JPH08214892A - Production of partial glyceride containing highly unsaturated fatty acid - Google Patents

Abstract

PURPOSE: To obtain the subject oil and fat for medicines, foods, etc., for health containing a highly unsaturated fatty acid in high concentration by reacting an oil and fat with a lipase in the presence of glycerol and carrying out the reaction while lowering temperature step by step under specific conditions. CONSTITUTION: Oil and fat (e.g. tuna oil) is reacted with a lipase such as of Pseudomonas fluorescens and the reaction is carried out at 30 deg.C for 5hr and at room temperature for 16hr and at 5 deg.C for 24hr under stirring while step by step lowering the temperature from 30 deg.C to 5 deg.C to provide the objective partial glyceride containing a highly unsaturated fatty acid comprising a monoglyceride and/or a diglyceride, extremely excellent in emulsion dispersibility to water system and stability and widely utilizable for emulsifier, medicines, foods for specific health and general food, etc., because of its biological activity.

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 a partial glyceride having a high content of polyunsaturated fatty acids and an application of the glyceride.

[0002]

2. Description of the Related Art In recent years, eicosapentaenoic acid (hereinafter referred to as "E
PA), docosahexaenoic acid (hereinafter "DHA")
It has been reported that polyunsaturated fatty acids such as) have many physiologically active effects such as a preventive effect against adult diseases such as arteriosclerosis and thrombosis, an anticancer effect, and a learning effect enhancing effect. , Has been attracting attention as a material for dietary supplements and foods for specified health uses.

Recently, in addition to dietary supplements containing DHA in gelatin capsules, many developments have been made on DHA-enriched foods added to general foods such as ham, sausage, chikuwa, miso, canned foods, candies and soft drinks. Has been. However, when adding DHA-containing oils and fats to soft drinks, it is extremely difficult to emulsify them into an aqueous system because the DHA-containing oils and fats raw material is tuna, bonito-derived fish oil and the like. For this reason, a considerable amount of emulsifier must be added to disperse and stabilize, but the amount of emulsifier added is, for example, about 15 mg for a 350 ml drink.
The limit is (only about 3.8 mg included as DHA). In this, it is not possible to include an amount of DHA having physiological activity.

Therefore, an amount of D which can be expected to have a physiologically active effect
In order to add HA to soft drinks, it is necessary to develop a DHA-containing oil / fat having excellent emulsion stability. by the way,
It has been conventionally known that monoglycerides have an emulsifying action, and methods for producing such monoglycerides have been studied for a long time. That is, at present, industrially, a mixture of oil and fat and glycerin is mainly mixed with about 0.1% of a metal catalyst and reacted at a high temperature of 200 to 240 ° C with stirring to synthesize a monoglyceride. Method is used.

However, since the reaction is carried out at a considerably high temperature in the above-mentioned synthetic method, monoglycerides of saturated fatty acids such as palmitic acid and stearic acid and monoglycerides such as oleic acid and erucic acid which are monounsaturated fatty acids can be produced with good quality. On the other hand, since highly unsaturated fatty acids such as DHA and EPA have many double bonds and are susceptible to polymerization by oxidation or heat and are very unstable, monoglycerides containing these highly unsaturated fatty acids are It is difficult to produce by the above glycerolysis method.

Therefore, recently, attention has been focused on a method for producing a monoglyceride by hydrolyzing natural fats and oils using an enzyme under mild reaction conditions. According to this method, it is possible to accumulate a certain amount of monoglyceride in the hydrolysis process of fats and oils. However, as the decomposition progresses, the monoglyceride is also hydrolyzed, and it is difficult to produce a sufficient amount of monoglyceride commensurate with the industrial cost.

On the other hand, Yamane et al performed a glycerolysis reaction using lipase derived from microorganisms such as the genus Chromobacterium in the reaction mixture consisting of beef tallow and glycerol (Chromobacterium) and Pseudomonas (Pseudomonas), about 70
% Monoglyceride accumulation reported (JAOCS, Vo
l.67, no.11, 1990, JAOCS, Vol.68, no.1, 1991), but monoglyceride is produced by the glycerolysis reaction method from fish oil containing highly unsaturated fatty acids such as DHA and EPA. It's unclear if they can.

[0008]

The present invention uses fish oil, glycerin and a small amount of water as raw materials and has an excellent emulsifying action,
Moreover, it aims at providing the manufacturing method of the partial glyceride which contains highly unsaturated fatty acid in high concentration.

[0009]

Means for Solving the Problems The present inventors have focused on the fact that partial glycerides have an excellent emulsifying power, and are concerned with a method for producing partial glycerides using fats and oils containing highly unsaturated fatty acids in high concentrations as raw materials. As a result of earnest research, by reacting a solution containing fats and oils and glycerin with a lipase, it is possible to produce a highly unsaturated fatty acid-rich partial glyceride that can be added to foods, has excellent emulsion stability, and has physiological activity. And succeeded in completing the present invention.

That is, the present invention is characterized in that fats and oils are allowed to react with lipase in the presence of glycerin to carry out the reaction while gradually lowering the temperature from 30 ° C. to 5 ° C. It is a method for producing glycerides.
Furthermore, the present invention hydrolyzes oils and fats with lipase, obtains a highly unsaturated fatty acid-rich glyceride from the resulting reaction product, and causes lipase to act on the glyceride in the presence of glycerin to give a glycerin content of 30 ° C to 5 ° C. A method for producing a partial glyceride containing a high degree of polyunsaturated fatty acid, characterized in that the reaction is carried out while gradually lowering the temperature to obtain a partial glyceride.

Furthermore, the present invention hydrolyzes oils and fats with lipase to obtain a glyceride containing a high content of highly unsaturated fatty acids from the reaction product obtained, and diglyceride obtained by fractionating the glyceride in the presence of glycerin. A method for producing a partial glyceride having a high polyunsaturated fatty acid content, characterized in that a monoglyceride is obtained by reacting with lipase and gradually lowering the temperature from 30 ° C to 5 ° C.

Here, the partial glyceride is a monoglyceride and / or a diglyceride, and the highly unsaturated fatty acid includes docosahexaenoic acid, eicosapentaenoic acid or arachidonic acid, or a combination thereof.
Examples of the oils and fats include fish oil or those extracted from krill, seaweed or fungi. Furthermore, the present invention is an emulsifier comprising the above-mentioned highly unsaturated fatty acid-rich partial glyceride.

Hereinafter, the present invention will be described in detail. In the present invention, first, lipase is added to a reaction mixture containing oils and fats such as fish oil, glycerin and a small amount of water, and a glycerolysis reaction is performed while gradually cooling gradually from 30 ° C to 5 ° C, whereby a highly unsaturated fatty acid content is increased. A partial glyceride containing is produced. The partial glyceride in the present invention means a glyceride other than triglyceride, that is, diglyceride or monoglyceride, or a mixture of diglyceride and monoglyceride. Further, a polyunsaturated fatty acid is a fatty acid having 20 or more carbon atoms having 3 or more double bonds, for example, arachidonic acid (20: 4), EPA (20: 5), and DHA.
(22: 6).

Further, the oil and fat according to the present invention means that the constituent fatty acids contain a high concentration of highly unsaturated fatty acids, and preferably DH.
A containing 30% or more. For example, in addition to fish oils such as tuna, bonito, sardines, mackerel, horse mackerel, saury, cod, and squid, oils and fats extracted from krill, seaweeds such as chlorella and spirulina, and fungi such as Mortierella can be mentioned.

Therefore, the highly unsaturated fatty acid-rich partial glyceride means a diglyceride or monoglyceride containing a highly unsaturated fatty acid in a high concentration as a constituent fatty acid, or a mixture thereof. In the present invention, a mixed liquid containing fats and oils, glycerin and a small amount of water can be used as a starting material.

As the lipase used in the present invention, for example, a lipase derived from a microorganism belonging to the genus Pseudomonas can be used. As such a lipase, a commercially available one can be used. For example, Pseudomonas fluorescens (Pseudomonas fluoresce
nce ) lipase (Amano Pharmaceutical Co., Ltd., Lipase AK),
Examples thereof include Pseudomonas sp. Lipase (Amano Pharmaceutical Co., Lipase P).

The use form of these lipases may be as it is, but a lipase immobilized on an immobilizing agent (for example, Celite, a ceramic carrier, etc.) may be used and is not particularly limited. The amount of lipase used may be appropriately determined depending on the reaction conditions and is not particularly limited. For example, add 2 to 5 mol of glycerin to 1 mol of oil (triglyceride), and add 20 mol per 1 g of the reaction mixture.
0 to 10,000 U of lipase can be used. Regarding the water content, for example, oil (triglyceride)
10 to 500 µl, preferably 10 to 200 µl can be added to 5 g.

The reaction is carried out as follows. That is, the lipase is allowed to act on the oil or fat in the presence of glycerin, and the temperature is lowered stepwise from 30 ° C to 5 ° C by a glycerolysis reaction. In this case, first, the reaction is performed at 30 ° C for 2 to 10 hours, and then at room temperature (15 to 25 ° C) for 16 hours. Further, the obtained reaction solution is cooled to 5 ° C. and reacted for 24 to 120 hours.

A partial glyceride rich in highly unsaturated fatty acids is extracted from the solution after the reaction. Extraction is the usual method,
For example, an alkali deoxidizing method, a solvent liquid distribution method, or the like is used. The analysis of the lipid composition is performed by, for example, gas chromatography, ear troscan method, or the like. In order to produce a partial glyceride containing a high concentration of DHA, first, an oil or fat is selectively hydrolyzed with a lipase to produce an oil or fat having a high DHA content, and this is subjected to glycerolysis using the lipase in the presence of glycerin.

That is, a lipase (for example, lipase of Candida cylindracea ) is allowed to act on fats and oils to carry out a hydrolysis reaction at 30 ° C to 40 ° C for 15 to 70 hours to give a highly unsaturated fatty acid-rich glyceride. To get Then, diglyceride is fractionated from the glyceride. Examples of the fractionation method include ordinary methods such as silica gel column chromatography, molecular distillation and vacuum precision distillation.

Next, the obtained highly-unsaturated fatty acid-rich glyceride or diglyceride fractionated from the glyceride is allowed to act again with lipase in the presence of glycerin to give 30
The glycerolysis reaction is carried out in the same manner as the above method, while gradually lowering the temperature from ℃ to 5 ℃. As the lipase used here, a lipase different from the above-mentioned lipase for hydrolysis, for example, lipase of Pseudomonas fluorescens (Amano Pharmaceutical Co., Ltd., Lipase AK), Pseudomonas genus ( Pseudomo )
nas sp.) lipase (Amano Pharmaceutical Co., Ltd., Lipase P)
Etc. are used.

Regarding lipid analysis and fatty acid composition analysis,
The same as above. The first hydrolysis gives a DHA-rich diglyceride containing 40 to 50% of DHA in the glyceride fraction. By performing the second lipase reaction using this diglyceride, a partial glyceride having a higher DHA content can be obtained. The partial glyceride produced by the present invention is subjected to treatment such as alkali washing, molecular distillation, vacuum precision distillation, steam distillation, solvent liquid-liquid distribution, low temperature crystallization fractionation, chromatography, etc., from the reaction mixture, or a combination thereof. Can be purified and fractionated.

The thus-obtained highly unsaturated fatty acid-rich partial glyceride is mainly composed of monoglycerides and / or diglycerides, and contains highly unsaturated fatty acids such as arachidonic acid, DHA, and EPA at high concentrations. Arachidonic acid, EPA, DHA and the like are essential fatty acids in the living body that are involved in the functions of the circulatory system and the central nervous system. Therefore, the highly unsaturated fatty acid-rich partial glyceride produced by the present invention is used as a drug or a health supplement. It is extremely useful as a food or a food for specified health use, and since the partial glyceride is an extremely excellent emulsifier, it is extremely useful as an emulsifier having the above physiologically active action.

[0024]

EXAMPLES The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples. [Example 1] Tuna oil (saponification value: 184.2, EPA: 5.
8%, DHA: 23.3%) 100 g, glycerin 20 g (molar ratio 1: 2), and distilled water 5 ml in the reaction system Pseudomonas fluorescens ( Pseudomonas fluorescens ) lipase (Amano Pharmaceutical Co., Ltd., Lipase AK, 26,800 U / g)
50,000 U (unit) was added and reacted with stirring. The reaction was carried out at 30 ° C. for 5 hours, room temperature for 16 hours and then at 5 ° C. for 24 hours.

An appropriate amount of the obtained reaction mixture was sampled, spotted on Chromalot S-III (manufactured by Yatron Co., Ltd.), and developed with a mixed solvent of benzene: chloroform: acetic acid (50: 20: 0.5 (v / v)). After that, the lipid composition was analyzed by using Iatroscan TH-10 (manufactured by Yatron Co., Ltd.). As a result, the lipid composition was triglyceride (TG) 26.1%, fatty acid (FFA) 6.8%,
Diglyceride (DG) 33.0%, Monoglyceride (MG) 34.1
% (% By weight).

It was thus found that about 70% of fish oil triglycerides can be converted into monoglycerides and diglycerides. The free fatty acid in the obtained reaction mixture was removed to the aqueous layer by the alkaline deoxidation method to obtain 90.8 g of a glyceride mixture consisting of triglyceride, diglyceride and monoglyceride. The fatty acid composition in the glyceride mixture was methyl esterified by a conventional method and then analyzed by capillary gas chromatography.

As a result, EPA was 5.7% and DHA was 24.6%. The obtained glyceride mixture has dramatically improved emulsification dispersibility and stability compared to the tuna oil used as the raw material, and 60 mg (as DHA without using other emulsifiers per 350 ml of the drink). 14.8 mg) could be added. This emulsifier did not show any oil dispersion or precipitate formation even after being sealed and stored at 5 ° C. and 25 ° C. for 60 days.
There was no fishy odor during storage at 5 ° C.

[Example 2] Tuna oil (saponification value: 184.2,
EPA: 5.8%, DHA: 23.3%) 100g, water 100g and Candida cylindrac
ea ) lipase (manufactured by Meito Sangyo Co., Ltd., Lipase OF, 360,
(000U / g) 16g at 30 ℃ while stirring the reaction mixture consisting of 0.2g.
The hydrolysis reaction was performed for a time. The reaction liquid after hydrolysis reached a sufficient equilibrium.

Then, the aqueous layer containing lipase was removed from the reaction solution to obtain a hydrolyzed oil. (The acid value of hydrolyzed oil is 13
2.1. ) Further, fatty acids liberated from the hydrolyzed oil were removed to the aqueous layer by an alkaline deoxidation method to obtain 33.5 g (yield 33.5%) of highly unsaturated fatty acid concentrated glyceride.
The acid value of this highly unsaturated fatty acid concentrated glyceride was 0.1. The fatty acid composition was analyzed by capillary gas chromatography after methyl esterification according to a conventional method.
As a result, EPA in constituent fatty acids was 5.1% and DHA was 4%.
It was 7.8%. Further, as a result of analyzing the lipid composition by an ear scan in the same manner as in Example 1, triglyceride
(TG) was 85.3%, diglyceride (DG) was 12.9%, and monoglyceride (MG) was 1.8%.

30 g of this glyceride mixture was purified and isolated by silica gel column chromatography to obtain 3.5 g of diglyceride fraction. This diglyceride was methyl-esterified by a conventional method and then analyzed by capillary gas chromatography. As a result, EPA was 4.1% and DHA was 53.2.
%Met.

Next, Pseudomonas fluorescens ( Pseudo ) was added to a reaction system containing 3 g of the obtained diglyceride, 0.4 g of glycerin (molar ratio 1: 1) and 150 μl of distilled water.
monas fluorescens) lipase (Amano Pharmaceutical Co. Ltd., Lipase AK, 26,800U / g) was added 1,500U (units) was allowed to react with stirring. The reaction was carried out at 30 ° C. for 5 hours, room temperature for 16 hours and then at 5 ° C. for 24 hours.

The reaction mixture obtained was analyzed for lipid composition by an ear scan in the same manner as in Example 1. The lipid composition was TG 18.8%, FFA 6.5%, DG 49.0%, MG 25.7%.
(% By weight). Thus, diglyceride isolated and purified by silica gel column chromatography from highly unsaturated fatty acid-rich fats and oils obtained by selectively hydrolyzing tuna oil with lipase, about 25% monoglyceride (MG ) Could be converted to.

The free fatty acid in the obtained reaction mixture was removed to the aqueous layer by the alkaline deoxidation method, and 2.8 g of a glyceride mixture consisting of triglyceride, diglyceride and monoglyceride.
I got The fatty acid composition in the glyceride mixture was analyzed by capillary gas chromatography after methyl esterification according to a conventional method.

As a result, the EPA was 4.5% and the DHA was 53.8%. The resulting glyceride mixture has dramatically improved emulsification dispersibility and stability compared to the tuna oil used as the raw material, and 60 mg (32.5 as DHA 32.5 ml) for 350 ml of the drink without using any emulsifier. (mg) could be added. This emulsifier did not show any oil dispersion or precipitate formation even after being sealed and stored at 5 ° C. and 25 ° C. for 60 days.
There was no fishy odor during storage at 5 ° C.

[0035]

INDUSTRIAL APPLICABILITY The present invention can provide a method for efficiently producing a partial glyceride having a high content of highly unsaturated fatty acids. Efficient production of highly unsaturated fatty acid-rich partial glycerides that have extremely excellent emulsifying dispersibility and stability in aqueous systems and that have physiological activity is widely used for pharmaceuticals, cosmetics, foods for specified health uses, and general foods. It is possible and extremely useful in industry.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kazuki Maruyama 16-2 Wadai, Tsukuba-shi, Ibaraki Maruha Stock Company Central Research Institute (72) Inventor Chikako Shiina 16-2 Wadai, Tsukuba-shi, Ibaraki Maruha Stock Company Central Research Institute (72) Inventor Hide Nakayama 16-2 Wadai, Tsukuba, Ibaraki Maruha Stock Company Central Research Laboratories

Claims (7)

[Claims] 1. A method for producing a partial glyceride having a high content of highly unsaturated fatty acids, which comprises reacting fats and oils with lipase in the presence of glycerin to carry out the reaction while gradually lowering the temperature from 30 ° C. to 5 ° C. . 2. A fat or oil is hydrolyzed with a lipase to obtain a highly unsaturated fatty acid-rich glyceride from the resulting reaction product, and the glycerin is allowed to act with lipase in the presence of glycerin to give a step from 30 ° C. to 5 ° C. A method for producing a partial glyceride having a high polyunsaturated fatty acid content, characterized in that a partial glyceride is obtained by carrying out a reaction while lowering the temperature. 3. An oil or fat is hydrolyzed with a lipase to obtain a glyceride having a high content of polyunsaturated fatty acids from the resulting reaction product, and the diglyceride obtained by fractionating the glyceride is reacted with the lipase in the presence of glycerin. A method for producing a partial glyceride having a high content of highly unsaturated fatty acids, characterized in that the reaction is carried out while gradually lowering the temperature from 30 ° C to 5 ° C to obtain a monoglyceride. 4. The method for producing a partial glyceride having a high polyunsaturated fatty acid content according to claim 1, wherein the partial glyceride is a monoglyceride and / or a diglyceride. 5. The method for producing a partial glyceride rich in highly unsaturated fatty acid according to claim 1, wherein the highly unsaturated fatty acid is docosahexaenoic acid, eicosapentaenoic acid, arachidonic acid or a combination thereof. . 6. The method for producing a partial glyceride having a high polyunsaturated fatty acid content according to claim 1, wherein the oil or fat is extracted from fish oil, krill, seaweed or fungi. 7. An emulsifier comprising a highly unsaturated fatty acid-rich partial glyceride obtained by the method for producing a highly unsaturated fatty acid-rich partial glyceride according to any one of claims 1 to 3.