JPH06287594A - Production of triglyceride containing docosahexaenoic acid - Google Patents

Abstract

PURPOSE:To facilitate the formation of triglyceride having a residue of docosahexaenoic acid useful as a material for a medicine or food at the position 2 of glycerol. CONSTITUTION:A fat or oil having a residue of docosahexaenoic acid at the position 2 of a triglyceride and an arbitrary selected fatty acid or its ester are used as starting materials, and these materials are treated with lipase acting specifically on the positions 1 and 3 of a triglyceride to form a triglyceride having a residue of docosahexaenoic acid at the position 2, and having residues of the fatty acid at the positions 1 and 3. This triglyceride is fractionated to produce a triglyceride having a residue of docosahexaenoic acid at the position 2.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to
The present invention relates to a method for producing a triglyceride containing one kind of arbitrarily selected fatty acid at the 3-position and docosahexaenoic acid at the 2-position.

[0002]

2. Description of the Related Art Recently, docosahexaenoic acid (hereinafter referred to as DH
It is becoming clear that it has many physiological activities such as a learning function improving effect, a carcinostatic effect, or a blood lipid lowering effect. In humans, a large amount of DHA is contained in brain gray matter, retina, nerves, heart, sperm, breast milk and the like. And as we get older, DH in the brain
It is presumed that it plays an important role in the nervous system because the A content decreases. Therefore,
It is considered necessary to add DHA to infant foods, especially foods for premature babies. In addition, it has been expected that the addition of DHA to foods will help improve brain function in the elderly.

It is known that a large amount of DHA is contained in fish oil of marine fish. Further, highly unsaturated fatty acids such as eicosapentaenoic acid (hereinafter abbreviated as EPA) and DHA have been separated and purified from fish oils such as sardine oil or highly unsaturated fatty acid-producing microorganisms. In particular, in the case of DHA, no microorganism that produces DHA has been found, and the use of fish oil is regarded as important in terms of price and image. Therefore, various methods for selectively separating and purifying DHA from fish oil having a complicated fatty acid composition have been tried. When separating and purifying highly unsaturated fatty acids from fish oil, it is considered necessary to carry out chemical treatment or enzymatic treatment in advance because of their complicated fatty acid composition. Usually, DHA obtained by hydrolyzing fish oil by a chemical method or an enzymatic method and obtained as a free fatty acid or an esterified product is subjected to a urea addition method, salt formation method, supercritical carbon dioxide extraction method, column chromatography, etc. Is purifying DHA. Since the enzymatic method can be treated under milder conditions than the chemical method, an enzymatic method can be used as a pretreatment for fats and oils containing unstable highly unsaturated fatty acids. As a method that is considered to be preferable and obtains DHA by utilizing the substrate specificity or regiospecificity of the enzyme, Candida
Methods such as selective hydrolysis using a lipase derived from cylideacea (Japanese Patent Laid-Open Nos. 58-165796 and 3-19693) have been proposed.

Further, a method in which a glyceride is converted to an ester of a lower alcohol and then subjected to a distillation or urea inclusion treatment for concentration and separation (JP-A-58-8037) and a method using reverse phase chromatography (JP-A-58). -88339 publication,
Japanese Unexamined Patent Publication (Kokai) No. 58-109444) is known, but it has a problem that it requires a multi-step process because it is a substance having weak oxidation stability and is extremely unstable, and that it requires a great deal of cost for purification. , The supply is not enough. as a result,
Actually, DHA ethyl ester is only marketed as a drug. Generally, it is said that the fats and oils of ethyl ester form and methyl ester form are inferior in digestive absorbability in the small intestine as compared with triglyceride form. Therefore, supply of DHA in the form of triglyceride is strongly desired in the pharmaceutical field and food field. In particular,
In order to use DHA in the food field, it is necessary to inexpensively supply a large amount of DHA in the form of triglyceride. Therefore, DH that is efficient and has oxidative stability taken into consideration.
Development of the manufacturing method of A was desired.

[0005]

DISCLOSURE OF THE INVENTION The inventors of the present invention have made extensive studies as to a method for separating fats and oils having a high DHA content from fish oil. As a result, an ester of lipase which specifically acts on the 1,3-positions of triglycerides. By the exchange reaction, a triglyceride containing a polyunsaturated fatty acid at the 2-position as a constituent fatty acid and one kind of arbitrarily selected fatty acid at the 1- and 3-positions is produced, thereby facilitating an ordinary separation and purification method. It was found that triglycerides having a high DHA content can be obtained and the present invention has been completed. Therefore, it is an object of the present invention to provide a method for producing a triglyceride containing DHA at the 2-position as a constituent fatty acid and one kind of fatty acid arbitrarily selected at the 1- and 3-positions. .

[0006]

The method for producing the DHA-containing triglyceride of the present invention will be described below. Examples of fats and oils containing DHA at the 2-position of triglyceride as a constituent fatty acid that can be used as a raw material in the present invention are:
If DHA is a triglyceride linked to the 2-position,
Any kind of fats and oils including the content thereof can be used, and for example, fats and oils having a DHA content of less than 10% may be used. Further, if the triglyceride is pure, no further purification treatment in the previous step is necessary. However,
It is disadvantageous to use as a raw material because the oil and fat having a lower second-order DHA content requires longer processing time.

As one kind of arbitrarily selected fatty acid or fatty acid ester, saturated acids such as myristic acid, palmitic acid and stearic acid and unsaturated acids such as oleic acid, linoleic acid, EPA and DHA, or Examples thereof include esters of those fatty acids with ethyl alcohol and methyl alcohol. Further, a hydrolyzate such as olive oil which is a high oleic acid-containing oil, a saponified product and the like can also be used.

In the present invention, first, an oil or fat containing DHA at the 2-position of triglyceride as a constituent fatty acid and one kind of arbitrarily selected fatty acid or fatty acid ester are used as raw materials, and the specific fatty acids at the 1- and 3-positions are specified. By carrying out a transesterification reaction by acting on a lipase that acts on to produce a triglyceride containing DHA at the 2-position as a constituent fatty acid and one kind of arbitrarily selected fatty acid at the 1- and 3-positions Let Examples of the lipase that can be used in the present invention that specifically acts at the 1- and 3-positions include porcine pancreatic lipase, Rhizopus genus, and Aspergill.
Those produced by microorganisms such as genus us can be exemplified. The use form of these lipases is not particularly limited, but it is desirable to use an immobilized enzyme or a chemically modified enzyme. The amount of lipase used may be appropriately determined depending on the reaction conditions and the properties of the raw material fat or oil used, and is not particularly limited.

The present invention particularly utilizes the fact that DHA is concentrated as a constituent fatty acid at the 2-position of triglyceride in fish oil, and uses a lipase that specifically acts at the 1- and 3-positions of triglyceride. , Replacing only the fatty acids at positions 1 and 3 while leaving DHA at position 2 of triglyceride,
A triglyceride containing DHA at the 2-position as a constituent fatty acid and one kind of arbitrarily selected fatty acid at the 1- and 3-positions is produced. That is, the fatty acid at the 1- and 3-positions in the fat or oil is arbitrarily selected by an acidolysis reaction between triglyceride and fatty acid or a transesterification reaction between triglyceride and lower alcohol fatty acid ester.
It is replaced with a different type of fatty acid. The produced free fatty acid, free fatty acid ester, monoglyceride, diglyceride and the like can be removed by performing treatments such as alkali washing, steam distillation, molecular distillation and solvent extraction. In this way, many molecular species consisting of the complex fatty acid composition of fish oil can be arranged and simplified while leaving the 2-position DHA of triglyceride.

Next, from the triglyceride containing one kind of fatty acid arbitrarily selected in the 1,3-position thus obtained, DHA is contained in the 2-position as a constituent fatty acid, and the 1,3-position is contained. A triglyceride containing one kind of fatty acid arbitrarily selected in is separated. For this separation, it is possible to use techniques such as crystallization, extraction, adsorption, chromatography and membrane separation which are usually performed, but it is advantageous to use chromatography in view of work efficiency and the like. . There are several known operation modes such as a batch type, a circulation type, a moving bed type, and a simulated moving bed type in the column chromatography which has been conventionally performed, but in particular, a simulated moving bed type chromatographic separation device (SMB) is used. Is preferably used.

For example, when fish oil and oleic acid are used as raw materials, first, from the produced triglyceride containing oleic acid at the 1,3-positions, 1,3-dioleo-2-e is subjected to column chromatography. Two triglycerides, icosapentaene and 1,3-dioleo-2-docosahexaene, are separated. Then, these two types of triglycerides can be purified by a simulated moving bed chromatographic separation device. The triglyceride thus obtained will contain 33% DHA.

By changing the elution conditions of the simulated moving bed chromatographic separation device, a composition containing triglyceride containing a highly unsaturated fatty acid such as EPA at the 2-position can be prepared. It is also possible to provide triglycerides with a controlled / EPA ratio.
Further, by appropriately selecting the fatty acid used as the raw material, the saturated fatty acid (S) / unsaturated fatty acid (U) ratio or n-3
The system fatty acid / n-6 system fatty acid ratio can also be freely controlled. Next, an example of the present invention will be specifically described.

[0013]

Example 1 Sardine oil (DHA content 6.51%) 100
g and oleic acid (purity 95%) 1,000 g as raw materials, and using lipase 40 g obtained by chemically modifying lipase derived from Rhizopus delemar with fatty acid, n-
The enzymatic reaction was carried out in hexane at 40 ° C. for 24 hours. After completion of the reaction, 80 g of triglyceride produced by extracting fatty acid using a solvent of ethanol: water (9: 1) (DHA
Content 5.48%) was obtained. 12. Add this triglyceride
After dissolving in a solvent of acetone: acetonitrile (1: 1) at a concentration of 5 g / l, ODS-AM120-S50 (YM
Column (diameter 25 mm, length 460) packed with C company
mm), and a fraction containing 1,3-dioleo-2-eicosapentaene and 1,3-dioleo-2-docosahexaene was confirmed by UV 206 nm and gas chromatography and collected. Next, 1,3-dioleo-2
12 g of a fraction containing eicosapentaene and 1,3-dioleo-2-docosahexaene was dissolved in a solvent of acetone: acetonitrile (2: 1) at a concentration of 12.5 g / l,
This was the SMB treatment liquid. SMB has a column diameter of 25 mm
Eight towers with a length of 460 mm, each with ODS-AM120-
S50 (manufactured by YMC) was filled, and a solvent of acetone: acetonitrile (1: 1) was used as a mobile phase. SMB
The operating conditions were as follows: processing solution supply rate 0.89 ml / min, eluent supply rate 2.53 ml / min, extract withdrawal rate 1.71 ml / min, raffinate withdrawal rate 1.71 m.
1 / min, column temperature 40 ° C., step time 114.
It was 89 minutes. As a result, 1844 ml of the target fraction was obtained in the extract. This extract was concentrated to dryness to give 5.4 g of 1,3-dioleo-2-docosahexaene.
(Purity 92.3%) was obtained.

[0014]

Example 2 Skipjack oil (DHA content 7.91%) 500
g and oleic acid (purity 95%) as raw materials, M
Ucor miehei- derived immobilized enzyme lipozyme (manufactured by Novo Co., Ltd.) 60 g was used in n-hexane to prepare 60
The enzyme reaction was performed at 6 ° C for 6 hours. After the reaction was completed, the lipozyme was filtered off, the fatty acid portion was removed by the alkaline deoxidation method, and 432 g of the produced triglyceride (DHA content 11.20) was obtained.
%) Was obtained. This triglyceride was dissolved in a solvent of acetone: acetonitrile (1: 1) at a concentration of 12.5 g / l, and then purified with a column (diameter 25 mm, length 460 mm) packed with ODS-AM120-S50 (YMC). The fraction containing 1,3-dioleo-2-eicosapentaene and 1,3-dioleo-2-docosahexaene was confirmed by UV 206 nm and gas chromatography and collected. Next, 86.4 g of a fraction containing 1,3-dioleo-2-eicosapentaene and 1,3-dioleo-2-docosahexaene was dissolved in a solvent of acetone: acetonitrile (2: 1) at a concentration of 12.5 g / l. And SMB
It was used as a treatment liquid. SMB has a column diameter of 25 mm and a length of 4
60mm 8 tower type, ODS-AM120-S50
(Manufactured by YMC) was filled, and a solvent of acetone: acetonitrile (2: 1) was used as a mobile phase. The operating conditions of the SMB are as follows: processing liquid supply rate 0.89 ml / min, eluent supply amount 2.53 ml / min, extract withdrawal amount 1.71.
The raffinate withdrawal rate was 1.71 ml / min, the column temperature was 40 ° C., and the step time was 60.84 minutes. As a result, the target fraction 13,280 was added to the extract.
ml was obtained. Concentrate and dry this extract to 1,3
-Dioleo-2-docosahexaene 40.1 g (purity 9
5.1%) was obtained.

[0015]

INDUSTRIAL APPLICABILITY By the method of the present invention, DHA such as fish oil
DHA-containing triglyceride useful as a raw material for medicines and foods can be easily separated and recovered from oils and fats containing a large amount of.

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Claims (4)

[Claims] 1. An oil or fat containing docosahexaenoic acid at the 2-position of triglyceride as a constituent fatty acid and one kind of arbitrarily selected fatty acid or fatty acid ester are used as raw materials, and specifically at the 1- and 3-positions of triglyceride. A transesterification reaction is carried out by acting a lipase which acts to form a triglyceride containing docosahexaenoic acid at the 2-position as a constituent fatty acid and one kind of arbitrarily selected fatty acid at the 1- and 3-positions. And a docosahexaenoic acid-containing triglyceride. 2. The method for producing a glyceride containing docosahexaenoic acid according to claim 1, wherein the fat or oil containing docosahexaenoic acid at the 2-position of the triglyceride as a constituent fatty acid is fish oil. 3. The method for producing a docosahexaenoic acid-containing triglyceride according to claim 1 or 2, wherein one kind of arbitrarily selected fatty acid is oleic acid. 4. The simulated moving bed chromatographic separation device collects the glyceride containing docosahexaenoic acid.
5. The method for producing a triglyceride containing docosahexaenoic acid according to any one of 3 to 3.