JPH0889265A - Production of triglyceride containing highly unsaturated fatty acid - Google Patents

Abstract

PURPOSE: To obtain a triglyceride containing a highly unsaturated fatty acid and useful for the prevention, treatment, etc., of degenerative diseases in high efficiency and yield by reacting a monoglyceride, etc., containing a highly unsaturated fatty acid as a constituent fatty acid with lipase. CONSTITUTION: This triglyceride containing a highly unsaturated fatty acid is produced by reacting a mono and/or a diglyceride containing a highly unsaturated fatty acid such as docosahexaenoic acid or eicosapentaenoic acid as a constituent fatty acid with one or more kinds of lipases selected from a lipase originated from Geotrichum candidum, a lipase originated from Candida cylindrasie, a lipase originated from Chromobacterium viscosum, a lipase originated from Pseudomonas sp. and a lipase originated from Rhizopus delemar.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing highly unsaturated fatty acid-containing triglyceride using an enzymatic reaction.

[0002]

2. Description of the Related Art Oils and fats containing ω3 polyunsaturated fatty acids are often contained in fish oils in the form of triglycerides. ω
Docosahexaenoic acid (hereinafter abbreviated as DHA) is a type 3 polyunsaturated fatty acid, which has a learning function improving action, an anti-atherogenic action, an antitumor action, an immunostimulating action, an antiallergic action, and the like, and eicosapentaenoic acid. (Hereinafter, abbreviated as EPA) is known to have useful physiological activities such as anti-atherogenic effect and anti-tumor effect. Naturally occurring fats and oils have at most 2 as constituent fatty acids, DHA and EPA.
The content is about 0 to 30%, but in addition to this, a large amount of saturated fatty acids such as palmitic acid and stearic acid, and highly unsaturated ω6 fatty acids represented by linoleic acid are contained. Therefore, when such natural fats and oils are used as health foods and pharmaceuticals, there has been a problem of excess lipid. In order to eliminate this inconvenience, fats and oils reduced in fatty acids other than DHA were prepared by an enzymatic method (Yukihisa).
Tanaka et al. , Journal of
American Oil Chemical Soc
iety, 69, (1992), 1210-121.
4). Further, there has been reported a high-concentration DHA oil and fat prepared by utilizing an ester synthesis reaction by lipase using free high-purity DHA and glycerin as raw materials and a transesterification reaction of high-purity DHA ethyl ester by lipase ( Yukihisa Tanaka et al., Oil Chemistry, Volume 41, 199.
2 years, pp. 563-567 and JP-A-5-331105.
Issue).

[0003]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention
a Tanaka et al. , Journal o
f American Oil Chemical S
ociety, 69, (1992), 1210-12.
The method described in 14 makes use of the difference in the hydrolysis specificity of lipase with respect to the fatty acids constituting fats and oils.
Although the HA concentration was raised to about 50%, it was insufficient in terms of DHA concentration. Further, the method described in Yukihisa Tanaka et al., Oil Chemistry, Vol. 41, 1992, p. Although the reaction is performed with glycerin to synthesize an ester, strict control of the water concentration in the reaction system is essential. Further, the free acid of DHA, which is a raw material used in this reaction, has a drawback that the stability against air oxidation is inferior to that of glycerin ester. Therefore, it is an object of the present invention to provide a method for producing an oil or fat (ie, triglyceride) containing a high concentration of highly unsaturated fatty acid, which overcomes the drawbacks of the conventional techniques.

[0004]

Means for Solving the Problems As a result of intensive studies, the present inventors have utilized the enzymatic reaction of lipase to convert highly unsaturated fatty acids from mono- and / or diglycerides into highly unsaturated fatty acids. The inventors have found a method for producing contained triglycerides and completed the present invention.

That is, the present invention provides a method for producing a highly unsaturated fatty acid-containing triglyceride, which comprises allowing a lipase to act on a mono- and / or diglyceride having a polyunsaturated fatty acid as a constituent fatty acid. In the production method of the present invention, using a mono- and / or diglyceride having a polyunsaturated fatty acid as a constituent fatty acid as a substrate, a hydrolysis reaction with lipase and a transesterification (synthesis) reaction of the polyunsaturated fatty acid simultaneously proceed, Triglycerides containing highly unsaturated fatty acids as constituent fatty acids are produced.

The present invention will be described in detail below. In the present specification, “mono- and / or diglyceride having polyunsaturated fatty acid as a constituent fatty acid” means a monoester of glycerol and highly unsaturated fatty acid, a diester of glycerol and highly unsaturated fatty acid, and glycerol and highly unsaturated. It is intended to include fatty acids and diesters with other fatty acids.

As the "mono- and / or diglyceride having a polyunsaturated fatty acid as a constituent fatty acid" used in the present invention, a chemical synthetic reaction between glycerin and a polyunsaturated fatty acid, glycerin and a polyunsaturated fatty acid are used. In addition to those obtained by a synthetic reaction with lipase, the one obtained by reacting phospholipid C, which is a phospholipid hydrolase, with a phospholipid containing a polyunsaturated fatty acid can be used. , Diglyceride alone or a mixture thereof. Mono- and / or polyunsaturated fatty acids as constituent fatty acids
Alternatively, the diglyceride is preferably a mono- and / or diglyceride having DHA and / or EPA as constituent fatty acids, which have particularly useful physiological activity.

The lipase used in the present invention can be used irrespective of its origin, but an enzyme derived from a microorganism which is stable in terms of supply, for example, Candida (Can
genus Dida), Geotrichum
Yeast-derived lipases such as genus Chromobacterium (Ch
genus Rhomobacterium, Pseudomonas (P
A lipase derived from a bacterium such as the genus Seudomonas and a lipase derived from a filamentous fungus such as the genus Rhizopus are preferable, and Geotricum candy dam (Geo)
trichum candidum) -derived lipase,
Candida cyrus
lipase derived from Chromobacteria (Chromobacteria)
viscosum) -derived lipase, Pseudomonas
Lipase derived from Pseudomonas sp., And Rhizopus
More preferred is a lipase derived from (delemer). The above enzymes can be used alone or in combination.

The enzyme reaction with lipase may be carried out in the presence of a sufficient amount of water for expressing the activity of the enzyme, and the amount thereof is, for example, a mono-unsaturated fatty acid as a constituent fatty acid. And / or 1 for diglyceride
0 to 4000% by weight, preferably 50 to 2000
It is about% by weight. In order to keep the pH of the enzyme reaction constant, a buffer solution such as an acetate buffer solution, a phosphate buffer solution, and Good's buffer solution may be used instead of water. The pH of the reaction may be selected within a range in which the enzyme used is not inactivated, but is preferably pH 3.5 to 7.5, and more preferably pH 4 to
It is 6. Further, in order to accelerate the enzymatic reaction, a divalent cation such as calcium or magnesium, a dispersant such as gum arabic or polyvinyl alcohol, or the like may be added to the reaction solution.

The amount of the enzyme to be used varies depending on the concentration of mono- and / or diglyceride containing a highly unsaturated fatty acid as a substrate as a constituent fatty acid, the reaction temperature, the reaction pH, and the reaction time, but is 2 to 10,000 units per 1 g of the reaction solution. (U) is suitable, and preferably about 10 to 1000 units (U). Further, the concentration of mono- and / or diglyceride containing highly unsaturated fatty acid as a substrate as a constituent fatty acid is appropriately 5 to 90%, preferably 10 to 50%. The reaction temperature can be appropriately selected within the range where the lipase is not inactivated, but it is preferably 20 to 50 ° C.

The enzymatic reaction proceeds without problems even in the presence of air, but generally, polyunsaturated fatty acids are easily oxidized, so the reaction is carried out in an environment in which oxygen is blocked using an inert gas such as nitrogen or argon gas. It is preferable.

The reaction time can be appropriately set while analyzing the amount of the reaction product by thin layer chromatography using a silica gel plate or by an ear tro scan capable of quantitative analysis using a silica gel rod, but the amount of the reaction product becomes maximum. It is preferable to set as follows. When the reaction product amount is analyzed using thin-layer chromatography, chloroform: acetone: acetic acid (96: 4: 1) can be used as a developing solvent, and the reaction product amount is analyzed using an earloscan. In some cases, benzene: chloroform:
Acetic acid (50: 20: 0.5) can be used.

The main product after the reaction is a highly unsaturated fatty acid-containing triglyceride, but some free fatty acids and mono- and / or diglycerides may be mixed in. In this case, a commonly used means such as an alkali deoxidation method, a molecular distillation method, a steam distillation method, a membrane separation method, a fractionation by adsorption chromatography using an ion exchange resin or various adsorption carriers is used. , It suffices to separate these by-products and raw materials,
The method is not particularly limited.

The polyglyceride containing a polyunsaturated fatty acid obtained as described above is a triglyceride containing a high concentration of a polyunsaturated fatty acid, which includes a triester of glycerol and a polyunsaturated fatty acid, and glycerol. Includes polyunsaturated fatty acids and triesters with other fatty acids.

[0015]

INDUSTRIAL APPLICABILITY According to the method of the present invention, lipase is used to produce mono- and / or polyunsaturated fatty acids as constituent fatty acids.
Alternatively, triglycerides containing useful highly unsaturated fatty acids such as EPA and DHA at high concentrations can be efficiently produced from diglycerides. The triglyceride containing a high concentration of this polyunsaturated fatty acid can be used for prevention and treatment of adult diseases.

[0016]

The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

[Reference Example 1] Synthesis of monoglyceride and diglyceride Monoglyceride was prepared by dissolving 20 g of glycerin and 50 mg of sodium hydroxide in 60 ml of dimethylformamide, and then dissolving DHA ethyl ester (Maruha Co., Ltd.) and EPA ethyl ester in this solution. 7 g of each ester (manufactured by Maruha Co., Ltd.) was added, and transesterification was performed while stirring at 70 ° C. for 5 hours. After the reaction, extraction with n-hexane was performed, and this extract was subjected to a silica gel 60 column (3 × 25 cm, silica gel 6 manufactured by Merck & Co., Inc.).
0) and unreacted ethyl ester and diglyceride were eluted and removed with benzene: ethyl acetate (8: 2), and then monoglyceride was eluted with benzene; ethyl acetate (6: 4). DHA monoglyceride 5.8g,
6.7 g of EPA monoglyceride was obtained.

Diglyceride was synthesized from glycerin and free fatty acid by an enzymatic method. That is, 4.6 g of glycerin, 16 g of DHA (manufactured by Maruha Co., Ltd.), and 16 g of EPA (manufactured by Maruha Co., Ltd.) were mixed, and then 2500 units of lipase derived from Chromobacterium (manufactured by Asahi Kasei Kogyo Co., Ltd., product Including the name LPT-01).
5 ml of an aqueous solution was added, and the reaction was carried out while stirring at 50 ° C. for 70 hours. After the reaction, the glyceride fraction extracted with n-hexane was applied to a silica gel column (3 × 25 cm, packed with Merck silica gel 60), the triglyceride fraction was eluted with benzene, and then diglyceride was added with benzene: ethyl acetate (9: 1). Was eluted. 1.4 g of DHA diglyceride and 1.3 g of EPA diglyceride
Obtained.

Example 1 Production of Triglyceride from Monoglyceride 2 ml of 2 g of DHA monoglyceride produced in Reference Example 1
Suspended in purified water of Geotricum candy dam (Geotricum candy dam ATCC 34614
(200 strains of lipase) was added, and the reaction was carried out at 30 ° C. for 20 hours with stirring. The pH of the reaction solution was not particularly adjusted, but the pH remained around 4.9. After the reaction, oil and fat was extracted with 100 ml of n-hexane, and triglyceride was eluted under the silica gel column conditions described in Reference Example 1 to obtain 1.3 g of DHA triglyceride.

Example 2 Production of Triglyceride from Monoglyceride 2 ml of 2 g of EPA monoglyceride produced in Reference Example 1
Suspended in purified water of Geotricum candy dam (Geotricum candy dam ATCC 34614
200 units of lipase obtained from K.K.) was added, and the reaction was carried out for 16 hours while stirring at 30 ° C. 100 ml after reaction
Extraction of fats and oils with n-hexane from Example 1
The triglyceride was eluted under the silica gel column conditions described in 1. to obtain 0.9 g of EPA triglyceride.

[Example 3] Production of triglyceride from diglyceride 1 g of each of DHA diglyceride and EPA diglyceride produced in Reference Example 1 was suspended in 3 ml of purified water, and Geotricum candy dam (Geotricum candydam AT
200 units of lipase obtained from CC34614 strain) was added and reacted at 30 ° C. for 16 hours. 100m after reaction
The oil and fat was extracted with 1 n-hexane, and the triglyceride was eluted under the silica gel column conditions described in Reference Example 1 to obtain 710 mg of DHA triglyceride, 590
Obtained mg EPA triglyceride.

Example 4 Production of Triglyceride from Monoglyceride 500 mg of EPA monoglyceride produced in Reference Example 1 was suspended in 3.5 ml of purified water, and 200 units of various lipases (the lipase derived from Geotricum candydam was Geotricum candydam). From ATCC 34614 strain,
The lipase derived from Rhizopus duller was obtained from Rhizopus duller ATCC34612 strain. Also,
The lipase derived from Chromobacterium viscosum is LPT-01 manufactured by Asahi Kasei Kogyo Co., Ltd., the lipase derived from Pseudomonas sp. Is CENT-18 manufactured by Asahi Kasei Kogyo Co., Ltd. It was -8525. ) Is added and 16 at 30 ° C.
The reaction was carried out with stirring for a time. After extracting the oil and fat with n-hexane, a purified EPA triglyceride was obtained by the method described in Reference Example 1. The results are shown in Table 1.

[0023]

[Table 1]

Example 5 Production of Triglyceride from Diglyceride 200 mg of DHA diglyceride produced in Reference Example 1 was suspended in 3.8 ml of purified water, and 200 units of various lipases (the lipase derived from Geotricum candydam was Geotrichum candydam). From ATCC 34614 strain,
The lipase derived from Rhizopus duller was obtained from Rhizopus duller ATCC34612 strain. Also,
The lipase derived from Chromobacterium viscosum is LPT-01 manufactured by Asahi Kasei Kogyo Co., Ltd., the lipase derived from Pseudomonas sp. Met. ) Was added and the reaction was carried out at 30 ° C. with stirring. After extracting the oil and fat with n-hexane, a purified DHA triglyceride was obtained by the method described in Reference Example 1. Table 2 shows the results.

[0025]

[Table 2]

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tetsuo Sugihara 87-thousand Senjo, Itami City, Hyogo Prefecture (72) Inventor Yuji Shimada 2-32 Higashi 4-chome, Kushiya-cho, Sakai City, Osaka Prefecture (72) Ikki Maruyama Ibaraki 16-2 Wadai, Tsukuba, Japan Inside the Central Research Institute, Maruha Co., Ltd. (72) Inventor: Chikako Shiina, 16-2, Wadai, Tsukuba, Ibaraki, Central Research Laboratory, Maruha Co., Ltd. (72) Hide Nakayama, Wa, Tsukuba, Ibaraki No. 16-2 Central Research Institute of Maruha Co., Ltd. (72) Inventor Shigeyuki Imamura 1 at 2 Samejima, Fuji City, Shizuoka Prefecture Asahi Kasei Corporation (72) Inventor Toshio Shimizu 1 at 2 Shimajima, Fuji City, Shizuoka Asahi Kasei Industry Within the corporation

Claims (3)

[Claims] 1. A process for producing a triglyceride containing a polyunsaturated fatty acid, which comprises reacting a mono- and / or diglyceride having a polyunsaturated fatty acid as a constituent fatty acid with a lipase. 2. A lipase derived from Geotricham candy dam, a lipase derived from Candida syrindrache, a lipase derived from Chromobacterium viscosum, a lipase derived from Pseudomonas sp.,
The method according to claim 1, wherein the method is at least one selected from the group consisting of and lipase derived from Rhizopus dermer. 3. The polyunsaturated fatty acid is docosahexaenoic acid and / or eicosapentaenoic acid.
Or the method described in 2.