JP6715586B2 - Method for producing highly unsaturated fatty acid - Google Patents

Description

The present invention relates to a method for producing highly unsaturated fatty acids.

With the recent increase in health orientation, many studies have been conducted on the function of fatty acids in fats and oils. Above all, the physiological activity of polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (C20:5, EPA) and docosahexaenoic acid (C22:6, DHA), which are the constituents of fish oil, has been attracting attention. , Is actively used as a raw material for medicines and health foods.

Most of the industrially used highly unsaturated fatty acids are produced from fats and oils rich in them, such as fish oil, by utilizing an enzymatic reaction. Generally, first, the fatty acid selectivity of lipase is used to release a fatty acid having a small number of carbon atoms from a fat or oil containing a polyunsaturated fatty acid, and a glyceride fraction enriched with the polyunsaturated fatty acid is obtained, followed by further hydrolysis. Degradation is performed to separate out fatty acids containing a high concentration of highly unsaturated fatty acids.
Therefore, various techniques for increasing the hydrolysis rate and the concentration of highly unsaturated fatty acids have been studied, and for example, fats and oils containing highly unsaturated fatty acids are hydrolyzed using a lipase derived from the genus Candida to produce highly unsaturated fatty acids. After obtaining a fatty acid-concentrated oil and fat, it is hydrolyzed using a lipase derived from the genus Penicillium, and a method for collecting a fatty acid containing a high concentration of highly unsaturated fatty acid (Patent Document 1), using a lipase. After hydrolyzing fats and oils containing polyunsaturated fatty acids to obtain concentrated fats and oils of polyunsaturated fatty acids, two specific lipases are simultaneously acted on to hydrolyze them to increase the concentration of polyunsaturated fatty acids. A method for obtaining the contained fatty acid (Patent Document 2) and the like have been reported.

Publication of JP-A-7-51075 JP 2004-208546 A

In the said patent document 2, since the lipase which acts on triglyceride and partial glyceride lipase are made to act simultaneously with respect to the fats and oils which concentrated the polyunsaturated fatty acid, the highly unsaturated fatty acids concentrated on all the triglyceride, diglyceride, and monoglyceride in fats and oils. Saturated fatty acids can be used, and highly unsaturated fatty acids can be obtained in a higher yield than in Patent Document 1.
However, the lipase used in Patent Document 2 has a problem that it takes a long time to hydrolyze the fats and oils in which the highly unsaturated fatty acid is concentrated.
Therefore, the present invention seeks to provide a new method for producing a highly unsaturated fatty acid in a short time and in a high yield.

The present inventor has conducted diligent research in view of the above problems and found that fats and oils containing highly unsaturated fatty acids as constituent fatty acids are lipases and partial glyceride lipases that preferentially decompose the 1,3-position derived from the genus Alcaligenes. It was found that the hydrolysis proceeds in combination with and the reaction proceeds in a short time, and highly unsaturated fatty acid can be obtained in a high yield.

That is, the present invention hydrolyzes fats and oils containing a polyunsaturated fatty acid as a constituent fatty acid by simultaneously acting a lipase and a partial glyceride lipase that preferentially decompose the 1,3 position derived from the genus Alcaligenes. And a step of obtaining a fatty acid containing a highly unsaturated fatty acid, the method for producing a highly unsaturated fatty acid is provided.

According to the present invention, oils and fats containing highly unsaturated fatty acids can be efficiently hydrolyzed in a short time, and highly unsaturated fatty acids can be obtained in high yield.

The method for producing a polyunsaturated fatty acid of the present invention comprises a lipase and a partial glyceride lipase that preferentially decompose the 1,3-position derived from the genus Alcaligenes with respect to an oil or fat containing a polyunsaturated fatty acid as a constituent fatty acid. A step of simultaneously acting and hydrolyzing to obtain a fatty acid containing a polyunsaturated fatty acid.

In the present invention, a polyunsaturated fatty acid (PUFA) means a fatty acid having 3 or more double bonds and having 20 or more carbon atoms. Specific examples thereof include eicosapentaenoic acid (C20:5, EPA), docosahexaenoic acid (C22:6, DHA), arachidonic acid (C20:4, AA), and the like. Acids and docosahexaenoic acid are preferred.

The oil or fat containing a polyunsaturated fatty acid as a constituent fatty acid is a glyceride in which at least one polyunsaturated fatty acid is added to glycerin through an ester bond, and is triglyceride, monoglyceride, diglyceride or a mixture of two or more thereof. is there. Incidentally, glycerides to which polyunsaturated fatty acids are not bound may be included as constituent fatty acids.
Examples of oils and fats containing highly unsaturated fatty acids as constituent fatty acids generally include microbial oils such as fish oil and algae oil, and these may be used alone or in combination of two or more kinds. Fish oil is a marine animal oil and fat, and can be collected from raw materials such as sardines, herring, saury, mackerel, bonito, tuna, whale, squid, cod liver and the like. Further, algal oil can be collected from algae belonging to the classes Chlorophyta, Diatoms, and the like. Prior to the enzymatic reaction, it is preferable to appropriately perform known separation means and purification means such as liquid-liquid separation, filtration, and centrifugation in order to remove impurities.
Further, a so-called highly unsaturated fatty acid concentrated oil in which the ratio of highly unsaturated fatty acids in the fatty acids constituting the fats and oils is increased may be used. Examples of the method for increasing the ratio of highly unsaturated fatty acids in the constituent fatty acids include conventionally known methods, for example, a method of preferentially releasing and removing fatty acids other than highly unsaturated fatty acids using lipase, a solvent fractionation method, and the like. And either method can be used.
Further, commercially available fats and oils containing highly unsaturated fatty acids and highly unsaturated fatty acid concentrated oils may be used.

In fats and oils containing a polyunsaturated fatty acid as a constituent fatty acid, the content of DHA with respect to the total fatty acids constituting the fats and oils is preferably 10 to 75% by mass from the viewpoint of increasing the DHA concentration in the glyceride, and further 15 It is preferably -65% by mass, more preferably 20-55% by mass. In addition, the fatty acid amount in this specification is a free fatty acid conversion amount.

Oils and fats containing highly unsaturated fatty acids as constituent fatty acids are oils and fats containing 0.1 to 75% by mass, further 0.5 to 65% by mass, and further 1 to 60% by mass of diglyceride. It is preferable from the viewpoint of making it easier.
In addition, in fats and oils containing a polyunsaturated fatty acid as a constituent fatty acid, the content of triglyceride is 25 to 99.8% by mass, further 35 to 99.5% by mass, and further 40 to 99% by mass. From the viewpoint of.
In addition, the content of monoglyceride in the oil and fat containing highly unsaturated fatty acid as a constituent fatty acid is 0.01 to 10% by mass, further 0.1 to 7% by mass, and further 0.5 to 5% by mass. , Are preferable from the same point.
Further, the content of the free fatty acid or its salt is preferably 0.01 to 10% by mass in the oil and fat containing the highly unsaturated fatty acid as a constituent fatty acid, and further 0 from the viewpoint of industrial productivity of the oil and fat and suppression of deterioration. 0.02 to 5 mass% is preferable.

The lipase that preferentially decomposes the 1,3-position used in the present invention is a lipase originating from the genus Alcaligenes, and shows specificity at the sn-1 position and the sn-3 position of triglycerides.
Examples of commercially available lipases that preferentially decompose the 1,3-position derived from the genus Alcaligenes include Lipase QLM (manufactured by Meito Sangyo Co., Ltd.).

As the lipase from the genus Alcaligenes that preferentially decomposes at the 1- and 3-positions, it is preferable to use an immobilized lipase in which the lipase is immobilized on a carrier, from the viewpoint of effective use of lipase activity and cost. The immobilization carrier for immobilizing lipase and the immobilization method are the same as those for the partial glyceride lipase described later.

The partial glyceride lipase used in the present invention is a lipase which hydrolyzes monoglyceride and diglyceride but hardly hydrolyzes triglyceride. Therefore, by allowing a partial glyceride lipase to act on an oil or fat containing a highly unsaturated fatty acid as a constituent fatty acid, the partial glyceride in the oil or fat is mainly decomposed.
Examples of the partial glyceride lipase include monoglyceride lipase or diglyceride lipase derived from animal organs such as rat small intestine and porcine adipose tissue; Bacillus sp. H-257 derived monoglyceride lipase (J. Biochem., 127, 419-425, 2000), Pseudomonas sp. LP7315-derived monoglyceride lipase (Journal of Bioscience and Bioengineering, 91(1), 27-32, 2001), Penicillium syropium-derived lipase (J. Bim. -211, 1980), Penicillium camemberti (Penicillium camemberti) U-150-derived lipase (J. Fermentation and Bioengineering, 72(3), 162-167, 1991) and the like. Among them, a partial glyceride lipase derived from Penicillium camemberti is preferable.
Examples of commercially available products include "Monoglyceride lipase (MGLPII)" (Asahi Kasei Corp.), "Lipase G "Amano"50" (Amano Enzyme Corp.) and the like.

As the partial glyceride lipase, it is preferable to use an immobilized partial glyceride lipase obtained by immobilizing the lipase on a carrier, because the lipase activity can be effectively used.
As the immobilizing carrier, Celite, diatomaceous earth, kaolinite, silica gel, molecular sieves, porous glass, activated carbon, calcium carbonate, inorganic carriers such as ceramics, ceramic powder, polyvinyl alcohol, polypropylene, chitosan, ion exchange resin, hydrophobic adsorption Examples thereof include organic polymers such as resins, chelate resins, and synthetic adsorption resins. Of these, ion exchange resins are preferable because of their high water retention capacity. Further, among the ion exchange resins, the ion exchange resin is preferably porous because it has a large surface area so that the adsorption amount of the enzyme can be increased.

The particle size of the resin used as the immobilization carrier is preferably 50 to 2000 μm, more preferably 100 to 1000 μm. The pore size is preferably 10 to 150 nm, more preferably 10 to 100 nm. Examples of the material include phenol formaldehyde-based, polystyrene-based, acrylamide-based, divinylbenzene-based, and the like, and phenol-formaldehyde-based resin (for example, Duolite A-568 manufactured by Rohm and Haas) is preferable from the viewpoint of improving lipase adsorption.
At this time, the amount of lipase used is preferably 10 to 300% by mass, more preferably 30 to 200% by mass, and further preferably 50 to 150% by mass based on the mass of the carrier from the viewpoint of industrial productivity. At the time of immobilization, the lipase is brought into a solution state, but it is preferable to use it after adjusting the pH to 3 to 7 using a buffer. The temperature at the time of immobilization is preferably 0 to 60°C, more preferably 3 to 40°C.

In order to enhance the activity of the immobilized lipase, a treatment for adsorbing the fat-soluble fatty acid or its derivative on the carrier may be performed before the immobilization of the lipase. Examples of the treatment method include a method in which the fat-soluble fatty acid or its derivative is once dispersed and dissolved in an organic solvent such as chloroform, hexane, and ethanol, and then added to a carrier dispersed in water.
Examples of the fat-soluble fatty acid used include saturated or unsaturated, linear or branched, fatty acids having 8 to 18 carbon atoms, which may be substituted with a hydroxyl group. Specific examples include capric acid, lauric acid, myristic acid, oleic acid, linoleic acid, α-linolenic acid, and ricinoleic acid. Examples of the derivatives include esters of these fatty acids with monohydric or polyhydric alcohols, phospholipids, and derivatives obtained by adding ethylene oxide to these esters. Specific examples thereof include methyl ester, ethyl ester, monoglyceride, diglyceride of the above fatty acids, ethylene oxide adducts thereof, polyglycerin ester, sorbitan ester, sucrose ester and the like. Two or more kinds of these fat-soluble fatty acids or derivatives thereof may be used in combination.

The lipase that preferentially decomposes the 1,3 position derived from the genus Alcaligenes has a titer of 100 U/g or more, further 1,000 to 1,000,000 U/g, further 10,000 as measured by the Dole method. It is preferably in the range of up to 500,000 U/g.
The partial glyceride lipase preferably has a fat digestion power measured by the LV emulsification method of 100 U/g or more, more preferably 1,000 to 1,000,000 U/g, and further preferably 10,000 to 500,000 U/g.

The amount (dry mass) of the lipase and the partial glyceride lipase that preferentially decomposes the 1,3-position derived from the genus Alcaligenes can be appropriately determined in consideration of the activity of the lipase, but Alcaligenes The lipase that preferentially decomposes the 1,3-position derived from the genus is 0.01 to 20% by mass, further 0.02 to 10% by mass, and further 0% with respect to the fat or oil containing a highly unsaturated fatty acid as a constituent fatty acid. It is preferable to use 0.05 to 5 mass% from the viewpoint of industrial productivity. In the case of immobilized lipase, industrial production is performed by using 0.1 to 35% by mass, 1 to 25% by mass, and further 3 to 15% by mass based on the fat or oil containing highly unsaturated fatty acid as a constituent fatty acid. It is preferable from the viewpoint of sex.
Further, the partial glyceride lipase is used in an amount of 0.01 to 20% by mass, further 0.02 to 10% by mass, and further 0.05 to 5% by mass based on the fat or oil containing the polyunsaturated fatty acid as a constituent fatty acid. Is preferable from the viewpoint of industrial productivity. In the case of the immobilized partial glyceride lipase, it is industrially used in an amount of 0.1 to 35% by mass, further 1 to 25% by mass, and further 3 to 15% by mass based on the fat or oil containing a highly unsaturated fatty acid as a constituent fatty acid. It is preferable from the viewpoint of productivity.

In order to allow the lipase to act on the fat or oil containing a polyunsaturated fatty acid as a constituent fatty acid, it is sufficient to bring the fat and oil into contact with the lipase. As the contact means, dipping, stirring, a column filled with the immobilized enzyme is used. For example, passing a liquid through a pump or the like. In the case of stirring, the immobilized lipase does not settle, the crushing is suppressed, and the hydrolysis reaction proceeds efficiently, although it depends on the diameter of the reaction tank, 10 to 1000 r/min is preferable, and further 20 to It is preferably 700 r/min, more preferably 30 to 500 r/min.

In the present invention, the hydrolysis of fats and oils can be carried out batchwise, continuously or semi-continuously. The supply of oil and water into the device may be either a parallel flow type or a counter flow type. As the oil and fat and water supplied to the hydrolysis reaction device, oil and fat that have been degassed or deoxygenated in advance may be used as necessary.

The hydrolysis reaction of fats and oils is controlled by the fatty acid concentration in the obtained hydrolyzed oil, and can be terminated when a predetermined fatty acid concentration is reached. The "free fatty acid concentration" in the present invention refers to a value obtained according to the method described in [Analysis Method] (iii) described below.
In the present invention, the hydrolysis reaction of fats and oils containing a polyunsaturated fatty acid as a constituent fatty acid has a free fatty acid concentration of 82 to 99% by mass, further 86 to 98% by mass, since the DHA concentration and yield can be increased. It is preferable to carry out until it becomes 91 to 97 mass %.

In the hydrolysis reaction, the amount of water in the reaction system is 5 to 500% by mass, and further 10 to 300% by mass with respect to the fat or oil containing the highly unsaturated fatty acid as a constituent fatty acid, from the viewpoint of industrial productivity. Furthermore, it is preferable to set it to 20 to 200 mass %.

The reaction temperature of hydrolysis can be determined by the characteristics of lipase, but from the viewpoint of improving the reaction rate and suppressing the inactivation of the enzyme, it is 0 to 80°C, further 10 to 70°C, and further 20 to 60. C is preferred.

From the viewpoint of industrial productivity, the reaction time is preferably 1 to 200 hours, more preferably 2 to 100 hours, and further preferably 2.5 to 50 hours. In the method of the present invention, it is possible to efficiently hydrolyze in a short time by using the two types of lipases at the same time.

The hydrolysis reaction is preferably carried out in the presence of an inert gas such as nitrogen so that contact with air is avoided as much as possible.

The hydrolyzed oil obtained by the hydrolysis reaction contains unreacted oils and fats, that is, triglycerides, diglycerides, and monoglycerides, in addition to free fatty acids including highly unsaturated fatty acids. Therefore, after removing the aqueous layer containing lipase, glycerin, etc. from the hydrolysis reaction product, for example, chromatography, molecular distillation, liquid-liquid partitioning, crystal fractionation, deoxidation, etc. It is preferable to separate the free fatty acid fraction containing saturated fatty acids. The distillation conditions are not particularly limited. Next, adsorbent treatment, filtration and the like can be further performed.

The fatty acid obtained by the above treatment has a high content of highly unsaturated fatty acid. The DHA content of the highly unsaturated fatty acid in the fatty acid is preferably 10 to 75% by mass, more preferably 20 to 65% by mass, further 30 to 55% by mass, and further preferably 45 to 55% by mass from the viewpoint of quality.

The yield of highly unsaturated fatty acids from fats and oils containing highly unsaturated fatty acids as constituent fatty acids is 84 to 99.9% by mass, further 87 to 99% by mass, and further 91 to 98% by mass. It is preferable from the point of view.

[Raw oils and fats]
As the raw material fat and oil, the fat and oil containing the highly unsaturated fatty acid shown in Table 1 as a constituent fatty acid (DD Oil DHA-46, manufactured by Nippon Suisan Kaisha, Ltd.) was used.

[Analysis method]
(I) Measurement of constituent fatty acid composition According to "Methyl esterification method (boron trifluoride methanol method) (2.4.1.2-1996)" in "Standard Oil and Fat Analysis Test Method 2003 Edition" edited by Japan Oil Chemists' Society. It was measured.
A fatty acid methyl ester was prepared from a sample using trihenicosanoin (manufactured by Wako Pure Chemical Industries, Ltd.) as an internal standard, and the obtained sample was subjected to gas chromatography (GLC) to analyze the constituent fatty acids.

(Ii) Measurement of glyceride composition The “glyceride composition” was measured by adding 10 mg of a sample and 0.5 mL of trimethylsilylating agent (“silylating agent TH”, manufactured by Kanto Kagaku) to a glass sample bottle and sealing the bottle at 70° C. Heated for 15 minutes. Distilled water (1.0 mL) and hexane (2.0 mL) were added to this, and after mixing, the hexane layer was subjected to gas chromatography (GLC) to analyze the glyceride composition.

(Iii) Calculation of Free Fatty Acid Concentration The free fatty acid concentration was obtained by measuring the acid value and fatty acid composition of the hydrolyzed oil and using the following formula (1) in accordance with the knowledge of oil and fat products (Koushobo Co., Ltd.).
Free fatty acid concentration (mass %)=x×y/56.1/10 (1)
(X=acid value [mg-KOH/g], y=average molecular weight determined from fatty acid composition)

(Iv) Measurement of Acid Value The acid value was measured according to “Acid Value (2.3.1-1996)” in “Standard Oil and Fat Analysis Test Method 2003 Edition” edited by Japan Oil Chemists' Society.

[Immobilized lipase QLM]
First, to 100 g of anion exchange resin Duolite A-568 (manufactured by Rohm & Haas), 1000 mL of 0.1 mol/L sodium hydroxide aqueous solution was added, and the mixture was stirred for 1 hour and washed with an alkali. Then, the immobilized carrier was washed with 1000 mL of distilled water for 1 hour, and the pH was equilibrated with 1000 mL of 500 mM phosphorus buffer solution (pH 7) for 2 hours. Subsequently, the pH was equilibrated twice with 1000 mL of 50 mM phosphorus buffer (pH 7) for 2 hours each. After the completion, ethanol replacement was performed with 500 mL of ethanol for 30 minutes. After filtering, 500 mL of ethanol containing 100 g of low-melting fatty acid obtained by removing saturated fatty acid from soybean fatty acid was added, and adsorption operation was performed for 30 minutes. Then, after recovering the carrier, the carrier was recovered by washing with 500 mL of 50 mM phosphorus buffer (pH 7) four times to remove ethanol and filtering.
Then, 100 g of the recovered carrier and 100 g of lipase (lipase QLM, manufactured by Meito Sangyo Co., Ltd., titer 103000 U/g) that preferentially decomposes the 1,3-position derived from a commercially available genus Alcaligenes is added to a 50 mM acetate buffer ( Immobilization was performed by contacting with 1900 mL of the enzyme solution dissolved in 1800 mL of pH 7) for 2 hours. The lipase that preferentially decomposes the 1,3 position was used in 100% by mass based on the mass of the carrier. The recovered immobilized enzyme was washed with 500 mL of 50 mM acetate buffer (pH 7) to remove the enzyme and protein that were not adsorbed. All of the above operations were performed at normal pressure of 20°C. Then, 400 g of rapeseed oil was added, and the mixture was stirred at 40° C. for 2 hours, filtered and separated from the rapeseed oil to obtain an immobilized enzyme.
The immobilized lipase QLM thus obtained was washed with a raw material fat which is a substrate on which an actual reaction was carried out before use.

[Method for producing immobilized lipase AY]
Immobilized lipase by the same production method as immobilized lipase QLM except that lipase QLM was changed to lipase AY "Amano" 30-SD (manufactured by Amano Enzyme, fat digestion power 37900 U/g) derived from the genus Candida. I got AY.

[Method for producing immobilized lipase G]
Lipase QLM was changed to Lipase G "Amano" 50 (Amano Enzyme, fat digestion 54300 U/g) derived from Penicillium camemberti, and phosphate buffer (pH 7) was changed to acetate buffer (pH 5). Immobilized lipase G was obtained by the same production method as that of immobilized lipase QLM except for the above.

[Distillation method]
The hydrolyzed oil obtained by the hydrolysis reaction was subjected to a fraction of free fatty acid (FFA) and monoglyceride (MG) and a triglyceride (TG) and diglyceride ( Fractionation into a residue consisting of DG). The distillation conditions were a temperature of 230° C., a pressure of 2 Pa or less, and an oil supply flow rate of 100 mL/hr.

[Example 1]
An enzyme hydrolysis reaction was performed on fats and oils containing the highly unsaturated fatty acids shown in Table 1 as constituent fatty acids (hereinafter referred to as raw fats and oils). As the enzyme, a powder of lipase QLM (QLM, manufactured by Meito Sangyo Co., Ltd., titer 103000 U/g) and lipase G “Amano” 50 (G, manufactured by Amano Enzyme, fat digestion 54300 U/g) were used in combination. Regarding the charging conditions, 30 g of raw material oil and fat, 30 g of water, and 0.3 g of each enzyme were charged in a 200 mL four-necked flask. The hydrolysis reaction conditions were normal pressure, temperature 40° C., stirring speed 300 r/min, and reaction time 48 hr.
After the reaction was completed, the acid value and glyceride composition of the hydrolyzed oil were analyzed. Then, the DHA concentration in the (DG+TG) fraction (=residue) and the (FFA+MG) fraction (=distillate) separated by the above-mentioned distillation method was analyzed by gas chromatography. Here, in this system, the MG concentration in the hydrolyzed oil was 6% by mass or less. Therefore, all the fractions obtained by the distillation operation were regarded as free fatty acids. The DHA yield (%) of the fraction was obtained from the formula (2).

Fraction DHA yield (%) = {fraction DHA concentration x fraction weight}/{[fraction DHA concentration x fraction weight] + [residue DHA concentration x residue weight]} ... ( 2)

From the results shown in Table 2, the free fatty acid concentration reached 89.3% by mass, and the hydrolysis from the raw material fat proceeded. Further, the DHA concentration of the fraction was 45.2 mass% with respect to the DHA concentration of 45.6% in the raw material oil and fat. At that time, the DHA yield was 90.1%.

[Example 2]
As the enzyme, immobilized lipase QLM and immobilized lipase G were used in combination. The charging conditions were 200 g of water and 200 g of immobilized enzyme for each 200 g of raw material oil and fat. The hydrolysis reaction conditions were normal pressure, temperature 40° C., stirring speed 300 r/min, and reaction time 48 hr.
After the reaction was completed, the acid value and glyceride composition of the hydrolyzed oil were quantified. Then, the DHA concentration in each fraction separated by distillation was quantified by gas chromatography.
As a result, the free fatty acid concentration reached 94.5% by mass, and it was revealed that the hydrolysis proceeded as compared to when the QLM was used alone. Further, the DHA concentration of the fraction was 47.3% by mass, which was higher than the DHA concentration in the raw material fat and oil, and DHA was selectively decomposed. The DHA yield at that time was 96.8%, indicating that DHA can be almost released.

[Comparative Example 1]
As the enzyme, powder of lipase QLM (manufactured by Meito Sangyo) was used. The charging conditions were 30 g of water and 30 g of enzyme with respect to 30 g of raw material oil and fat. The hydrolysis reaction conditions were normal pressure, temperature 40° C., stirring speed 300 rpm, and reaction time 48 hr.
After the reaction was completed, the acid value and glyceride composition were quantified. Then, the DHA concentration in each fraction separated by the column was quantified by gas chromatography. The free fatty acid concentration was 76.51% by mass. The DHA concentration of the fraction was 42.7% by mass. At that time, the DHA yield was 76.4%.

[Comparative Example 2]
The enzyme used was immobilized lipase QLM. The charging conditions were 200 g of water and 200 g of enzyme with respect to 200 g of raw material oil and fat. The hydrolysis reaction conditions were normal pressure, temperature 40° C., stirring speed 300 r/min, and reaction time 48 hr.
After the reaction was completed, the acid value and glyceride composition of the hydrolyzed oil were quantified. Then, the DHA concentration in each fraction separated by distillation was quantified by gas chromatography. The free fatty acid concentration was 73.8% by mass. The DHA concentration of the fraction was 44.5% by mass. At that time, the DHA yield was 75.9%.

[Comparative Example 3]
The enzyme used was immobilized lipase AY. The charging conditions were 200 g of water and 200 g of enzyme with respect to 200 g of raw material oil and fat. The hydrolysis reaction conditions were normal pressure, temperature 40° C., stirring speed 300 r/min, and reaction time 48 hr.
After the reaction was completed, the acid value and glyceride composition of the hydrolyzed oil were quantified. Then, the DHA concentration in each fraction separated by the column was quantified by gas chromatography. The free fatty acid concentration was 45.8% by mass, and hydrolysis did not proceed as compared with Lipase QLM. The DHA concentration of the fraction was a relatively low value of 28.0% by mass. The DHA yield at that time was 31.1%.

[Comparative Example 4]
As the enzyme, immobilized lipase AY and immobilized lipase G were used in combination. Regarding the charging conditions, 200 g of water was used for 200 g of raw material oil and fat, and the amount of each enzyme was 20 g. The hydrolysis reaction conditions were atmospheric pressure, a temperature of 40° C., a stirring speed of 300 r/pm, and a reaction time of 48 hours.
After the reaction was completed, the acid value and glyceride composition of the hydrolyzed oil were quantified. Then, the DHA concentration in each fraction separated by the column was quantified by gas chromatography. The free fatty acid concentration reached 81.1% by mass. The DHA concentration of the fraction was 43.0% by mass. At that time, the DHA yield was 83.4%.

The results of Examples and Comparative Examples are shown in Table 2.

As is clear from Table 2, Example 1 in which a lipase that preferentially decomposes the 1,3 position derived from the genus Alcaligenes and a partial glyceride lipase were simultaneously acted on had a high free fatty acid concentration and a short time. DHA could be obtained in good yield. Furthermore, as shown in Example 2, by immobilizing the above lipase, the free fatty acid concentration was improved and DHA could be obtained in a high yield.

Claims (4)

For fats and oils containing polyunsaturated fatty acids as constituent fatty acids, lipases that preferentially decompose the 1,3-position derived from the genus Alcaligenes and partial glyceride lipases are simultaneously immobilized as immobilized lipases on a carrier. A method for producing a highly unsaturated fatty acid, which comprises a step of acting to hydrolyze for 2.5 to 50 hours to obtain a fatty acid containing a highly unsaturated fatty acid. The method for producing a polyunsaturated fatty acid according to claim 1, wherein the partial glyceride lipase is a partial glyceride lipase derived from Penicillium camemberti. The method for producing a highly unsaturated fatty acid according to claim 1 or 2 , wherein the oil or fat containing a highly unsaturated fatty acid as a constituent fatty acid is an oil or fat containing 10 to 75% by mass of docosahexaenoic acid in all the fatty acids constituting the oil or fat. The method for producing a highly unsaturated fatty acid according to claim 1, wherein the oil or fat containing a highly unsaturated fatty acid as a constituent fatty acid is an oil or fat containing 0.1 to 75% by mass of diglyceride.