JP2018082676A - Production method of constituent oils and fats - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a structural fat / oil containing docosahexaenoic acid selectively in diacylglycerol by suppressing the production of triacylglycerol containing docosahexaenoic acid. SOLUTION: This is a method for producing a structural fat and oil containing 85% by mass or more of docosahexaenoic acid in a constituent fatty acid in diacylglycerol, and the fatty acid containing docosahexaenoic acid and having an acid value of 170 to 185 mgKOH / g. , A production method comprising an esterification reaction of glycerin with a 1,3-position selective lipase derived from Rhizomucour mieehei. [Selection diagram] None

Description

  The present invention relates to a method for producing a structural fat containing docosahexaenoic acid.

  Ω3 polyunsaturated fatty acids such as eicosapentaenoic acid (C20: 5, EPA) and docosahexaenoic acid (C22: 6, DHA), which are constituents of fish oil, are attracting attention for their bioactivity, and the use of oils and fats containing them is hoped for. It is rare.

  Conventionally, an esterification reaction between a fatty acid and glycerin is known as a method for producing diacylglycerol containing docosahexaenoic acid (for example, Patent Documents 1 and 2). The esterification reaction is roughly classified into a chemical method using an alkali catalyst or the like and an enzyme method using an enzyme such as lipase, but an enzyme method in which the reaction is performed under mild conditions is preferable.

JP 2004-208539 A JP 2004-222595 A

In general, docosahexaenoic acid is difficult to recognize as a substrate for lipases used in enzymatic methods, and in particular, the reactivity of 1,3-position selective lipase showing specificity at the sn-1 and sn-3 positions of glycerol is low (patent document) 1 and 2) For the production of diacylglycerol containing docosahexaenoic acid, a partial glyceride lipase that specifically acts on partial glycerides has been used.
However, it has been found that when docosahexaenoic acid and glycerin are esterified using a partial glyceride lipase, not only diacylglycerol but also triacylglycerol containing docosahexaenoic acid is produced as a by-product.
Here, in order to more effectively bring out the physiological function expression of docosahexaenoic acid, diacylglycerol is preferable to triacylglycerol as the structural oil. In other words, triacylglycerol requires a larger amount than diacylglycerol for equivalent physiological function expression. And since docosahexaenoic acid has many unsaturated bonds, its stability with respect to heat and light is extremely low, and fats and oils rich in these easily generate deteriorated odors and off-flavors. Therefore, when the amount of docosahexaenoic acid is increased, more additives such as antioxidants and masking agents for suppressing the generation of deteriorated odors and off-flavors are required. desirable. On the other hand, if diacylglycerol is selectively produced instead of triacylglycerol, the amount of such an additive can be reduced compared to the reverse case.

  Therefore, the subject of this invention is suppressing the production | generation of the triacylglycerol containing docosahexaenoic acid, and providing the method of manufacturing the structure fats and oils which contain docosahexaenoic acid selectively in diacylglycerol.

  The present inventor conducted intensive studies in view of the above-described problems, and as a result, using a 1,3-position selective lipase derived from Rhizomucor miehei, fatty acids containing docosahexaenoic acid having a high acid value, glycerin, and If the esterification reaction is carried out, the reaction proceeds unexpectedly, and the by-product of triacylglycerol containing docosahexaenoic acid is suppressed, and a structural fat or oil containing docosahexaenoic acid selectively in diacylglycerol can be obtained. I found it.

That is, the present invention is a method for producing a structural fat or oil containing 85% by mass or more of docosahexaenoic acid in constituent fatty acids in diacylglycerol,
Including a step of esterifying a fatty acid containing docosahexaenoic acid and having an acid value of 170 to 185 mg KOH / g and glycerin using a 1,3-position selective lipase derived from Rhizomucor miehei. A manufacturing method is provided.

  According to the present invention, there are few triacylglycerols containing docosahexaenoic acid, and structural oils and fats containing a large amount of docosahexaenoic acid in diacylglycerol can be obtained.

The production method of the present invention is a method for producing a structural fat or oil containing 85% by mass or more of docosahexaenoic acid in a constituent fatty acid in diacylglycerol,
A step of esterifying a fatty acid containing docosahexaenoic acid and having an acid value of 170 to 185 mg KOH / g and glycerin using a 1,3-position selective lipase derived from Rhizomucor miehei. .
In this specification, "oil" is synonymous with "oil", and substances constituting the oil (oil) include not only triacylglycerol (TAG) but also monoacylglycerol (MAG) and diacylglycerol (DAG). . That is, fats and oils (oil) contain at least one of monoacylglycerol, diacylglycerol, and triacylglycerol.

[Fatty acids]
The fatty acids used in the present invention contain docosahexaenoic acid and have an acid value of 170 to 185 mg KOH / g.
Fatty acids may contain acyl glycerol (triacyl glycerol, diacyl glycerol, monoacyl glycerol) and the like in addition to fatty acids.
The acid value (AV) of the fatty acids is 170 to 185 mgKOH / g, but is preferably 184 mgKOH / g or less, more preferably 172 mgKOH / g from the viewpoint of reaction efficiency and the ability to increase DHA binding to diacylglycerol. g or more, preferably 174 mgKOH / g or more, more preferably 176 mgKOH / g or more.

The fatty acids preferably contain 38% by mass or more of docosahexaenoic acid, and more preferably 43 to 58% by mass so that docosahexaenoic acid can easily act in the esterification reaction.
Moreover, 40 mass% or more is preferable from the same point, and, as for content of (omega) 3 type | system | group highly unsaturated fatty acid in fatty acids, 45-60 mass% is more preferable.
In the present specification, the ω3 highly unsaturated fatty acid is a long chain fatty acid having 18 or more carbon atoms, preferably 20 or more carbon atoms, and 3 or more, preferably 5 or more unsaturated bonds. In addition to docosahexaenoic acid, for example, eicosapentaenoic acid can be mentioned.

In the present invention, it is preferable to obtain fatty acids by hydrolyzing fats and oils.
Here, the fats and oils to be hydrolyzed may be vegetable oils or animal fats, but fats and oils containing ω3 highly unsaturated fatty acids are preferred as constituent fatty acids. Examples of such fats and oils include microbial oils such as fish oil and algae oil, and animal oils such as seal oil, and these may be used alone or in combination of two or more. Fish oil is a marine animal fat, and can be collected from raw materials such as sardines, herring, saury, mackerel, skipjack, tuna, whales, squid and cod liver. Algae oil can be collected from algae belonging to the green alga class, the diatom class, and the like.
Moreover, you may use what is called omega-3 type | system | group highly unsaturated fatty acid concentrated oil which raised the ratio of the omega-3 type | system | group highly unsaturated fatty acid in the fatty acid which comprises fats and oils. As a method for increasing the ratio of the ω3 highly unsaturated fatty acid in the constituent fatty acids, a conventionally known method, for example, a method or solvent for preferentially releasing or removing fatty acids other than the ω3 highly unsaturated fatty acid using lipase Examples include a fractionation method, and any method can be used.

The content of docosahexaenoic acid with respect to all fatty acids constituting the oil to be hydrolyzed is preferably 10% by mass or more, and more preferably 13 to 41 from the viewpoint that docosahexaenoic acid can easily act in the esterification reaction. It is preferable that they are mass%, 16-38 mass%, and also 18-36 mass%.
Moreover, it is preferable that it is 10 mass% or more from the same point with respect to the total fatty acid which comprises fats and oils in fats and oils, It is preferable that it is 10 mass% or more, Furthermore, 15-43 mass%, Furthermore, 20-38 mass. % Is preferred.

Examples of the method for hydrolyzing fats and oils include a high-temperature and high-pressure decomposition method and an enzyme decomposition method.
The high-temperature and high-pressure decomposition method is a method for obtaining fatty acid and glycerin by adding water to fat and oil and reacting under conditions of high temperature and high pressure. The enzymatic decomposition method is a method for obtaining fatty acid and glycerin by adding water to fat and oil and reacting under conditions of low temperature using fat and oil hydrolase as a catalyst. Among these, from the viewpoint of suppressing translation of ω3 highly unsaturated fatty acid, an enzymatic decomposition method using an oil and fat hydrolase is preferable.
As the oil hydrolyzing enzyme, lipase is preferable, and it is not particularly limited, and lipases derived from animals, plants and microorganisms can be used. For example, the genus Rhizopus, the genus Aspergillus, the genus Mucor, the genus Rhizomucor, the genus Pseudomonas, the genus Geotrichum and the genus C And lipases of the origin.
Among these, from the viewpoint of hydrolysis efficiency, it is preferable to use a so-called non-selective lipase having no position / chain length selectivity, and further, a non-selective lipase produced by Candida cylindracea is used. Is preferred. For example, there is lipase AY “Amano” 30SD-K (manufactured by Amano Enzyme Co., Ltd.).

As the fat hydrolase, it is preferable to use an immobilized fat hydrolase in which the enzyme is immobilized on a carrier from the viewpoint that the enzyme activity can be effectively used.
Immobilization carriers include celite, diatomaceous earth, kaolinite, silica gel, molecular sieves, porous glass, activated carbon, calcium carbonate, ceramics and other inorganic carriers, 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. Among these, an ion exchange resin is preferable from the viewpoint of high water retention. Of the ion exchange resins, a porous material is preferable because it has a large surface area and can increase the amount of adsorbed enzyme.

The particle diameter of the resin used as the immobilization carrier is preferably 50 to 2000 μm, more preferably 100 to 1000 μm. The pore diameter is preferably 10 to 150 nm, more preferably 10 to 100 nm. Examples of the material include phenol formaldehyde, polystyrene, acrylamide, divinylbenzene, and the like, and phenol formaldehyde resin (for example, Duolite A-568 manufactured by Dow Chemical Company) is preferable from the viewpoint of improving lipase adsorption.
At this time, the amount of the oil and fat hydrolase used is preferably 10 to 300% by mass, more preferably 30 to 200% by mass, and further preferably 50 to 150% by mass with respect to the mass of the carrier, from the viewpoint of industrial productivity. At the time of immobilization, the enzyme is put into a solution state, but it is preferably adjusted to pH 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 increase the activity of the immobilized lipase, a treatment for adsorbing the fat-soluble fatty acid or a derivative thereof to the carrier in advance may be performed before the lipase is immobilized. Examples of a method for performing the treatment include a method in which a fat-soluble fatty acid or a derivative thereof 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 to be 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, ricinoleic acid and the like. Examples of the derivatives include esters of these fatty acids with mono- or polyhydric alcohols, phospholipids, and derivatives obtained by adding ethylene oxide to these esters. Specific examples include methyl esters, ethyl esters, monoacylglycerols, diacylglycerols, ethylene oxide adducts thereof, polyglycerol esters, sorbitan esters, and sucrose esters of the above fatty acids. Two or more of these fat-soluble fatty acids or derivatives thereof may be used in combination.

  After the hydrolysis of the fats and oils, the free fatty acids are preferably removed. The method for removing the free fatty acid is not particularly limited, such as alkali decomposition, distillation, etc. From the viewpoint of industrial productivity, the distillation treatment is preferably performed using a thin film evaporator. Examples of the thin film evaporator include a centrifugal thin film distillation apparatus, a falling film distillation apparatus, and a wiped film evaporation apparatus, depending on the method for forming a thin film.

It is preferable to further hydrolyze the distilled oil from which free fatty acids have been removed by distillation after the fats and oils are hydrolyzed. As the oil and fat hydrolase, lipase is preferable as described above. Among them, from the viewpoint of hydrolysis efficiency, a lipase that preferentially hydrolyzes the first and third positions produced by a non-selective lipase produced by Candida cylindracea or Alcaligenes sp. Is preferably used.
From the viewpoint of efficiency, partial glyceride lipase produced by Penicillium camembertii may be used in combination. Partial glyceride lipase is a lipase that hydrolyzes monoacylglycerol and diacylglycerol, but hardly hydrolyzes triacylglycerol.

The hydrolysis reaction can be performed according to a conventional method.
After the hydrolysis, the fatty acids may be used in an esterification reaction described later without performing a distillation treatment, but the distillation treatment is preferably performed under conditions satisfying the above-described acid value (AV) range.
As in the above, the distillation treatment is preferably performed using a thin film evaporator from the viewpoint of industrial productivity.
The pressure is preferably under reduced pressure from the viewpoint of reducing equipment costs and operating costs, increasing the distillation capacity, and optimally selecting the distillation temperature, and more preferably 0.5 to 200 Pa, and more preferably 2 to 100 Pa.
The temperature is preferably 180 to 280 ° C, more preferably 190 to 260 ° C, and further preferably 195 to 250 ° C from the viewpoint of suppressing isomerization of fatty acids.
The residence time is preferably 5 to 120 seconds, more preferably 10 to 90 seconds, and further preferably 15 to 60 seconds from the viewpoint of suppressing isomerization of the fatty acid.

[Glycerin]
The glycerin used in the present invention preferably has a purity of 95% by mass or more from the viewpoint of esterification reactivity.

[1,3-position selective lipase]
The 1,3-position selective lipase derived from Rhizomucor miehei used in the present invention is a lipase exhibiting specificity at the sn-1 and sn-3 positions of triacylglycerol. As the 1,3-position selective lipase, it is preferable to use an immobilized lipase obtained by immobilizing the lipase on a carrier from the viewpoint of effective utilization of lipase activity and cost.
The immobilization carrier is preferably an acrylic resin from the viewpoint of improving the reactivity of the ω3 highly unsaturated fatty acid, particularly docosahexaenoic acid. Examples of the immobilized 1,3-position selective lipase include Novozym 40086 (manufactured by Novozyme Japan).

[Esterification reaction]
In the present invention, the method of esterifying fatty acids and glycerin using a 1,3-position selective lipase can be performed according to a conventional method.
The amount of the immobilized lipase used for the ester reaction can be appropriately determined in consideration of the activity of the enzyme. From the viewpoint of improving the reaction rate, 1 to 1 part by mass relative to 100 parts by mass of the total amount of fatty acids and glycerol. 30 mass%, Furthermore, 2-20 mass% is preferable.

The ratio [FA / GLY] of the number of moles of fatty acid groups to the number of moles of glycerin groups in the esterification reaction is 3.0 or less from the viewpoint of suppressing by-production of triacylglycerols containing ω3 highly unsaturated fatty acids. Further, it is preferably 2.5 or less, more preferably 2.3 or less, and from the viewpoint of improving the reaction rate and the distillation residue ratio, 0.5 or more, 1.0 or more, and 1.5 or more. It is preferable to do this.
The ratio [FA / GLY] of the number of moles of fatty acid groups to the number of moles of glycerin groups is represented by the following formula.
FA / GLY = (moles of fatty acid + moles of monoacylglycerol + moles of diacylglycerol × 2 + moles of triacylglycerol × 3) / (moles of glycerin + moles of monoacylglycerol + diacylglycerol) Number of moles + number of moles of triacylglycerol)

The reaction temperature of the esterification reaction is preferably 0 to 100 ° C., more preferably 20 to 80 ° C., and further preferably 30 to 60 ° C. from the viewpoint of improving the reaction rate and suppressing the deactivation of the enzyme.
The reaction time is preferably within 15 hours, more preferably 1 to 12 hours, and more preferably 2 to 10 hours from the viewpoint of inhibiting the transfer reaction to triacylglycerol and industrial productivity.

  The esterification reaction is preferably performed while removing the reaction product water from the reaction system. For example, it is preferably removed out of the system by a method such as reduced pressure; use of an absorbent such as zeolite or molecular sieve; and ventilation of a dry inert gas into the reaction vessel.

  Examples of the means for contacting the 1,3-position selective lipase and the raw materials (fatty acids and glycerin) include a method of passing through a column filled with immersion, stirring, and immobilized lipase with a pump or the like. In the case of stirring, 10 to 1000 r / min is preferable, 50 to 700 r / min, and further 100 to 600 r / min are preferable from the viewpoint of production efficiency and suppression of crushing of lipase.

  The pressure in the reaction system is preferably under reduced pressure, preferably 1 to 10000 Pa, more preferably 10 to 5000 Pa, and further preferably 100 to 3000 Pa.

The reaction oil after the esterification reaction contains fats and oils, that is, triacylglycerol, diacylglycerol, and monoacylglycerol, and fatty acids as unreacted substances.
The acid value (AV) of the reaction oil is 20 to 60 mgKOH / g, more preferably 25 to 55 mgKOH / g, and further 30 to 50 mgKOH / g from the viewpoint of increasing DHA binding to diacylglycerol and industrial production. It is preferable.
In addition, the total content of diacylglycerol and triacylglycerol in the reaction oil is preferably 45 to 67% by mass, more preferably 48 to 65% by mass, from the viewpoint of physiological effects and industrial productivity.

In the present invention, after the esterification reaction, it is preferable to remove the monoacylglycerol and the fatty acid from the esterification reaction oil by evaporating the light fraction to obtain a triacylglycerol and diacylglycerol as a residue.
The distillation treatment is preferably performed using a thin film evaporator.
The pressure is preferably reduced under reduced pressure from the viewpoint of removing volatile odorous components, reducing equipment costs and operating costs, increasing the distillation capacity, and optimally selecting the distillation temperature. 200 Pa, more preferably 2 to 100 Pa are preferred.
The temperature is preferably 180 to 280 ° C., more preferably 190 to 260 ° C., and further preferably 195 to 250 ° C. from the viewpoint of removing volatile odorous components and improving the flavor.
The residence time is preferably 5 to 120 seconds, more preferably 10 to 90 seconds, and further preferably 15 to 60 seconds from the viewpoint of removing volatile odorous components and improving the flavor.

As a result of the treatment of the present invention, the production of triacylglycerol containing docosahexaenoic acid is suppressed, and a structured fat / oil containing docosahexaenoic acid selectively in diacylglycerol is obtained. The structural fats and oils of the present invention are expected to exhibit high physiological functions with a small amount of use as compared with conventional fats and oils containing docosahexaenoic acid.
In the structured fats and oils of the present invention, 85% by mass or more of docosahexaenoic acid in the constituent fatty acid is contained in diacylglycerol. The proportion of docosahexaenoic acid contained in this diacylglycerol is a constituent fatty acid from the viewpoint of physiological function expression. 87 mass% or more of the docosahexaenoic acid in it, and also 89 mass% or more are preferable.
The ratio of docosahexaenoic acid contained in diacylglycerol is a percentage of the ratio of docosahexaenoic acid constituting diacylglycerol to the total amount of docosahexaenoic acid in the fatty acids constituting the fats and oils. Details are described in Examples below.

The content of docosahexaenoic acid in the fatty acids constituting the structural fats and oils is 18 to 68% by mass, more preferably 23 to 58% by mass, and more preferably 28 to 48% by mass from the viewpoint of working favorably for the manifestation of physiological functions and oil and fat characteristics. preferable.
Moreover, it is preferable that content of the (omega) 3 type | system | group highly unsaturated fatty acid in the fatty acid which comprises structural fats and oils is 20-70 mass%, and also 25-60 mass% from the point which acts advantageously on physiological function expression.

In the structured fat and oil of the present invention, the content of diacylglycerol is preferably 60 to 95% by mass, and more preferably 65 to 90% by mass, from the viewpoint of physiological function expression.
In the structured fat of the present invention, the content of triacylglycerol is preferably 5 to 40% by mass, more preferably 10 to 35% by mass, from the viewpoint of manifesting physiological functions.

  The structural fats and oils obtained by the method of the present invention can be used in the same manner as general edible fats and oils by performing a purification step as necessary.

In the following examples, “%” means “mass%”.
[Raw oil]
Tuna crude oil shown in Table 1 was used as the raw material fat. The content of docosahexaenoic acid (DHA), the acid value (AV), and the glyceride composition were measured by the following methods.

[Analysis method]
(I) Measurement of DHA content According to “Methyl esterification method (boron trifluoride methanol method) (2.4.1.2-1996)” in “Standard oil analysis method 2003 edition” edited by Japan Oil Chemists' Society The sample was subjected to fatty acid methyl ester, and the obtained sample was subjected to gas chromatography (GLC). A calibration curve of DHA (manufactured by Larodan Fine Chemicals) was prepared using trihenicosanoin (manufactured by Wako Pure Chemical Industries) as an internal standard substance. Next, an internal standard substance was added to the sample for analysis, and the DHA content was determined from the peak of the internal standard substance and a calibration curve.

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

(Iii) Measurement of glyceride composition “Glyceride composition” is obtained by adding 10 mg of a sample and 0.5 mL of a trimethylsilylating agent (“silylating agent TH”, manufactured by Kanto Chemical Co., Ltd.) to a glass sample bottle and sealing it at 70 ° C. Heated for 15 minutes. Distilled water (1.0 mL) and hexane (2.0 mL) were added thereto, and after mixing, the hexane layer was subjected to gas chromatography (GLC) to analyze the glyceride composition.

(Iv) Separation method of TAG and DAG in distillation residue 20 mL of methanol was passed through a solid phase column (Sep-Pak C18 5 g, manufactured by Waters) for conditioning. Thereafter, a sample solution in which 0.2 g of sample was dissolved in 2 mL of acetonitrile was loaded onto a solid phase column. Next, 100 mL of methanol was loaded onto the solid phase column to obtain a DAG fraction. The DAG fraction was desolvated and weighed, and the DHA content was analyzed. Furthermore, 30 mL of acetone was loaded onto a solid phase column to obtain a TAG fraction. Similarly, the TAG fraction was desolvated and weighed and analyzed for DHA content.

[Calculation of DHA ratio]
The DHA ratio (%) was calculated from the following formula (1).
DHA ratio (mass%)
= (DAG [%] in residue × DHA [%] in DAG) / (DAG [%] in residue × DHA [%] in DAG + TAG [%] in residue × DHA [%] in TAG) × 100 (1)

[Immobilized 1,3-position selective lipase]
Novozym 40086 (manufactured by Novozyme Japan) derived from Rhizomucour miehei was used.

[Preparation of immobilized lipase AY]
Duolite A-568 (manufactured by Sasaki Chemical Co., Ltd.) was used as a carrier for immobilizing lipase. 1000 g of support was stirred in 10 L of N / 10 NaOH solution for 1 hour and filtered. Thereafter, the mixture was stirred for 1 hour in 10 L of ion-exchanged water, filtered, and then equilibrated with 10 L of 500 mM phosphate buffer (pH 7) for 2 hours and filtered. Thereafter, the pH was equilibrated with 10 L of 50 mM phosphate buffer (pH 7) for 2 hours, and the operation of filtration was performed twice. Then, ethanol substitution with 5 L of ethanol was performed for 30 minutes, followed by filtration. Thereafter, 5 L of ethanol containing 1000 g of soybean fatty acid excluding the high melting point component precipitated at −3 ° C. was added, and the fatty acid was adsorbed on the carrier for 30 minutes, followed by filtration. Thereafter, the substrate was washed with 5 L of 50 mM phosphate buffer (pH 7) four times for 30 minutes, ethanol was removed, and the carrier was recovered by filtration. Thereafter, 1000 g of lipase derived from commercially available Candida cylindracea (lipase AY “Amano” 30SD-K, manufactured by Amano Enzyme) was contacted with an enzyme solution dissolved in 9000 g of 50 mM phosphate buffer (pH 7) for 5 hours. The lipase was immobilized. Then, the enzyme and protein which were not fix | immobilized were removed by filtering and wash | cleaning the support | carrier with which the lipase was fix | immobilized by 10 L of 50 mM phosphate buffer (pH7). Thereafter, 4000 g of tuna crude oil was added, stirred for 12 hours, and filtered to obtain immobilized lipase AY. All the above operations were performed at 20 ° C. Then, it filtered, wash | cleaned fats and oils with hexane, removed the solvent, and obtained immobilized lipase AY.

[Preparation of immobilized lipase QLM]
In the same production method as immobilized lipase AY, the type of lipase was changed to a lipase derived from the genus Alcaligenes (Lipase QLM, manufactured by Meisei Sangyo) to obtain immobilized lipase QLM.

[Preparation of immobilized lipase G]
Phosphate buffer (pH 7) was applied to a partial glyceride lipase (lipase G “Amano” 50, manufactured by Amano Enzyme) derived from Penicillium camemberti using the same production method as immobilized lipase AY. Immobilized lipase G was obtained by changing the acetate buffer (pH 5).

[Preparation of raw fatty acid 1]
<1. Enzymatic hydrolysis reaction 1>
While adding 2000 g of tuna crude oil shown in Table 1 and 2000 g of distilled water and stirring at a temperature of 40 ° C. and 400 r / min, 200 g of immobilized lipase AY was added, and a hydrolysis reaction was performed by batch stirring reaction for 2 hours. The immobilized enzyme was filtered off, and then centrifuged (manufactured by Hitachi Koki, rotor R9A, 8000 r / min × 10 min) to separate sweet water. Thereafter, the oil phase was dehydrated under reduced pressure to obtain a tuna crude oil cracked oil. This operation was repeated twice to obtain a tuna crude oil cracked oil.

<2. Distillation 1>
Thin-film distillation treatment of the tuna crude oil decomposed oil obtained in 1 above using a wipe film evaporator (type 2-03: manufactured by Shinko Environmental Solution Co., Ltd.) at a temperature setting of 230 ° C., a vacuum <2 Pa, and a flow rate of 150 mL / h. Then, free fatty acids were distilled off to obtain glycerides in which DHA and EPA were concentrated in the residue.

<3. Enzymatic hydrolysis reaction 2>
1500 g of the residue distilled in 2 above and 1500 g of distilled water are charged into a four-necked flask, and while stirring at a temperature of 40 ° C. and 400 r / min, 150 g of immobilized lipase QLM and 150 g of immobilized lipase G are added, and batch stirring reaction The hydrolysis reaction was carried out for 24 hours. After the immobilized lipase was filtered off, the oil phase was dehydrated under reduced pressure.

<4. Distillation 2>
Thin-film distillation of the enzyme hydrolysis reaction oil obtained in 3 above using a wipe film evaporator (type 2-03: manufactured by Shinko Environmental Solution) under the conditions of temperature setting 230 ° C., vacuum <2 Pa, flow rate 150 mL / h. The raw material fatty acid 1 was obtained by removing the residual components MAG, DAG and TAG. Table 2 shows the analytical values.

[Preparation of raw fatty acid 2]
<Enzymatic hydrolysis reaction>
<1. Preparation of raw fatty acid 1> Enzymatic hydrolysis reaction 1> and <2. The same operation was performed until distillation 1> to obtain a glyceride composition in which DHA and EPA were concentrated. Charged 1500 g of distilled residue and 1500 g of distilled water into a four-necked flask, added 150 g of immobilized lipase AY and 150 g of immobilized lipase G while stirring at a temperature of 40 ° C. and 400 r / min, and hydrolyzed by batch stirring reaction The reaction was performed for 72 hours. After the immobilized lipase was filtered off, the oil phase was dehydrated under reduced pressure to obtain raw fatty acid 2. Table 2 shows the analytical values.

[Example 1]
<1. Esterification reaction>
Raw material fatty acid 1 derived from tuna oil was charged into a four-necked flask, and an immobilized 1,3-position selective lipase derived from Rhizomucor miehei was added 5% to the total of raw material fatty acid 1 and glycerin. The mixture was stirred at 50 ° C. and 400 r / min. Thereafter, glycerin was charged into a four-necked flask, and an esterification reaction was carried out under a vacuum degree of 400 Pa. The molar ratio of fatty acid to glycerin (FA / GLY) in the four-necked flask was 2. After 8 hours, the lipase was filtered off to obtain an esterification reaction oil.

<2. Distillation of esterification reaction oil>
Above 1. The esterification reaction oil obtained in the above was subjected to thin film distillation using a wipe film evaporator (2-03 type: manufactured by Shinko Environmental Solution Co., Ltd.) under the conditions of a temperature setting of 230 ° C., a vacuum of <2 Pa, and a flow rate of 120 mL / h. The separation ratio was determined from the mass of the residue (DAG + TAG) and the fraction (FA + MAG). DAG and TAG were separated from the distillation residue using a solid phase column, and each DHA content was measured.

[Example 2]
The esterification reaction and distillation were carried out under the same conditions as in Example 1 except that the raw material fatty acid 1 derived from tuna oil and glycerin were charged and the molar ratio of fatty acid to glycerin (FA / GLY) was 2.5. .

[Comparative Example 1]
<1. Esterification reaction>
Raw material fatty acid 2 derived from tuna oil is charged into a four-necked flask, 10% of immobilized lipase G (partial glyceride selectivity) is added to the total of raw material fatty acid 2 and glycerin, and stirred at a temperature of 40 ° C. and 400 r / min. did. Thereafter, glycerin was charged into a four-necked flask, and an esterification reaction was carried out under a vacuum degree of 400 Pa. The molar ratio of fatty acid to glycerin (FA / GLY) in the four-necked flask was 1.5. After 165 hours, the immobilized lipase G was filtered off to obtain an esterification reaction oil.

<2. Distillation of esterification reaction oil>
The esterification reaction oil obtained in 1 above was subjected to thin film distillation using a wipe film evaporator (type 2-03: manufactured by Shinko Environmental Solution Co., Ltd.) under conditions of a temperature setting of 230 ° C., a vacuum of <2 Pa, and a flow rate of 120 mL / h. . DAG and TAG were separated from the distillation residue using a solid phase column, and each DHA content was measured.

[Comparative Example 2]
The esterification reaction was carried out under the same conditions as in Comparative Example 1, except that the feed of fatty acid 2 derived from tuna oil and glycerin was changed and the molar ratio of fatty acid to glycerin (FA / GLY) was changed to 2 for 25 hours. And distilled.

[Comparative Example 3]
Esters were changed under the same conditions as in Comparative Example 1 except that the feed of fatty acid 2 and glycerin derived from tuna oil was changed and the molar ratio of fatty acid to glycerin (FA / GLY) was 2.5 and the esterification reaction was carried out for 18 hours. Reaction and distillation were carried out.

  Table 3 shows the esterification reaction conditions, the acid value of the reaction oil, the distillation separation ratio, and the analytical value of the distillation residue.

As is apparent from Table 3, when fatty acids containing DHA are esterified with 1,3-position selective lipase derived from Rhizomucor miehei, a distillation residue of 45 to 67% is obtained, and DHA is TAG. Rather, it was mainly included in DAG.
In contrast, when fatty acids containing DHA were esterified with immobilized lipase G (partial glyceride selectivity), there were many distillation residues, but DHA was not selectively contained in DAG, but was also contained in TAG. It was.

Claims (8)

A method for producing a structural fat or oil containing 85% by mass or more of docosahexaenoic acid in a constituent fatty acid in diacylglycerol,
Including a step of esterifying a fatty acid containing docosahexaenoic acid and having an acid value of 170 to 185 mg KOH / g and glycerin using a 1,3-position selective lipase derived from Rhizomucor miehei. ,Production method.   2. The method for producing a structured fat according to claim 1, wherein the fatty acids are distilled.   The method for producing a structural fat or oil according to claim 1 or 2, wherein the fatty acid contains 38 mass% or more of docosahexaenoic acid.   The method for producing a structural fat according to any one of claims 1 to 3, wherein the esterification reaction time is within 15 hours.   5. The esterification reaction of fatty acids and glycerin in a range in which the ratio of the number of moles of fatty acid groups to the number of moles of glycerin groups [FA / GLY] is 3.0 or less, according to claim 1. Manufacturing method of structural fats and oils.   The manufacturing method of the structural fats and oils of any one of Claims 1-5 which obtain the reaction oil whose acid value is 20-60 mgKOH / g by esterification reaction.   The method for producing a structured fat according to any one of claims 1 to 6, wherein the structured fat contains 18 to 68% by mass of docosahexaenoic acid in the fatty acid constituting the fat.   The method for producing structured fats and oils according to any one of claims 1 to 7, wherein the structured fats and oils contain 60 to 95% by mass of diacylglycerol.