JP2516860B2 - Method for producing concentrated highly unsaturated fatty acid-containing fats and oils - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a concentrated polyunsaturated fatty acid oil / fat, and more particularly to a method for producing a concentrated ω-3 highly unsaturated fatty acid-containing oil / fat. In the present specification, the ω-3 highly unsaturated fatty acid is a double bond in the third and fourth carbons from the terminal methyl of the carbon chain.
It also means an unsaturated fatty acid having 2 or more total double bonds in the carbon chain. Further, the highly unsaturated fatty acid-containing fat or oil means a fat or oil containing an unsaturated fatty acid triglyceride having a total number of double bonds of 2 or more.

[0002]

PRIOR ART AND PROBLEMS THEREOF ω-3 highly unsaturated fatty acid-containing fats and oils are different from beef tallow in which C ₁₈ fatty acid fats are the main component, and are often contained in marine foods such as various fish oils and liver oils. Recently, it has been said that it is necessary to take a well-balanced intake with ω-6 highly unsaturated fatty acids such as arachidonic acid and linoleic acid which are known as components of health foods. To balance this, ethyl esters of omega-3 highly unsaturated fatty acids have been used. However, the ethyl ester of this fatty acid has a lower degree of digestion and absorption as compared with the raw material oil and fat, that is, fatty acid triglyceride [eg Carol M. et al. Bio by Wojenshi et al.
chem. Biophys. Acta 1081 (19
91) 33-38]. On the other hand, as a means for hydrolyzing raw material fats such as fish oil to concentrate and produce ω-3 highly unsaturated fatty acids, a method using an enzyme is considered, and the decomposition rate of unsaturated fatty acids and saturated fatty acids when lipase is used. Method of using the difference of [T. Hoshino, T .; Yaman
e and S.E. Shimizu et al., Agric. Bio
l. Chem. 54 (1990), 1459-146.
7] Alternatively, after selectively hydrolyzing the ω-3 highly unsaturated fatty acid oil and fat using a lipase derived from Candida, the fatty acid and glycerin are treated by the lipase derived from a bacterium of the genus Chromobacterium. Method of concentrating ω-3 highly unsaturated fatty acids by reacting to synthesize triglyceride ["Practical bioreactor" by Tanaka, Imamura, and Kosuge et al., Food Industry Bioreactor System Technology Research Association Proceedings (1990) 391st to 41st
1 page] is known, but in these methods, ω-3
All of the processes of concentrating highly unsaturated fatty acids are placed only in the step of selectively hydrolyzing the raw fat or oil by the action of lipase, and it cannot be said that the method as a whole is efficient. Furthermore, as a method using lipase, hydrolyzing sardine oil,
A method of concentrating ω-3 highly unsaturated fatty acids and then using glycerin to synthesize triglycerides of these fatty acids by the action of a lipase also derived from a bacterium of the genus Chromobacterium [Nagata, Takahashi, Hatano and Hosokawa et al. Water magazine 57 (1991), pp. 119-125] is known, but the yield of triglyceride by this method is 5
It is less than 0% and cannot be said to be an efficient method. As described above, ω-3 highly unsaturated fatty acid-containing fats and oils are desired to be ingested as health foods, but these are easily oxidized at the unsaturated bond portion in the molecule, and double bond transfer and isomerization are also likely to occur. Is likely to occur and is very expensive in price. Therefore, it is desired to obtain an oil / fat containing a higher proportion of ω-3 highly unsaturated fatty acid.

[0003]

DISCLOSURE OF THE INVENTION The present invention efficiently hydrolyzes highly unsaturated fatty acid-containing fats and oils, particularly ω-3 highly unsaturated fatty acid-containing fats and oils without causing modification of this fatty acid, and further hydrolyzes It is an object of the present invention to provide a method for producing a highly concentrated triglyceride of highly unsaturated fatty acid by concentrating this highly unsaturated fatty acid from a decomposition product and then allowing glycerin to act.

[0004]

The present inventors have completed the present invention as a result of intensive investigations to solve the above-mentioned problems.

According to the present invention, a highly unsaturated fatty acid-containing oil or fat or an alcohol ester of a highly unsaturated fatty acid is hydrolyzed using an immobilized lipase containing a lipase produced by Pseudomonas sp. Step (A), Step (B) of concentrating higher unsaturated fatty acid from the obtained hydrolysis product using a non-alcoholic light solvent, from the obtained concentrate and its 8-10 wt% glycerin A method for producing a concentrated highly unsaturated fatty acid-containing oil or fat, which comprises the step (C) of contacting a mixture of the following with an immobilized lipase containing a lipase produced by a Candida genus, and converting the mixture into triglyceride: Provided. Since the polyunsaturated fatty acids are chemically unstable, it is preferable to carry out the above steps in an atmosphere of an inert gas.

The present invention selectively hydrolyzes highly unsaturated fatty acid-containing fats and oils by the action of a lipase produced by Pseudomonas, concentrates the obtained ω-3 highly unsaturated fatty acid, and then lipase derived from a microorganism. It is intended to obtain concentrated ω-3 highly unsaturated fatty acid-containing oil or fat by reacting with glycerin to give triglyceride.

The fats and oils used as a raw material in the present invention are highly unsaturated fatty acid triglycerides, particularly fats and oils containing a high proportion of ω-3 highly unsaturated fatty acid triglycerides, for example, 10% by weight or more. Organisms that eat planktonic or freshwater plankton, such as fish oils and fats, generally called fish oils, are fish oils such as sardines, mackerel, and the like, or liver oils of these fishes. Also, an alcohol ester obtained by alcoholyzing an oil or fat containing ω-3 highly unsaturated fatty acid such as sardine oil with an alcohol such as ethanol can be used as a raw material of the present invention.

The hydrolysis of the fat or oil in the present invention is carried out by biochemical means using lipase produced by Pseudomonas sp., Without using chemical means such as alkali. This lipase hydrolyzes an acyl group of a highly unsaturated fatty acid-containing oil or fat to produce a fatty acid and glycerin. Pseudomonas lipase used in the present invention is preferably used by fixing it to a carrier usually, and among them, lipase obtained by fixing the lipase derived from Pseudomonas bacteria to an anion exchange resin is a reaction product of hydrolysis. It is stable for a long time even at a temperature of 50 to 60 ° C, which has fluidity, and has good decomposability of oils and fats such as fish oil. To generate. The chemical composition during decomposition of fish oil is undecomposed triglyceride, partially decomposed diglyceride and completely decomposed fatty acid, and the ratio of monoglyceride is extremely small.
This monoglyceride is not preferable because it has a large surface-active effect and thus makes it difficult to separate the produced fatty acid, but a small amount of this monoglyceride increases the separation efficiency. In addition, eicosampentaenoic acid tends to be concentrated in undegraded glyceride. The hydrolysis rate of fats and oils is expressed by weight% of fats and oils that have been decomposed into fatty acids,
It is 66% or more, preferably 80 to 99% by weight.

In order to carry out this hydrolysis, a light feedstock fat and oil is countercurrently contacted with water, which is filled with a lipase derived from Pseudomonas fixed in a reactor. It is preferable that the container be of an upright type so that the flow containing the fatty acid and the flow containing glycerin and water are well separated, and that these flows flow countercurrently by gravity.
The reaction temperature at this time is generally 40 to 70 ° C. and 50 to 70 ° C.
It is preferable to maintain the temperature at 60 ° C. When carrying out this hydrolysis reaction, it is preferable to maintain the reactor in an atmosphere of an inert gas, and as the inert gas, nitrogen, argon, carbon dioxide, neon or the like can be used, and the air in the reactor is Substitute with one of these inert gases. Since carbon dioxide and argon have large specific gravities, they are gases convenient for substitution treatment. By making the reaction zone an atmosphere of an inert gas, it is possible to prevent the double bond in the highly unsaturated fatty acid from forming a peroxide bond due to the action of oxygen and increasing the peroxide value. In addition, in this reaction, a non-alcoholic water-immiscible light solvent can be used in order to promote separation of light components such as fatty acids and unreacted glycerides from water. As used herein, “light” means smaller in specific gravity than water, that is, lighter than water. This water-immiscible light solvent includes, for example, isooctane, octane,
Hydrocarbons such as pentane, heptane, hexane and other hydrophobic solvents are mentioned. This solvent may be added to the fat or oil before the reaction, or may be added to the reaction system at any time during the reaction. The addition of this solvent facilitates the separation of products which are separated from water as low specific gravity products. Especially when isooctane is used, oil-water separation is extremely good, and if isooctane is added before the reaction,
It is more preferable because the hydrolysis rate is improved. The reaction can be carried out continuously or discontinuously, and when the hydrolysis reaction is carried out batchwise, it generally requires 15 to 20 hours. The product containing polyunsaturated fatty acids is separated from the high density product containing water and glycerin as a low density product.

The low specific gravity product containing the highly unsaturated fatty acid obtained by the hydrolysis reaction and separated from the high specific gravity product is dissolved in a light solvent, separated and, if necessary, purified. Concentrate fatty acids. A light solvent is used as a solvent for the dissolution, separation, and purification treatments. As such a light solvent, a non-alcoholic light solvent which does not have an alcoholic OH group which may generate a fatty acid ester in the molecule, for example, acetone, a ketone such as methyl ethyl ketone, pentane, hexane, peptane,
Hydrocarbons such as octane and isooctane, and other ethers are used. The presence of alcohol ester of highly unsaturated fatty acid in the hydrolysis product lowers the yield when converting the highly unsaturated fatty acid to triglyceride in the final step. Therefore, as a light solvent, an alcoholic OH group is included in the molecule. Use a solvent as described above that does not have.

Concentration of the polyunsaturated fatty acid is carried out by a method known per se, for example, by dissolving the low specific gravity product in a light solvent, evaporating the solvent from the obtained solution, and then performing low temperature fractional crystallization. Method, salt formation method, urea addition method,
An adsorption separation method or the like is used. It is desirable that this concentration step is also performed in an atmosphere of an inert gas to prevent denaturation of highly unsaturated fatty acids due to oxidation.

When a water-immiscible light solvent is used during hydrolysis, the solvent used for concentration is preferably the same solvent as that used for concentration in order to simplify the recovery of the solvent. Of course, it is possible to use different solvents for the concentration and the concentration. In this concentration step, the concentration of highly unsaturated fatty acid can be concentrated to about 2 to 6 times the concentration before concentration.

The highly unsaturated fatty acid concentrate obtained by evaporating the solvent is then reacted (esterified) with glycerin to convert it into triglycerides. The water generated in this triglyceride synthesis is removed by dehydration at the same time as esterification. This synthetic reaction is carried out by a catalytic reaction with immobilized lipase containing lipase produced by Candida. This immobilized lipase can maintain its activity even in a substantially anhydrous state having a water content of 100 ppm or less, and is suitable for the synthesis of highly unsaturated fatty acid triglyceride. Examples of such immobilized lipase include those in which lipase derived from Candida is immobilized on acrylic resin.
When a lipase derived from a bacterium belonging to the genus Mucor, which has been conventionally known to be effective for conversion into triglyceride, is used, synthesis of docosahexaenoic acid triglyceride is inferior. The present invention is extremely excellent in terms of conversion efficiency into triglycerides, particularly in terms of conversion efficiency of docosahexaenoic acid into triglycerides, since lipase produced by Candida is used as the lipase. When alcohol esters are present in the polyunsaturated fatty acid concentrate, the substrate specificity of the lipase results in a poor yield of triglyceride as compared with the case where free fatty acids are used. Therefore, care must be taken in selecting the solvent used in the hydrolysis step and the concentration step in the present invention.

The glycerin used in the synthesis of this triglyceride is near the stoichiometric amount of this reaction.
Glycerin is used in an amount of 8 to 10% by weight of the polyunsaturated fatty acid concentrate used, calculated from the molecular weight of the fatty acids, taking into account that the polyunsaturated fatty acids are modified during the reaction.

The dehydration treatment carried out at the same time as the catalytic reaction of the immobilized lipase in the present invention employs a vacuum dehydration method or a dry inert gas aeration method. At this time, the water present in the reaction system and the water produced by the reaction between the glycerin and the polyunsaturated fatty acid concentrate are dehydrated together by the action of the solidified lipase, so that the water concentration in the reaction system is almost anhydrous.
It is set to 00 ppm or less. If alcohol ester is present in this triglyceride synthesis reaction, the yield of triglyceride is reduced due to the substrate specificity of lipase as described above, and it is necessary to pass the by-produced alcohol through a column packed with activated carbon for dealcoholization.

In the above triglyceride synthesis reaction,
The reaction temperature is preferably low in order to prevent migration of double bonds in the polyunsaturated fatty acid, but in view of the enzymatic reaction, the reaction temperature is 30 to 60 ° C, preferably 40 ° C.
~ 60 ° C. Although the time required for the reaction depends on the temperature and other conditions, it is generally 24 to 48 hours.

The polyunsaturated fatty acid triglyceride obtained by the above reaction can be purified by various methods such as column treatment. It is convenient to use ether or hexane as a solvent and perform purification with a column packed with basic alumina or a column packed with silica gel activated with magnesium to obtain triglyceride from which peroxide is also removed. 80% or more of the products obtained by the above triglyceride synthesis reaction are highly unsaturated fatty acid triglycerides. The product was treated with a column packed with basic alumina using ether as a solvent, treated with an ether eluate from which adsorbed substances were removed, and the eluate was evaporated to about 99%.
% Or more of highly pure highly unsaturated fatty acid triglyceride is obtained. When alcohol ester is mixed in the polyunsaturated fatty acid, the yield of triglyceride is deteriorated, and it becomes necessary to carry out dealcoholization treatment for triglyceride synthesis at the same time, which complicates the reaction operation. It is desirable to carry out this step in an inert gas atmosphere.

According to a preferred example of the method of the present invention, by the action of the immobilized lipase produced by immobilizing a lipase derived from a bacterium of the genus Pseudomonas on an anion exchange resin,
By hydrolyzing an oil / fat containing ω-3 highly unsaturated fatty acid such as sardine oil to a decomposition rate of 80% or more, a low specific gravity decomposition product containing 80% or more of free ω-3 highly unsaturated fatty acid can be obtained. At this time, undegraded glyceride did not contain monoglyceride having large surface activity and eicosapentaenoic acid was concentrated. In addition, if the raw material ω-3 highly unsaturated fatty acid-containing fats and oils is dissolved in isooctane as a water-immiscible light solvent and hydrolyzed, the separation of oil and water is promoted without adversely affecting the enzymatic fat and oil hydrolysis. In the case where the steps are continuously carried out, the process of recovering the low specific gravity decomposition product, which is a product, becomes easy. Esterification of the fatty acid can be prevented by dissolving the product containing the ω-3 highly unsaturated fatty acid thus obtained in hexane, which is a solvent having no alcoholic OH group, and performing concentration treatment.
Next, a immobilized lipase produced by immobilizing a lipase produced by a Candida genus on, for example, an acrylic resin is used to react concentrated ω-3 highly unsaturated fatty acid with glycerin to produce ω-3 highly unsaturated docosahexaenoic acid. Unsaturated fatty acids can also be efficiently synthesized into triglycerides.

[0019]

EXAMPLES The method of the present invention will now be described in more detail with reference to examples.

Example 1 Pseudomonas Fluorescens (Pseudomon)
us fluorence) Lipase produced by biotype I is immobilized on anion exchange resin (Enchiron PF, manufactured by Rakuto Kasei Co., Ltd.) 1 g, sardine oil 1 g
Then, 1 g of water was placed in a 50 ml Erlenmeyer flask, a silicon stopper was attached, and the mixture was shaken at 50 ° C. The fatty acid fraction was analyzed by gas chromatography (WCOT CP-Si) at each reaction time.
Table 1 shows the results of fatty acid analysis by applying 188 Chrompack. The decomposition rate was obtained from the ratio of the acid value and the saponification value. Looking at Table 1, if the decomposition rate is 66% or more, eicosapentaenoic acid (C20: 5) having 5 carbons and 20 double carbons, which has the same carbon number as methanol decomposition, and 22 carbons, is formed. It was found that docosahexaenoic acid having 6 (C22: 6) was obtained.

[0021]

[Table 1]

Example 2 The reactor shown in FIG. 1 was prepared. This reactor is a countercurrent contact type two-phase system reactor. The partition plate 5 and the emulsion destruction device 10 are stainless steel mesh partition plates having 75 micron stitches. Although not shown in the drawing, the reaction tank is surrounded by a mantle, and the temperature is kept constant by introducing and discharging a constant temperature liquid. Conical stationary tanks 2 and 8 are provided at the upper and lower ends, a low specific gravity product discharge pipe 1 is provided at the upper end of the stationary tank 2 at the upper end, and a high specific gravity product discharge pipe is provided at the stationary tank 8 at the lower end. There is 9. Among the six stirring tanks in the middle, a high specific gravity substrate supply pipe 3 is provided in the upper stirring tank, and a low specific gravity substrate supply pipe 7 is provided in the lower stirring tank. The diameter of each tank is 50 mm and the height is 26-30
The volume in the reactor including the upper and lower stationary tanks in mm is 421.
It was ml. The total amount of immobilized enzyme (ENTILON PF) added to the six stirring tanks 4 was 64.6 g. The high specific gravity product discharge pipe 9 is connected to a joint having a branch pipe divided into three directions in the direction of 120 degrees through one of the branch pipes, and the other one of the branch pipes 11 is connected to the pulse generator 6. A liquid feed pump is connected to the other branch pipe 12 connected to the other. The pulse generator 6 is designed so that a pulsating flow flows 15 times per minute. The size of one pulsating flow is about 15 ml. This pulsating flow has the function of mixing the contents of the stirring tank. The high specific gravity product discharge pipe 9 is closed, water is supplied from the high specific gravity substrate supply pipe 3, and sardine oil is filled from the low specific gravity substrate supply pipe 7 in the reactor such that the ratio of both is about 1: 1. Two-phase separation is achieved by moving only the pulse generator 6 overnight while it is stopped. Although not shown, the substrate is saturated with nitrogen gas and then stored in a container under pressure of nitrogen gas. Low specific gravity substrate supply pipe 7 while operating the pulse generator 6
More sardine oil was supplied at 5 ml / hour, water was supplied at 2.5 ml / hour from the high specific gravity substrate supply pipe 3, and high specific gravity products containing 120 to 190 mg / ml of glycerin were discharged from the high specific gravity product discharge pipe 1 to 1 to 1. When it was collected at 3 ml / hour, low specific gravity products were collected at 3 to 5 ml / hour. The low specific gravity product was stored in a container at −90 ° C. in which the atmosphere in the container was replaced with nitrogen. After operating the reactor for 840 hours under the above conditions, the conditions were changed to 30% as a water-immiscible light solvent in sardine oil.
% Hexane was added for 296 hours, 30% isooctane was added for 240 hours, and the same conditions as at the beginning but with a reaction time of 96 hours. The results are shown in Table 2.

[0023]

[Table 2]

TG in Table 2 is unreacted triglyceride, DG
Represents diglyceride, MG represents monoglyceride, FA represents fatty acid, and HPLC column (SHODEX GPC Kf
-802 It was calculated by Showa Denko KK 300x3). When isooctane was added, the decomposition rate was increased by 6 to 7% as compared with the case where no solvent was used, and oil-water separation was also good. In addition, the immobilized lipase produced less monoglyceride having a strong surface-active effect which adversely affects oil-water separation.

Table 3 below shows the fatty acid composition in sardine oil and solvent-free decomposition products (decomposition rate 81%). For the glyceride and fatty acid fractions, the degradation products were fractionated by thin layer chromatography. Looking at Tables 2 and 3, the undegraded diglyceride moiety (DG) was found to be ω-3 eicosapentaenoic acid (C20: 5) and docosapentaenoic acid (C22:
5), and docosahexaenoic acid (C22: 6) are concentrated but are recovered in the low specific gravity product. In Table 3, the underlined numbers are for ω-3 highly unsaturated fatty acids.

[0026]

[Table 3]

Example 3 A low specific gravity product with the addition of isooctane of Example 2 (decomposition rate 8
190 g of hydrolyzate obtained by evaporating isooctane to 1%)
In order to concentrate by the salt forming method, 1330 g of 7 times amount of acetone and 202 ml of 4N potassium hydroxide methanol solution were added as a light solvent and stirred for 30 minutes at room temperature.
It was left at 0 ° C. for 16 hours. Then, 582 g of acetone was added to 58.2 g of the filtrate except the portion precipitated as a potassium salt, and the mixture was allowed to stand at -20 ° C for 16 hours. After removing the precipitated portion, the filtrate was acidified with 4N hydrochloric acid to evaporate acetone, and then hexane was added to the solution and the solution was washed with water until the pH became 7. Hexane was evaporated to give 24.4 g of ω-3 highly unsaturated fatty acid concentrate. To 1 g of this concentrate, 0.09 g of glycerin and 0.1 g of immobilized lipase sp382
10 to 20 Torr at 60 ° C. for 24 hours
The mixture was degassed, and the mixture was shaken at 60 ° C. with vacuum drying. Immobilized lipase sp382 (manufactured by Novo) is obtained by immobilizing lipase produced by Candeda antactica on an acrylic resin. The composition of this reaction product after 24 hours was as follows: triglyceride 68.3%, diglyceride 2
4.7%, monoglyceride 3.7% and fatty acid 3.3
%Met. This reaction product was dissolved in ethyl ether, the immobilized enzyme was filtered off, and then purified by a basic alumina column. Table 4 shows the composition of each stage and the fatty acid composition.

[0028]

[Table 4]

In the table, the underlined numbers are for ω-3 highly unsaturated fatty acids. According to this method, in the case of eicosapentaenoic acid and docosahexaenoic acid, it was found that both were concentrated about 5 times by the concentration treatment. Further, since lipase derived from a bacterium belonging to the genus Candida is used as an enzyme for the synthesis of triglyceride, the incorporation of docosahexaenoic acid (C ₂₂ : 6) into triglyceride was good, and the amount of triglyceride synthesized was 68.3%. .

Comparative Example 3 To the solventless low specific gravity product (decomposition rate 81%) of Example 2, hexane was added and dehydrated with anhydrous sodium sulfate, and 35 g of the decomposition product obtained by evaporating hexane was concentrated by the low temperature fractional crystallization method. Therefore, add 7 times the amount of acetone as a light solvent, and
The mixture was left at 0 ° C overnight, filtered, washed with acetone at -50 ° C, and the filtrate and acetone in the washing liquid were evaporated to remove the primary concentrate 1
8.72 g was obtained. Add 7 times more acetone again-80
After standing overnight at 0 ° C., 10.31 g of a secondary concentrate was obtained in the same manner. Glycerin (0.0714 g) and ribozyme IM60 (0.1 g) were added to the secondary concentrate (1 g) to obtain 10 to 20 Torr (To
The mixture was degassed to rr) and dried under vacuum, and a shaking reaction was performed at 60 ° C. Lipozyme IM60 (manufactured by Novo) is obtained by immobilizing lipase produced by Mucor Mihay on an anion exchange resin having macropores. The composition of the reaction product after 48 hours was as follows: triglyceride 61.2%, diglyceride 22.0%, monoglyceride 2.8% and fatty acid 1
It was 3.9%. This reaction product was dissolved in ethyl ether and the immobilized enzyme was filtered off, followed by a basic alumina column (aluminum oxide 90 (product number 107 manufactured by Merck).
6) and the alkaline deoxidation method. Table 5 shows the composition of each stage and the fatty acid composition. In the table, all underlined numbers are for ω-3 highly unsaturated fatty acids, and as a result, in the case of eicosapentaenoic acid and docosapentaenoic acid, the results of the concentration by the low temperature fractional crystallization method with acetone, It can be seen that each of the secondary concentrates was twice concentrated, but these fatty acids show poor uptake of docosahexaenoic acid (C ₂₂ : 6) into triglycerides due to reaction with glycerin.

[0031]

[Table 5]

[0032]

EFFECT OF THE INVENTION ω-3 highly unsaturated fatty acids have important physiological significance such as a preventive effect on cardiovascular diseases and an effect of improving learning ability of rats. According to the present invention, an oil and fat such as fish oil which is rich in ω-3 highly unsaturated fatty acid and can be supplied and obtained in a large amount is used as a raw material, and the enzyme reaction is used to once hydrolyze the oil and fat containing ω-3 highly unsaturated fatty acid. By decomposing it into fatty acid, separating and concentrating it from other fatty acids by a concentrating method such as low temperature fractional crystallization method, and again reacting with glycerin using enzymatic reaction, ω-3 highly unsaturated fatty acid triglyceride It can be obtained efficiently. The raw material fats and oils at this time are inexpensive, all the reactions can be carried out under mild conditions, and the ω-3 highly unsaturated fatty acids can be highly concentrated without causing substantial alteration. Therefore, the present invention not only serves as a functional food but also contributes to medical and pharmaceutical applications.

[Brief description of drawings]

FIG. 1 is an explanatory sectional view of a countercurrent contact type two-phase system immobilized enzyme packed reactor.

[Explanation of symbols]

 1. Low specific gravity product discharge pipe 2. Top standing layer 3. High specific gravity substrate supply pipe 4. Stirring tank 5. Partition plate 6. Pulse generator 7. Low specific gravity substrate supply pipe 8. Bottom layer at bottom 9. High specific gravity product discharge pipe 10. Emulsion breaker 11. Branch pipe connected to pulse generator 12. Branch pipe connected to metering pump

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Naoki Higashi 2-171-1, Hinode 2-chome, Funabashi City, Chiba Prefecture Hiroshi Taniguchi, Examiner, Bosso Yushi Co., Ltd. (56) Reference JP-A-62-278991 (JP, A) ) JP-A-62-91188 (JP, A)

Claims (5)

(57) [Claims] 1. A step (A) of hydrolyzing a highly unsaturated fatty acid-containing oil or fat or an alcohol ester of a highly unsaturated fatty acid using an immobilized lipase containing a lipase produced by Pseudomonas sp. A step (B) of concentrating the higher unsaturated fatty acid from the obtained hydrolysis product using a non-alcoholic light solvent, a mixture comprising the obtained concentrate and 8 to 10% by weight of glycerin, A method for producing a concentrated highly unsaturated fatty acid-containing oil or fat, which comprises the step (C) of bringing into contact with an immobilized lipase containing a lipase produced by a Candida bacterium to convert it into triglyceride. 2. The method for producing a concentrated highly unsaturated fatty acid-containing fat according to claim 1, wherein the highly unsaturated fatty acid is an ω-3 highly unsaturated fatty acid. 3. The method for producing a concentrated highly unsaturated fatty acid-containing fat according to claim 1, wherein the step (A) is performed in the presence of a non-alcoholic water-immiscible light solvent. 4. The method for producing a highly unsaturated fatty acid-containing oil or fat according to claim 3, wherein isooctane is used as the water-immiscible light solvent. 5. The highly unsaturated fatty acid-containing fat according to claim 1, wherein at least one of the steps (A), (B) and (C) is carried out in an atmosphere of an inert gas. Production method.