4 and n-7 polyunsaturated fatty acids by microorganisms capable of producing arachidonic acid 2 mL of liquid medium containing one platinum loop of microorganism having the ability to produce arachidonic acid in a 10 mL Erlenmeyer flask (2% glucose, 1% yeast extract, 1% palmitoleic acid or none) was inoculated and cultured with shaking at 28 ° C. and 120 rpm for 7 days. After cultivation, the cells were collected by filtration and dried. According to a conventional method, the fatty acid in the microbial cells was 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. The results are shown in Table 1.

(1) Mortierella alpina IFO8568
(2) Mortierella hygrophila IFO5941
(3) Echinosporangium transversale NRRL3116
(4) Saprolegnia lapponica CBS313.81

Example 3 Effect of palmitoleic acid derivative 2 mL of liquid medium (2% glucose, 1% yeast extract, palmitoleic acid derivative 1 shown in Table 2) in which a platinum loop of Mortierella alpina IFO8568 is placed in a 10 mL Erlenmeyer flask %) And cultured with shaking at 28 rpm at 120 rpm for 7 days. After the culture, methyl esterification was performed in the same manner as in Example 2, and the obtained fatty acid methyl ester was analyzed by gas chromatography. The results are shown in Table 2.

Example 4 Production method using a reduced fatty acid chain extension activity Morterella alpina SAM 2268 (FERM P-17762) one platinum loop in a 10 mL Erlenmeyer flask 2 mL of liquid medium (glucose 4%, yeast extract 1%) and cultured with shaking at 28 ° C. at 120 rpm for 6, 10, 12, and 14 days. After the culture, methyl esterification was performed in the same manner as in Example 2, and the obtained fatty acid methyl ester was analyzed by gas chromatography. In addition, other cells cultured for 6, 10, 12 and 14 days under the same conditions were collected, dried, and then extracted with a chloroform-methanol extraction method.

  Further, according to a conventional method, the extracted lipid was fractionated by thin layer chromatography and silicic acid column chromatography to obtain triglyceride. The obtained triglyceride was methyl esterified by the above method and analyzed by gas chromatography. The changes with time are shown in Tables 3 and 4. The composition of the lipid extracted from the cells cultured for 14 days was 90% by weight of neutral lipid (triglyceride was 89% by weight of the extracted lipid) and 10% by weight of polar lipid.

Example 5 FIG. Effect of addition of substrate 2 mL of liquid medium (2% glucose, 1% yeast extract, 1%) in a 10 mL Erlenmeyer flask with one platinum loop of Mortierella alpina SAM 2268 (FERM P-17762) The indicated compound (1%) was inoculated and cultured with shaking at 28 rpm at 120 rpm for 7 days. After the culture, methyl esterification was performed in the same manner as in Example 2, and the obtained fatty acid methyl ester was analyzed by gas chromatography. The results are shown in Table 5.

Example 6 Method for producing lipid containing n-7 polyunsaturated fatty acid and not containing n-4 polyunsaturated fatty acid Mortierella alpina IFO8568 or Mortierella alpina deficient in Δ12 desaturation reaction (Mortierella alpina) Inoculate one platinum loop of SAM 1861 (No. 3590, Kikuken Kenjo No. 3590) into 2 mL of liquid medium (glucose 5%, yeast extract 1%, palmitoleic acid 1%) in a 10 mL Erlenmeyer flask. The culture was shaken at 12 to 28 ° C. and 120 rpm for 7 days. After the culture, methyl esterification was performed in the same manner as in Example 2, and the obtained fatty acid methyl ester was analyzed by gas chromatography. The results are shown in Table 6.

  When the SAM 1861 strain, which is a Δ12 desaturation-deficient strain, is used, no conversion from the n-7 system to the n-4 system occurs, so the lipid obtained from this strain contains n-7 highly unsaturated fatty acids. Was included, but n-4 polyunsaturated fatty acids were not included.

Example 7 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 20000 cps. Next, after crushing the dried microbial cells obtained by the method described in Example 3, hexane extracted oil extracted with hexane or triglyceride obtained by deoxidizing, deodorizing and degumming this was added 3 wt% vitamin E oil. The contents were prepared by mixing. Using these, capsules were molded and dried according to a conventional method to produce soft capsules containing 180 mg of content per grain.

Claims (7)

  An n-4 system characterized by culturing a microorganism having a reduced or deficient activity for extending the fatty acid chain from palmitic acid to stearic acid, obtained from a microorganism having an ability to produce arachidonic acid, and collecting lipids from the culture And / or a method for producing a lipid containing an n-7 polyunsaturated fatty acid.   Cultivating microorganisms with reduced or lacking fatty acid chain extension activity from palmitic acid to stearic acid obtained from microorganisms capable of producing arachidonic acid, collecting lipids from the culture, and separating and purifying triglycerides from the lipids The manufacturing method of the triglyceride containing n-4 type | system | group and / or n-7 type | system | group highly unsaturated fatty acid characterized by the above-mentioned.   Culturing a microorganism having a reduced or deficient activity of extending the fatty acid chain from palmitic acid to stearic acid obtained from a microorganism capable of producing arachidonic acid, collecting lipid from the culture, A method for producing n-4 series and / or n-7 series highly unsaturated fat, comprising separating and purifying n-7 series highly unsaturated fat.   n-4 and / or n-7 polyunsaturated fatty acids are 7,10,13,16-eicosatetraenoic acid, 5,8,11,14-octadecatetraenoic acid, 8,11,14 -At least one selected from octadecatrienoic acid, 7,10,13-eicosatrienoic acid, 5,8,11-octadecatrienoic acid, and 8,11-octadecadienoic acid The manufacturing method of any one of Claims 1-3 to do.   Microorganisms capable of producing arachidonic acid include the genus Mortierella, the genus Conidiobolus, the genus Pythium, the genus Phytophthora, the genus Penicillium, the genus Cladosporium, A microorganism belonging to the genus Mucor, Fusarium, Aspergillus, Rhodotorula, Entomophthora, Echinosporangium, or Saprolegnia The manufacturing method of any one of Claims 1-4 characterized by these.   The method according to any one of claims 1 to 5, wherein the microorganism having the ability to produce arachidonic acid is a microorganism belonging to the genus Mortierella genus Mortierella.   The method according to any one of claims 1 to 6, wherein the microorganism having the ability to produce arachidonic acid is a microorganism belonging to the species Mortierella alpina.