JP2007089522A - Method for producing fatty acid composition containing specific highly unsaturated fatty acid in concentrated state - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for producing a fatty acid composition containing a specific highly unsaturated fatty acid in concentrated state. <P>SOLUTION: The fatty acid composition is produced by removing saturated fatty acids by a urea inclusion method, specifically esterifying and removing unsaturated fatty acids other than the objective specific highly unsaturated fatty acid with a lipase, and repeating the specific esterification step twice. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing a fatty acid composition in which a specific highly unsaturated fatty acid is concentrated, and a fatty acid composition produced by this method.

  Eicosapentaenoic acid (hereinafter referred to as “EPA”) and docosahexaenoic acid (hereinafter referred to as “DHA”), which are highly unsaturated fatty acids, are particularly effective in preventing and anti-cancer and learning for adult diseases such as arteriosclerosis and thrombosis. It is known that it has many physiological functions, such as a potentiating action, and various attempts have been made for use in pharmaceuticals and foods for specified health use. Recently, however, the physiological functions of these other polyunsaturated fatty acids have attracted attention.

  Arachidonic acid, a type of polyunsaturated fatty acid, accounts for about 10% of fatty acids that make up vital organs such as blood and liver (for example, arachidone is a fatty acid composition ratio in phospholipids of human blood). 11% for acid, 1% for eicosapentaenoic acid and 3% for docosahexaenoic acid). It is involved in the regulation of membrane fluidity as a major component of cell membrane, and exhibits various functions in metabolism in the body, while prostaglandins Plays an important role as a direct precursor. Particularly recently, it has been attracting attention as a constituent fatty acid of endogenous cannabinoids (2-arachidonoyl monoglycerol, anandamide) that exhibit a role of arachidonic acid as infant nutrition and a neuroactive activity. Normally, when foods rich in linoleic acid are ingested, it is converted to arachidonic acid, but in ill patients and their reserves, infants and the elderly, the activities of enzymes involved in biosynthesis decrease, and these arachidonic acids tend to be deficient. Therefore, it is desirable to take it directly as fats and oils (triglyceride constituent fatty acids).

  EPA and DHA have abundant sources of fish oil, but dihomo-γ-linolenic acid (DGLA), arachidonic acid (ARA) and 5,8,11-eicosapentaenoic acid (20: 3 n-9) Can hardly be obtained from conventional oil sources, and at present, fats and oils (triglycerides) comprising polyunsaturated fatty acids obtained by fermenting microorganisms as constituent fatty acids are generally used. For example, a method has been proposed in which various microorganisms capable of producing fats and oils (triglycerides) containing arachidonic acid as a constituent fatty acid are cultured to obtain fats and oils (triglycerides) containing arachidonic acid as a constituent fatty acid. Of these, it is known that oils and fats (triglycerides) with a high content of arachidonic acid can be obtained by using microorganisms belonging to the genus Mortierella (JP-A 63-44891, JP-A 63-12290). Furthermore, a technique for producing dihomo-γ-linolenic acid, arachidonic acid and 5,8,11-eicosapentaenoic acid (20: 3 n-9) using the same mutant of the microorganism belonging to the genus Mortierella is disclosed ( JP-A-5-91887, JP-A-5-91888).

  Up to now, polyunsaturated fatty acids have shown various effects, but even for fats and oils made with microorganisms, 40% of highly unsaturated fatty acids such as arachidonic acid contained in these fats and oils (triglycerides). Degree. In order to spread this usefulness, it is necessary to increase the concentration of highly unsaturated fatty acids so that they can be used in small amounts. For this reason, highly pure highly unsaturated fatty acids are expected.

  As a means of industrially increasing the purity of these highly unsaturated fatty acids, a method using large-scale high performance liquid chromatography (HPLC) (Y. Yamamura, et.al., J. Am. Oil Chem. Soc., 74 , 1435 (1997)) and a method using a complex formation with silver (Yamaguchi et al., Oil Chemistry 40, 959 (1991)) has been devised. However, it has not been adopted as an industrial purification method from the viewpoint of production cost. Since polyunsaturated fatty acids are very unstable to heat and oxidation, the use of enzymes (lipases) that react under mild conditions is also attracting attention. 90% of tuna oil is a combination of two lipases. Of DHA (Y. Shimada, et.al., J. Am. Oil Chem. Soc., 74, 1441 (1997)) from borage oil to 98% pure γ-linolenic acid (18: 3 n-6) (Y. Shimada, et.al., J. Am. Oil Chem. Soc., 72, 1323 (1995); Y. Shimada, et.al., J. Am. Oil Chem. Soc., 75, 1581 ( 1998)). As for arachidonic acid, Shimada et al. (Shimada et al. Science and Industry 73, 125-130 (1999)) are examining non-selective hydrolysis of lipase and selective esterification of lipase. As a result, n-6 polyunsaturated fatty acids were concentrated and purified to 96%, but arachidonic acid remained at a purity of 81%. This is because other n-6 polyunsaturated fatty acids other than arachidonic acid mixed in the raw oil and fat were concentrated at the same time. Some n-6 highly unsaturated fatty acids and n-9 highly unsaturated fatty acids have not yet been studied for a method of concentrating only one kind of highly unsaturated fatty acid.

Japanese Patent Laid-Open No. 5-91887 Japanese Patent Laid-Open No. 5-91888 Y. Yamamura, et.al., J. Am. Oil Chem. Soc., 74, 1435 (1997) Yamaguchi et al., Oil Chemistry 40, 959 (1991) Y. Shimada, et.al., J. Am. Oil Chem. Soc., 74, 1441 (1997) Y. Shimada, et.al., J. Am. Oil Chem. Soc., 72, 1323 (1995) Y. Shimada, et.al., J. Am. Oil Chem. Soc., 75, 1581 (1998) Shimada et al. Science and Industry 73, 125-130 (1999)

Thus, sufficient examination has not yet been made about a method in which a single type of highly unsaturated fatty acid has high purity.
On the other hand, in the market, there is a demand for fats and oils containing a single type of highly unsaturated fatty acid of high purity in order to reduce the amount used, avoid mixing of extra fatty acids, and design a compact product. That is, the examination of the method of making a single kind of highly unsaturated fatty acid highly pure, and the fats and oils containing the highly unsaturated fatty acid obtained by the method have been desired.

In order to realize such a compact product design, the present invention selects a lipase and highly purifies highly unsaturated fatty acid typified by arachidonic acid by a combination of lipase reactions, and a composition containing the same And foods and health foods containing them.
In order to obtain a fatty acid composition containing a single type of highly unsaturated fatty acid with high purity, for example, an oil and fat produced by fungi and containing 20% or more of a highly unsaturated fatty acid containing 20 or more carbon atoms and 2 or more double bonds (Triglyceride) was used as a raw material, and it could be obtained by combining several stages of reaction, focusing on the esterification reaction of lipase with strict selectivity.

  In other words, it is important to determine the reactivity of lipase to fatty acids, and it is mainly to select lipases that have a large difference in reactivity between fatty acids that are mixed and removed and fatty acids that are to be concentrated. The production method of fats and oils containing a single highly polyunsaturated fatty acid with high purity was completed by combining triglyceride degradation with non-specific lipase and urea inclusion method.

  Therefore, the present invention can be obtained by combining several stages of reaction, focusing mainly on esterification of lipase with strict selectivity, using, for example, fats and oils (triglycerides) produced by microorganisms (particularly fungi) as raw materials. Provided are a method for producing fats and oils containing a single type of highly unsaturated fatty acid having a purity, and a composition containing these fats and oils.

Therefore, the present invention provides a method for producing a fatty acid composition in which the ratio of a specific highly unsaturated fatty acid is 50% or more,
(1) A lipase (specific lipase) having a specificity for the specific polyunsaturated fatty acid is lower than that for the other polyunsaturated fatty acid, and the specific polyunsaturated fatty acid in the presence of a fatty alcohol. Selectively esterifying fatty acids other than the specific polyunsaturated fatty acids by acting on the fatty acid composition comprising;
(2) removing the ester produced in the step (1) to obtain a fatty acid composition enriched with the specific polyunsaturated fatty acid; and (3) the same lipase as the lipase used in the step (1). Or go through different lipases and repeat steps (1) and (2) above;
A method comprising the above is provided.

In the above method, the fatty acid composition used in the step (1) is, for example, the following step:
(A) preparing a fatty acid composition containing the specific highly unsaturated fatty acid;
(B) A saturated fatty acid is removed by the urea inclusion method to obtain a fatty acid composition enriched with highly unsaturated fatty acids.

  In the above method, the fatty acid composition as a raw material in the step (a) is obtained by lipase decomposition or saponification of an ester containing the specific highly unsaturated fatty acid. The ester is, for example, a glyceride selected from the group consisting of monoglycerides, diglycerides and triglycerides containing the specific highly unsaturated fatty acid, and is typically triglyceride. The lipase used in the step (a) is, for example, a lipase with little difference in substrate specificity between the specific polyunsaturated fatty acid and other fatty acids.

The fatty alcohol used for the specific esterification is preferably a fatty alcohol having 8 to 16 carbon atoms, such as lauryl alcohol having 12 carbon atoms.
The specific highly unsaturated fatty acid is, for example, an n-6 highly unsaturated fatty acid such as arachidonic acid, dihomo-γ-linolenic acid; an n-9 highly unsaturated fatty acid such as mead acid.

  The specific polyunsaturated fatty acid is, for example, arachidonic acid. In this case, the specific lipase used in the step (3) is derived from Burkholderia cepacia (formerly Pseudomonas sp.). ) Lipase. The ratio of arachidonic acid in the obtained highly unsaturated fatty acid composition is 85% or more, preferably 90% or more, and more preferably 95% or more.

  In another example, the specific polyunsaturated fatty acid is dihomo-γ-linolenic acid, in which case the specific lipase used in step (1) is a lipase derived from Pseudomonas aeruginosa and It is a lipase derived from Candida rugosa. The ratio of dihomo-γ-linolenic acid in the obtained highly unsaturated fatty acid composition is 50% or more, preferably 80% or more, and more preferably 89% or more.

  In yet another example, the specific polyunsaturated fatty acid is mead acid, and in this case, the specific lipase used in the step (1) is derived from Burkholderia cepacia (Pseudomonas space in the old classification). Lipase. The proportion of mead in the obtained highly unsaturated fatty acid composition is 50% or more, preferably 80% or more, more preferably 90% or more.

  The present invention further provides an arachidonic acid-containing fatty acid composition, dihomo-γ-linolenic acid-containing fatty acid composition, and mead acid-containing fatty acid composition obtained by the above method. The present invention further provides foods comprising the fatty acid composition, such as health foods and supplements.

In the present invention, polyunsaturated fatty acids are fatty acids having 18 to 24 carbon atoms and having two or more double bonds, preferably 20 to 22 carbon atoms, It means a fatty acid having 3 to 4 double bonds.
The fatty acid composition containing a specific (target) highly unsaturated fatty acid used as a starting material in the method of the present invention can be obtained, for example, by saponification or lipase decomposition of a fatty acid ester. The fatty acid ester is practically a glyceride, a monoglyceride, a diglyceride or a triglyceride, and most practically a natural fat / oil mainly composed of triglyceride.

Fats and oils (triglycerides) produced by microorganisms as raw oils and fats
A microorganism capable of producing oils and fats (triglycerides) comprising highly unsaturated fatty acids as constituent fatty acids is cultured. The microorganism here is mainly composed of at least one highly unsaturated fatty acid of n-6 series, n-9 series or n-3 series, which has 20 or more carbon atoms and 2 or more double bonds. In particular, microorganisms that produce triglycerides as constituent fatty acids are desirable.

  Further, as an n-6 series, n-9 series or n-3 series highly unsaturated fatty acid having 20 or more carbon atoms and two or more double bonds, dihomo-γ-linolenic acid (DGLA; 8, 11, 14-eicosatrienoic acid), arachidonic acid (ARA; 5,8,11,14-eicosatetraenoic acid), 7,10,13,16-docosatetraenoic acid (22: 4n-6), DPAω6 ( 4,7,10,13,16-docosapentaenoic acid), 8,11-eicosadienoic acid, mead acid (5,8,11-eicosatrienoic acid), 8,11,14,17-eicosatetraene Acid (20: 4 n-3), EPA (5,8,11,14,17-eicosapentaenoic acid), DPAn-3 (7,10,13,16,19-docosapentaenoic acid), and DHA ( 4,7,10,13,16,19-docosahexaenoic acid).

Therefore, in the present invention, any microorganism can be used as long as it can produce fats and oils (triglycerides) composed of highly unsaturated fatty acids as constituent fatty acids. For example, as the microorganism capable of producing fats and oils comprising arachidonic acid as a constituent fatty acid (triglyceride), Mortierella (Mortierella) genus, Koni audio bolus (Conidiobolus) genus Fichiumu (Pythium) genus Phytophthora (Phytophthora) genus, Penishiryumu (Penicillium) genus, Clos de sports Liu beam (Cladosporium) genus, Mucor (Mucor) genus, Fuzaryumu (Fusarium) genus Aspergillus (Aspergillus) genus, Rhodotorula (Rhodotorula) genus, Entomofutora (Entomophthora) genus, echinochrome aminocephalosporanic indium (Echinosporangium) genus And microorganisms belonging to the genus Saprolegnia .

In the Mortierella (Mortierella) belonging to the genus Mortierella (Mortierella) microorganisms belonging to the subgenus, for example Mortierella elongata (Mortierella elongata), Mortierella exigua (Mortierella exigua), Mortierella Figurofira (Mortierella hygrophila), Mortierella alpina ( Mortierella alpina ). More specifically, Mortierella elongata IFO8570, Mortierella exigua IFO8571, Mortierella hygrophila IFO5941, Mortierella alpina 221266266, CC41, 266266CC Examples include CBS219.35, CBS224.37, CBS250.53, CBS343.66, CBS527.72, CBS529.72, CBS608.70, CBS754.68 and the like.

For example, a microorganism capable of producing DHA, Crypthecodinium Kode hexafluorophosphate (Crypthecodenium) genus scan Lau Toki thorium (Thrautochytrium) genus Schizochytrium (Schizochytrium) genus Ulkenia (Ulkenia) genus, Japo eaves thorium (Japonochytrium) genus or needles Photo Squirrel Mention may also be made of microorganisms belonging to the genus ( Haliphthoros ).

  All of these strains are obtained from Osaka City Foundation for Fermentation (IFO), American Type Culture Collection (ATCC), and Centrralbureau voor Schimmelcultures (CBS) without any restrictions. can do. 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.

  In order to culture the strain used in the present invention, a spore, mycelium of the strain, or a seed culture solution obtained by culturing in advance or a cell recovered from the seed culture is inoculated into a liquid medium or a solid medium. Main culture. In the case of a liquid medium, as carbon sources, generally used ones such as glucose, fructose, xylose, saccharose, maltose, soluble starch, molasses, glycerol, mannitol, saccharified starch, etc. can be used. It is not limited to.

  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, particularly nitrogen sources obtained from soybeans, specifically soybeans, defatted soybeans, soybean flakes, edible soybean proteins, okara, soy milk, kinako flour, etc. However, especially those obtained by subjecting defatted soybeans to heat denaturation, more preferably heat treatment of defatted soybeans at about 70 to 90 ° C. and further removal of ethanol-soluble components, alone or in combination, or in combination with the nitrogen source Can be used.

  In addition to phosphate ions, potassium ions, sodium ions, magnesium ions, and calcium ions, metal ions such as iron, copper, zinc, manganese, nickel, and cobalt, vitamins, etc. can be used as trace nutrient sources as needed. . These medium components are not particularly limited as long as they do not impair the growth of microorganisms. In practice, the total amount of carbon source is generally 0.1 to 40% by weight, preferably 1 to 25% by weight, and the total amount of nitrogen source is 2 to 15% by weight, preferably 2 to 10% by weight. More preferably, the initial carbon source addition amount is 1 to 5% by weight, the initial nitrogen source addition amount is 3 to 8% by weight, and the carbon source and nitrogen source are flowed during the cultivation, and still more preferably only the carbon source is allowed to flow. And incubate.

  In order to increase the yield of unsaturated fatty acid, as a precursor of unsaturated fatty acid, for example, a hydrocarbon such as hexadecane or octadecane; a fatty acid such as oleic acid or linoleic acid or a salt thereof; Ethyl esters, glycerin fatty acid esters, sorbitan fatty acid esters; or oils and fats such as olive oil, soybean oil, rapeseed oil, cottonseed oil or coconut oil can be used alone or in combination. The amount of the substrate added is 0.001 to 10%, preferably 0.5 to 10%, based on the medium. These substrates may be cultured as the sole carbon source.

  The culture temperature of microorganisms producing highly unsaturated fatty acids varies depending on the microorganisms used, but is 5-40 ° C, preferably 20-30 ° C, and after culturing at 20-30 ° C to grow the cells. Unsaturated fatty acids can also be produced by continuing the culture at 5 to 20 ° C. Such a temperature control can also increase the proportion of highly unsaturated fatty acids in the produced fatty acids. The pH of the medium is 4 to 10, preferably 5 to 9, and aeration stirring culture, shaking culture, solid culture, or stationary liquid culture is performed. The main culture is usually performed for 2 to 30 days, preferably 5 to 20 days, more preferably 5 to 15 days.

  Although microorganisms belonging to the genus Mortierella subgenus Mortierella are known as microorganisms capable of producing fats and oils (triglycerides) composed mainly of arachidonic acid, the present inventors apply mutation treatment to the above strains. , A microorganism capable of producing a fat (triglyceride) comprising dihomo-γ-linolenic acid as a main constituent fatty acid (Japanese Patent Laid-Open No. 5-91887), and a fat (triglyceride) comprising a n-9 polyunsaturated fatty acid as a main constituent fatty acid Has been obtained (JP-A-5-91888). Furthermore, microorganisms (WO 98/39468) having resistance to a high concentration of carbon source have also been obtained, and these microorganisms are microorganisms of the genus Mortierella. However, the present invention is not limited to microorganisms belonging to the genus Mortierella genus Mortierella, and microorganisms capable of producing fats and oils (triglycerides) comprising highly unsaturated fatty acids as constituent fatty acids can be used.

  As a method for obtaining crude oil from microorganisms cultured as described above, after culturing, the culture solution is left as it is or after treatment such as sterilization, concentration, and acidification, and then natural sedimentation, centrifugation, and / or filtration, etc. The cultured cells are obtained by conventional solid-liquid separation means. In order to assist solid-liquid separation, a flocculant or a filter aid may be added. As the flocculant, for example, aluminum chloride, calcium chloride, algin, chitosan and the like can be used. As a filter aid, for example, diatomaceous earth can be used. The cultured cells are preferably washed, crushed and dried. Drying can be performed by freeze drying, air drying, fluidized bed drying, freeze drying, or the like.

  As a means for obtaining crude oil from dried cells, an extraction method with an organic solvent or a pressing method can be used, but extraction with an organic solvent is preferably performed under a nitrogen stream. As the organic solvent, ethanol, hexane, methanol, ethanol, chloroform, dichloromethane, petroleum ether, acetone, or the like can be used. Alternate extraction of methanol and petroleum ether or a single solvent of chloroform-methanol-water can also be used. it can. However, the extraction method used for obtaining the crude oil is not limited to the above-described method, and all methods for efficiently extracting the fats and oils in the cells can be used. For example, a supercritical extraction method or the like can also be used as an effective means.

The target crude oil can be obtained by removing the organic solvent and the supercritical fluid component from the extract extracted with the organic solvent and the supercritical fluid under conditions such as reduced pressure. Moreover, it can replace with said method and can extract using a wet microbial cell. In this case, a solvent compatible with water, such as methanol, ethanol, acetone, or the like, 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.
Moreover, not only crude oil but refined fats and oils can also be used as a substrate for transesterification. Conventional steps such as degumming, deoxidation, deodorization, decolorization, column treatment, molecular distillation, and wintering can be used as the oil refining step.

Preparation of fatty acid composition raw material from glyceride To prepare a fatty acid composition raw material from glycerides such as triglyceride, a method such as saponification or lipase decomposition may be used.
(A) Saponification Saponification may be performed according to a conventional method using an inorganic base such as sodium hydroxide.

(B) Lipase decomposition method The lipase used in this step can be used without any particular problem as long as it is an enzyme that efficiently decomposes fats and oils containing a large amount of highly unsaturated fatty acids and efficiently generates free fatty acids. The conditions for the hydrolysis reaction with lipase are as follows. The amount ratio of water and raw oil (TG) to be added to the reaction should be 1/2 or more of the raw oil and fat, more preferably between the equivalent amount and 5 times the amount, further preferably from the equivalent amount to 2 Double amount is good. The temperature for hydrolysis may be any temperature at which the lipase used can act. For example, the temperature range is 15 to 80 ° C. More preferably, it is 25-60 degreeC, More preferably, 25-50 degreeC is good. The amount of enzyme to be added is examined in advance, and may be any amount that can release the target fatty acid.

Removal of saturated fatty acids by the urea inclusion method The fatty acid composition raw material prepared as described above contains saturated fatty acids in addition to unsaturated fatty acids including specific unsaturated polyunsaturated fatty acids. Prior to concentrating the polyunsaturated fatty acid of interest for a particular purpose, the saturated fatty acid is removed. The method for this is not particularly limited. For example, the urea inclusion method (H. Traitler, et.al., J. Am. Oil Chem. Soc. 75, 755 (1998)). In this case, the solvent used is preferably an alcohol (ethyl alcohol, methyl alcohol, propanol, etc.).

Concentration of a specific highly unsaturated fatty acid of interest In order to concentrate a specific highly unsaturated fatty acid of interest, the unsaturated fatty acid composition raw material prepared as described above is used in the present invention. Of the fatty acids, unsaturated fatty acids other than the specific polyunsaturated fatty acid are selectively esterified, and the produced specific polyunsaturated fatty acids are liberated (unesterified) by removing the produced ester. Concentrate as fatty acid.

(A) Esterification substrate In addition to the fatty acid, alcohol is required as the esterification substrate. A fatty alcohol is used as this alcohol, preferably a fatty alcohol having 8 to 16 carbon atoms. Lauryl alcohol having 12 carbon atoms is preferred. In order to carry out the esterification in high yields with respect to fatty acids, it is necessary to use one or more molar amounts of alcohol relative to the fatty acid, for example twice the amount of fatty alcohol relative to the total fatty acid molar amount, such as lauryl alcohol Is used.

(B) Selection of specific lipases Lipids derived from Candida genus microorganisms, especially Candida rugosa, which are considered to have relatively high selectivity, have 18 carbon atoms or less and two double bonds. Since it specifically acts on the following fatty acids, this lipase cannot be used when the number of carbon atoms of the fatty acid to be removed is 20 or more and when the number of double bonds is 3 or more. Cannot be used in
Therefore, the present inventor screened a selective lipase in order to efficiently perform selective esterification of the lipase.

  That is, one of various available known food processing lipases is allowed to act on any one of various fatty acids and 2-fold molar amount of lauryl alcohol with respect to the fatty acid, and the residual fatty acid after the reaction for 2 hours. The composition was calculated, and the enzyme activity for oleic acid was taken as 100, and the relative value of enzyme activity for each fatty acid was determined. The lipases used were as follows:

(1) Lipase (GLM) derived from Alcaligenes sp.
(2) Lipase (PS) derived from Burkholderia cepacia (formerly Pseudomonas sp.) (Manufactured by Amano Enzyme)
(3) Pseudomonas aeruginisa derived lipase (LPL) (manufactured by TOYOBO)
(4) Lipase (TL) derived from Pseudomonas stutzeri (manufactured by Meito Sangyo Co., Ltd.)
(5) Lipase (SL) derived from Burkholderia cepacia (manufactured by Meito Sangyo Co., Ltd.)
(6) Lipase derived from Rhizopus oryzae (Talipase) (manufactured by Amano Enzyme)
(7) Candida rugosa-derived lipase (OF) (manufactured by Meito Sangyo Co., Ltd.)

The results are shown in Table 1 below.

  When the fatty acid composition of the fats and oils produced by the raw material microorganisms is measured in advance and it is clarified which fatty acid is desired to be removed, it is decided which kind of lipase from the lipases shown in the above table should be used. For example, microbial fats and oils containing a high amount of arachidonic acid contain DGLA and GLA in addition to arachidonic acid. The Candida rugosa lipase mentioned above has almost the same reactivity with arachidonic acid and DGLA and GLA, and DGLA and GLA cannot be separated from arachidonic acid by this reaction.

  On the other hand, the reactivity of lipase derived from Burkholderia cepacia (former name: Pseudomonas sp.) Is significantly different between arachidonic acid and DGLA and GLA. Is almost 1/10. By utilizing this difference, selectivity is born and high purity arachidonic acid can be obtained. Therefore, for the concentration of arachidonic acid, lipases derived from the genus Pseudomonas) and exhibiting higher specificity for DGLA and GLA than for arachidonic acid are preferred.

  Moreover, when DGLA is highly purified from DGLA-containing fats and oils produced by microorganisms, another lipase may be used. DGLA-containing fats and oils contain almost no arachidonic acid, and the fatty acids to be removed are GLA and linoleic acid. The lipase derived from Pseudomonas aeruginosa is different in the reactivity of DGLA and these fatty acids. Can be used for selective esterification to increase the purity of DGLA. Furthermore, lipase derived from Candida rugosa is also preferred for concentrating DGLA. Therefore, for enrichment of DGLA, a lipase derived from Pseudomonas aeruginosa and having a higher specificity for GLA and linoleic acid than for DGLA is preferred. Furthermore, lipases derived from the genus Candida rugosa are also preferred. These lipases are preferably used in combination.

  In concentrating mead acid, it is preferable to use a lipase derived from Burkholderia cepacia (formerly Pseudomonas space).

Removal of Esterified Fatty Acids Esterified fatty acids are used between esters and free fatty acids by taking advantage of the difference in physicochemical properties between the esterified fatty acids and the specific polyunsaturated fatty acids of interest. Conventional methods used for separation can be used. Specifically, distillation, solvent extraction, or the like can be used.
For example, fatty acids esterified by distillation can be removed by evaporation. Further, the ester can be selectively extracted from the esterification-reactive organism using an extraction solvent such as hexane.

Use of Concentrated Specific Polyunsaturated Fatty Acids Polyunsaturated fatty acids concentrated as described above are free and can be used in various molecular forms depending on the application. For example, when used as a perfume, it may be used after ethyl esterification, or when added to edible fats and oils, there is no problem even if it is added in the form of triglyceride. Furthermore, it can also be used as a raw material for phospholipids that have recently attracted functional attention.
The oils and fats of the present invention, for example, fats and oils containing high-purity arachidonic acid, DGLA, mead acid, etc., have unlimited possibilities, and can be used as raw materials and additives for foods, beverages, cosmetics and pharmaceuticals. it can. And there is no restriction on the purpose and amount of use.

Next, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to examples.
Example 1. Preparation of arachidonic acid concentrate (1) Degradation by lipase 200 g of water was added to 100 g of arachidonic acid-containing oil (SUNTGA40S, triacylglycerol) obtained from Mortierella alpina, and 1800 U of water was added. It was hydrolyzed with lipase (QLM: Meisho Sangyo) at 40 ° C for 72 hours. After hydrolysis, free fatty acids were extracted with hexane, and hexane was removed with an evaporator.

(2) Removal of saturated fatty acid by urea inclusion method The long-chain saturated fatty acid contained in the free fatty acid composition prepared in the above (1) was removed by the urea inclusion method. After removing the alcohol used for urea inclusion, it was subjected to a selective esterification reaction in the next step.
(3) Selective esterification (first time)
Selective esterification was performed in a two-step reaction using the same lipase and varying the enzyme titer. In the first selective enzyme reaction, double the amount (molar ratio) of lauryl alcohol and 20 U of selective lipase (Lipase PS) were added to the free fatty acid obtained in the previous urea inclusion, and then 30 ° C. The reaction was carried out for 16 hours to esterify unnecessary fatty acids.

(4) Removal of esterified fatty acid This fatty acid ester was removed by hexane extraction. The remaining aqueous layer was acidified with hydrochloric acid and extracted once more with hexane to obtain further purified free arachidonic acid. The purity of arachidonic acid at this time reached 85.7% as shown in Table 2.

実施例２. DGLA濃縮物の調製
（１）DGLA含有脂肪酸組成物原料の調製
モルティエレラ（Mortierella）の変異株から得られたDGLA含有油(SUNTGD, トリアシルグリセロール)100ｇを、40 gの水酸化ナトリウム、80 gの水、および800 mlのエタノール混液を60℃まで加温した後に加え、窒素気流下で、時々攪拌しながら60℃で20分間保温した。800 mlの水を添加した後、pHが２以下になるまで濃塩酸を加え、室温まで冷却した。ここに存在する遊離脂肪酸を3000 mlのヘキサンで抽出した後、ヘキサン層を回収し、エバポレーターで脱溶媒した結果、92.1 gの遊離脂肪酸が得られた。 (5) Selective esterification (second time)
Add lauryl alcohol twice as much (molar ratio) to the fatty acid containing the high purity arachidonic acid obtained in (4) above, this time selecting 60U, which has a higher titer than the first. Lipase (Lipase PS) was added and reacted at 30 ° C. for 16 hours. The esterified fatty acid was removed by hexane extraction as in the previous reaction, and purified arachidonic acid was obtained from the remaining aqueous layer by hexane extraction under acidic conditions. With a recovery rate of 48.9%, 96.6% high purity free arachidonic acid was obtained. Table 2 shows the fatty acid composition of the oil containing arachidonic acid purified in Example 1.

Example 2. Preparation of DGLA Concentrate (1) Preparation of DGLA-containing Fatty Acid Composition Raw Material 100 g of DGLA-containing oil (SUNTGD, triacylglycerol) obtained from a mutant strain of Mortierella was converted to 40 g of hydroxylated product. A mixture of sodium, 80 g of water, and 800 ml of ethanol was added after heating to 60 ° C., and the mixture was kept at 60 ° C. for 20 minutes with occasional stirring under a nitrogen stream. After adding 800 ml of water, concentrated hydrochloric acid was added until the pH was 2 or less, and the mixture was cooled to room temperature. The free fatty acid present here was extracted with 3000 ml of hexane, and then the hexane layer was recovered and desolvated with an evaporator. As a result, 92.1 g of free fatty acid was obtained.

(2) Removal of saturated fatty acids by urea addition
100 g of urea, 14 ml of water, 500 ml of methanol, and 92 g of the free fatty acid were mixed and stirred at 60 ° C. until the urea and free fatty acid were completely dissolved. The mixture was gradually cooled to 4 ° C. over 6 hours while stirring. After removing the precipitate by filtration, 500 ml of 0.1M aqueous hydrochloric acid was added to the resulting supernatant fraction. Furthermore, after extracting a free fatty acid with 2000 ml of hexane, the hexane layer was collect | recovered and the solvent was removed with the evaporator. As a result, 53.0 g of free fatty acid was obtained. The acid value at this time was 187.4 mg KOH / g. Table 3 shows the fatty acid composition of the raw oil, the oil after saponification decomposition, and the oil after urea addition.

  Saturated fatty acid content could be reduced by urea addition and DGLA content was increased to 59.9%. In the future, this free fatty acid after urea addition will be called FFA-DGLA60.

(3) Selective esterification of unwanted fatty acids with lipase LPL (first time)
A mixed solution consisting of 0.71 g of FFA-DGLA60, 0.89 g of lauryl alcohol (LauOH, 2 times mol to fatty acid), 0.4 g of water, and 400 U of lipase derived from Pseudomonas aeruginosa (manufactured by TOYOBO, lipase LPL) The reaction was allowed to stir at 30 ° C. Seven identical reaction solutions were made. After 2, 4, 6, 8, 10, 14, 17 hours, the oil layer was recovered by centrifugation of the reaction solution. The esterification rate was calculated from the decrease in acid value in the oil layer. Unreacted free fatty acids were recovered from the oil layer by hexane extraction, and the fatty acid composition was analyzed by gas chromatography. The results are listed in Table 4.

  As the esterification rate increased, C16: 0, C18: 0, C18: 1, C18: 2, GLA content decreased and DGLA content increased. This is because lipase LPL is easy to act on C16: 0, C18: 0, C18: 1, C18: 2, GLA and is difficult to act on DGLA, so DGLA is concentrated in the unreacted free fatty acid fraction. is there. When the esterification rate was about 60%, the DGLA content reached a maximum of about 82%.

  In a previous report (Shimada et al., Science and Industry, Vol.73, p.125-130, 1999), selective use of arachidonic acid (AA) -containing oil by Candida rugosa-derived lipase (manufactured by Meito Sangyo Co., Ltd., lipase OF) AA is purified by esterification. However, since the activity of lipase OF on AA, GLA and DGLA is almost the same, AA, GLA and DGLA were concentrated in the same unreacted free fatty acid fraction. Therefore, if lipase OF is allowed to act on FFA-DGLA60, it is presumed that GLA and DGLA are concentrated in the same unreacted free fatty acid fraction. However, since the lipase LPL used in this example distinguishes GLA and DGLA (works well for GLA but hardly acts on DGLA), it was possible to remove GLA.

  GLA could be removed by selective esterification using lipase LPL, but the removal efficiency of C16: 0, C18: 0, C18: 1 was not very good. The removal of these fatty acids was predicted to be more efficient with lipase OF than with lipase LPL. Therefore, after the unreacted free fatty acid fraction was collected by selective esterification using lipase LPL, it was decided to perform selective esterification using lipase OF.

(4) Selective esterification with lipase OF (second time)
Sample preparation: A mixture of 20 g FFA-DGLA60, 25 g LauOH, 11.3 g water, and 11250 U lipase LPL was reacted at 30 ° C. with stirring for 7 hours. The esterification rate at this time was 45.8%. Unreacted free fatty acid was extracted from the reaction mixture with hexane, and 11.6 g of free fatty acid was obtained by desolvation under reduced pressure. Since this sample had a DGLA content of 77.6 wt%, it will be referred to as FFA-DGLA78.

  A mixture consisting of 0.71 g of FFA-DGLA78, 0.89 g of LauOH, 0.4 g of water, and 400 U of lipase OF was reacted at 30 ° C. with stirring. Five identical reaction solutions were made. After 4, 12, 24, 48, and 72 hours, the oil layer was recovered by centrifugation of the reaction solution. The esterification rate was calculated from the decrease in acid value in the oil layer. Further, an unreacted free fatty acid fraction was recovered from the oil layer by hexane extraction, and the fatty acid composition was analyzed by gas chromatography. The results are listed in Table 5.

  Selective esterification with lipase OF removed C16: 0, C18: 0, C18: 1, C18: 2 and increased the DGLA content to about 90% (89.8%).

Example 3 Preparation of Mead Acid Concentrate (1) Preparation of MA-containing Fatty Acid Composition Raw Material 100 g of MA-containing oil (SUNTGM33, triacylglycerol) obtained from a mutant strain of Mortierella was added to 40 g of water. A mixture of sodium oxide, 80 g of water, and 800 ml of ethanol was added after heating to 60 ° C., and the mixture was kept at 60 ° C. for 20 minutes under nitrogen flow with occasional stirring. After adding 800 ml of water, concentrated hydrochloric acid was added until the pH was 2 or less, and the mixture was cooled to room temperature. The free fatty acid present therein was extracted with 3000 ml of hexane, and then the hexane layer was recovered and desolvated with an evaporator. As a result, 93.0 g of free fatty acid was obtained.

(2) Removal of saturated fatty acids by urea addition
100 g of urea, 14 ml of water, 500 ml of methanol, and 93 g of the above free fatty acid were mixed and stirred at 60 ° C. until the urea and free fatty acid were completely dissolved. The mixture was gradually cooled to 4 ° C. over 6 hours while stirring. After removing the precipitate by filtration, 500 ml of 0.1M aqueous hydrochloric acid was added to the resulting supernatant fraction. Furthermore, after extracting a free fatty acid with 2000 ml of hexane, the hexane layer was collect | recovered and the solvent was removed with the evaporator. As a result, 72.1 g of free fatty acid was obtained. The acid value at this time was 187.9 mg KOH / g. Table 6 shows the fatty acid composition of the raw oil, the oil after saponification decomposition, and the oil after urea addition.

  Saturated fatty acid content could be reduced by urea addition, increasing MA content to 41.2%. In the future, this free fatty acid after urea addition will be called FFA-MA41.

(3) Selective esterification with Liper PS (first time)
Consists of 0.71 g of FFA-MA41, 0.89 g of LauOH (2-fold mol to fatty acid), 0.4 g of water, and 400 U of Pseudomonas sp. Lipase (manufactured by Amano Enzyme, Lipase PS) The mixed solution was reacted at 30 ° C. with stirring. Seven identical reaction solutions were made. After 0.5, 1, 2, 3, 4, 6, 8 hours, the oil layer was recovered by centrifugation of the reaction solution. The esterification rate was calculated from the decrease in acid value in the oil layer. Unreacted free fatty acids were recovered from the oil layer by hexane extraction, and the fatty acid composition was analyzed by gas chromatography. The results are listed in Table 7.

  As the esterification rate increased, the contents of C16: 0, C18: 0, C18: 1, C18: 2, unknown 1, 2 decreased, and the MA content increased. This is because lipase PS tends to act on C16: 0, C18: 0, C18: 1, C18: 2, unknown 1, 2 and hardly acts on MA, so MA is concentrated in the unreacted free fatty acid fraction. Because. When the esterification rate was about 60%, the MA content reached a maximum of about 83%.

(4) Selective esterification with lipase PS (second time)
A mixed solution composed of 25 g of FFA-MA41, 31 g of LauOH, 14 g of water, and 14000 U of lipase PS was reacted at 30 ° C. with stirring for 3 hours. The esterification rate at this time was 57.6%. Unreacted free fatty acid was extracted from the reaction mixture with hexane, and 11.4 g of free fatty acid was obtained by desolvation under reduced pressure. Since the MA content of this sample was 79.2 wt%, it will be referred to as FFA-MA79.

  A mixed solution composed of 0.71 g of FFA-MA79, 0.89 g of LauOH, 0.4 g of water, and 400 U of lipase PS was reacted at 30 ° C. with stirring. After 1, 2, 4, 7, 14, 21 hours when 6 identical reaction solutions were prepared, the oil layer was recovered by centrifugation of the reaction solution. The esterification rate was calculated from the decrease in acid value in the oil layer. Further, an unreacted free fatty acid fraction was recovered from the oil layer by hexane extraction, and the fatty acid composition was analyzed by gas chromatography. The results are listed in Table 8.

  The second selective esterification with lipase PS removed C16: 0, C18: 0, C18: 1, C18: 2, unknown 1, 2 and increased the MA content to 92.1%. .

Claims (27)

In the method for producing a fatty acid composition in which the ratio of a specific highly unsaturated fatty acid is 50% or more,
(1) A lipase (specific lipase) having a specificity for the specific polyunsaturated fatty acid is lower than that for the other polyunsaturated fatty acid, and the specific polyunsaturated fatty acid in the presence of a fatty alcohol. Selectively esterifying fatty acids other than the specific polyunsaturated fatty acids by acting on the fatty acid composition comprising;
(2) removing the ester produced in the step (1) to obtain a fatty acid composition enriched with the specific polyunsaturated fatty acid; and (3) the same lipase as the lipase used in the step (1). Or repeat steps (1) and (2) using different lipases;
A method comprising that. The fatty acid composition used in the step (1) is the following step:
(A) preparing a fatty acid composition containing the specific highly unsaturated fatty acid; and (b) removing the saturated fatty acid by the urea inclusion method to obtain a fatty acid composition enriched in the highly unsaturated fatty acid;
The method of claim 1 obtained by:   The method according to claim 2, wherein the fatty acid composition in the step (a) is obtained by lipase decomposition or saponification of an ester containing the specific polyunsaturated fatty acid.   4. The method of claim 3, wherein the ester is a glyceride selected from the group consisting of monoglycerides, diglycerides and triglycerides containing the specific highly unsaturated fatty acid.   The method according to claim 3 or 4, wherein the glyceride is triglyceride.   The lipase used in the step (a) is a lipase having a small difference in substrate specificity between the specific polyunsaturated fatty acid and the other fatty acid. Method.   The method according to any one of claims 1 to 6, wherein the fatty alcohol is a fatty alcohol having 8 to 16 carbon atoms.   The method of claim 7, wherein the fatty alcohol is lauryl alcohol.   The method according to claim 1, wherein the specific highly unsaturated fatty acid is an n-6 highly unsaturated fatty acid.   The method according to any one of claims 1 to 8, wherein the specific polyunsaturated fatty acid is an n-9 polyunsaturated fatty acid.   The specific polyunsaturated fatty acid is arachidonic acid, and the specific lipase used in the step (1) is a lipase derived from Burkholderia cepacia. The method according to item.   12. The method according to claim 11, wherein the ratio of arachidonic acid in the obtained highly unsaturated fatty acid composition is 85% or more.   13. The method according to claim 12, wherein the ratio of arachidonic acid in the obtained highly unsaturated fatty acid composition is 90% or more.   14. The method according to claim 13, wherein the ratio of arachidonic acid in the obtained highly unsaturated fatty acid composition is 95% or more.   The specific polyunsaturated fatty acid is dihomo-γ-linolenic acid (DGLA), and the specific lipase used in the step (1) is a lipase derived from Pseudomonas aeruginosa and / or Candida rugosa. The method according to any one of claims 1 to 7, which is a lipase derived from (Candida rugosa).   16. The method according to claim 15, wherein the ratio of dihomo-γ-linolenic acid in the obtained highly unsaturated fatty acid composition is 50% or more.   17. The method according to claim 16, wherein the ratio of dihomo-γ-linolenic acid in the obtained highly unsaturated fatty acid composition is 80% or more.   18. The method according to claim 17, wherein the ratio of mead acid in the obtained polyunsaturated fatty acid composition is 89% or more.   The specific polyunsaturated fatty acid is mede acid, and the specific lipase used in the step (1) is a lipase derived from Burkholderia cepacia. 2. The method according to item 1.   20. The method according to claim 19, wherein the proportion of mead in the obtained highly unsaturated fatty acid composition is 50% or more.   21. The method according to claim 20, wherein the proportion of mead acid in the obtained highly unsaturated fatty acid composition is 80% or more.   The method according to claim 21, wherein the ratio of mead acid in the obtained polyunsaturated fatty acid composition is 90% or more.   An arachidonic acid-containing fatty acid composition obtained by the method according to any one of claims 12 to 14.   A dihomo-γ-linolenic acid-containing fatty acid composition obtained by the method according to any one of claims 15 to 17.   A medic acid-containing fatty acid composition obtained by the method according to any one of claims 18 to 22.   A food comprising the fatty acid composition according to any one of claims 23 to 25.   27. The food according to claim 26, which is a health food.