JP2004222595A - Method for producing diglyceride - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently producing a highly unsaturated fatty acid-containing diglyceride containing a high proportion of DHA or the like in high concentration. <P>SOLUTION: Glycerol is reacted with a highly unsaturated fatty acid-containing oil and fat in the presence of a catalyst and in the absence of water to carry out a glycerolysis reaction, and the obtained highly unsaturated fatty acid-containing partial glyceride is reacted with a fatty acid or a lower alkyl ester thereof in the presence of an immobilized partial glyceride lipase. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００３３】
【発明の効果】
本発明の製造方法によれば、ＤＨＡ等の比率が高く、かつジグリセリド濃度の高いＰＵＦＡ含有ジグリセリドが効率良く得られる。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for efficiently producing a highly unsaturated fatty acid-containing diglyceride having a high ratio of DHA or the like and a high concentration.
[0002]
[Prior art]
Diglycerides are used in the fields of additives for improving the plasticity of fats and oils, foods, medicines, cosmetics, and the like. Recently, foods that focus on the excellent physiological activity of diglycerides have been receiving attention. On the other hand, the physiological activity of highly unsaturated fatty acids (PUFA) such as arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid (DHA) has also been attracting attention. In particular, DHA has an inhibitory effect on platelet aggregation, a lowering effect on blood triglyceride, It is known that it has a medium cholesterol lowering effect, an anticancer effect, a brain function improving effect, and the like. For this reason, methods for producing fats and oils containing PUFA at a high concentration have been vigorously researched and developed.
[0003]
Generally, PUFA exists in the form of triglyceride in the natural world, but its content is low. Methods for separating and purifying PUFA include chromatography, urea addition, wintering, molecular distillation, liquid-liquid partitioning, addition to double bonds, supercritical extraction, and combinations thereof. A combined method is known. However, in these methods, the steps are complicated, the efficiency is low, and the cost is high.
[0004]
Therefore, as an easy and highly efficient method for directly concentrating the PUFA fraction in the form of triglyceride, there is an enzymatic method in which lipase is used to hydrolyze triglyceride. That is, since lipase generally has a weak action on PUFA, by utilizing this property, PUFA-containing triglyceride can be concentrated by selectively hydrolyzing ester bonds of constituent fatty acids other than PUFA in triglyceride and removing free acid. . For example, many methods have been studied for concentrating PUFA-containing triglycerides, such as a method for concentrating using a lipase derived from the genus Candida (see, for example, Patent Document 1).
[0005]
On the other hand, there are few examples of a method for producing a PUFA-containing diglyceride. As an example, there is a method for producing a PUFA-containing glyceride in which PUFA or a lower alcohol ester thereof is reacted with glycerin using a thermostable lipase (see Patent Document 2). The reaction is rather expensive, a special enzyme is used, and diglyceride cannot be obtained at a high concentration. Further, as a diglyceride production method using so-called chemical glycerolysis in which oil and fat are reacted with glycerin in the presence of a catalyst, preferably, after performing chemical glycerolysis at a temperature of 200 ° C. or higher, immobilization or the first and third positions in the cells A method is known in which a fatty acid or an ester thereof is added and reacted in the presence of a selective lipase (see Patent Document 3). However, since the enzyme has a weak effect on PUFA, when this method is applied to PUFA-containing fats and oils, the concentration of diglyceride obtained is not sufficient, and the temperature of chemical glycerolysis is extremely high. There is a problem that the production increases and the ratio of highly useful DHA or the like (all cis-forms) decreases.
[0006]
[Patent Document 1]
JP-A-58-165796 [Patent Document 2]
JP-A-62-91188 [Patent Document 3]
JP-A-10-234392
[Problems to be solved by the invention]
Therefore, an object of the present invention is to provide a method for efficiently producing a PUFA-containing diglyceride having a high ratio of DHA or the like and a high diglyceride concentration.
[0008]
[Means for Solving the Problems]
The present inventors have made it possible to produce a high-concentration PUFA-containing diglyceride by subjecting a PUFA-containing fat or oil to a chemical glycerolysis reaction and then reacting the fatty acid or an ester thereof in the presence of immobilized partial glyceride lipase. I found out.
[0009]
That is, the present invention provides a PUFA-containing oil and fat, in the presence of a catalyst and in the absence of water, glycerin is reacted to perform a glycerolysis reaction, and the obtained PUFA-containing partial glyceride and a fatty acid or a lower alkyl ester thereof, An object of the present invention is to provide a method for producing a PUFA-containing diglyceride which is reacted in the presence of an immobilized partial glyceride lipase.
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
〔First stage〕
In the first step of the method of the present invention, chemical glycerolysis, PUFA-containing fats and oils, a catalyst and glycerin are used. The polyunsaturated fatty acid (PUFA) as used herein means a fatty acid having 20 or more carbon atoms having 3 or more double bonds. Specifically, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid (AA) and the like are preferable. As PUFA-containing fats and oils, fish oils such as sardines, mackerel and tuna, natural oils such as algae and fungi, and fats and oils in which PUFAs are concentrated by selectively hydrolyzing them using lipase or the like can also be used.
[0011]
Examples of the catalyst include an alkali metal hydroxide, an alkaline earth metal hydroxide, and an alkoxide having 1 to 3 carbon atoms. As the alkali metal hydroxide, sodium hydroxide, potassium hydroxide, etc., as the alkaline earth metal hydroxide, calcium hydroxide, magnesium hydroxide, barium hydroxide, etc., as the alkoxide, sodium methylate, And sodium ethylate. Among them, calcium hydroxide is preferred.
[0012]
The reaction molar ratio of total fatty acid groups to total glyceryl groups [total fatty acid groups (mol) / total glyceryl groups (mol)] in the chemical glycerolysis reaction is 0.3 to 2.0 in order to obtain a high concentration of diglyceride. And particularly preferably 0.5 to 1.5. As long as the reaction molar ratio is within the above range, a fatty acid or a partial glyceride may be present in the reaction raw material in addition to the PUFA-containing fat and oil and glycerin. This reaction is carried out in the absence of water.Here, "in the absence of water" means that no water is added, and the presence of a trace amount of water originally contained in the raw material is also considered. It does not mean to eliminate.
[0013]
The reaction temperature in chemical glycerolysis is preferably from 120 ° C. to less than 200 ° C., particularly preferably from 150 to 180 ° C., from the viewpoint of obtaining a sufficient reaction rate and suppressing the generation of trans acid, and the reaction time is preferably a polymer or the like. It is preferably 6 hours or less, particularly preferably 4 hours or less, from the viewpoints of suppression of generation of methane and economic efficiency.
[0014]
[Second stage]
In the second step, the PUFA-containing partial glyceride obtained in the first step is reacted with a fatty acid or a lower alkyl ester thereof in the presence of an immobilized partial glyceride lipase. Here, as the PUFA-containing partial glyceride, a reaction mixture obtained by simply removing excess glycerin from the product obtained in the first step (including triglyceride) can be used as it is in the second step.
[0015]
The partial glyceride lipase used in the second step is a lipase that hydrolyzes partial glycerides of monoglyceride and diglyceride but does not hydrolyze triglyceride. 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; monoglyceride lipase derived from Bacillus sp. H-257 (J. Biochem., 127, 419-425, 2000), Pseudomonas sp. LP7315-derived monoglyceride lipase (Journal of Bioscience and Bioengineering, 91 (1), 27-32, 2001), Lipase from Penicillium cyclopium, 87 (e), Lipase derived from Penicillium cyclium, 87 (1), 87 (1), 87 (1) -211, 1980), Penicillium camemberti. U-0.99 lipase (J. Fermentation and Bioengineering, 72 (3), 162-167, 1991), and among them Penicillium (Penicillium) derived from the genus lipase is preferred. Examples of commercially available products include "Lipase G" Amano "50" (Amano Enzyme).
[0016]
The immobilized partial glyceride lipase to be used in the present invention may be an inorganic carrier such as celite, diatomaceous earth, kaolinite, silica gel, molecular sieves, porous glass, activated carbon, calcium carbonate, ceramics, etc .; cellulose powder, polyvinyl Alcohol, polypropylene, chitosan, ion-exchange resin, hydrophobic adsorption resin, chelate resin, is fixed on a carrier such as organic polymer such as synthetic adsorption resin, especially those immobilized on ion-exchange resin preferable.
[0017]
As the ion exchange resin, a porous anion exchange resin is preferable. The particle size of the resin is desirably 100 to 1000 μm, and the pore diameter is desirably 100 to 1500 °. These pores provide a large surface area for enzyme adsorption, and a larger adsorption amount can be obtained. Examples of the resin material include phenol formaldehyde, polystyrene, acrylamide, and divinylbenzene. Particularly, a phenol formaldehyde resin (trade name: Duolite A-568) is desirable.
[0018]
The immobilization temperature of the partial glyceride lipase may be any temperature at which the inactivation of the enzyme does not occur, and is preferably 0 to 60 ° C, particularly preferably 5 to 45 ° C. The pH of the aqueous enzyme solution used for immobilization may be in a range that does not cause denaturation of the enzyme, and is preferably from 3 to 9. In particular, when lipase whose optimal pH is acidified is used, the pH is preferably 4 to 6 in order to obtain the maximum activity. As a buffer used for the enzyme aqueous solution, a general acetate buffer, phosphate buffer, Tris-HCl buffer and the like can be used. The concentration of the partial glyceride lipase in the enzyme aqueous solution is desirably not more than the solubility of the enzyme and a sufficient concentration from the viewpoint of immobilization efficiency. If necessary, the insoluble portion may be removed by centrifugation, and the supernatant may be used. Further, the use ratio of the partial glyceride lipase and the immobilized carrier is preferably 0.05 to 10 parts by weight, particularly preferably 0.1 to 5 parts by weight, based on 1 part by weight of the immobilized carrier.
[0019]
In the present invention, as a pretreatment for immobilization, it is preferable to treat the immobilization carrier with a fat-soluble fatty acid or a derivative thereof in order to obtain an adsorption state that exhibits high activity. These may be used singly, but a combination of two or more of them can provide a further effect. As a method of contacting these fat-soluble fatty acids or derivatives thereof with the immobilized carrier, these may be added as they are in water or an organic solvent, but in order to improve dispersibility, the fat-soluble fatty acids or derivatives thereof are added to the organic solvent. Once dispersed and dissolved, it may be added to an immobilized carrier dispersed in water. Examples of the organic solvent include chloroform, hexane, and ethanol. The ratio of the fat-soluble fatty acid or its derivative to the immobilized carrier is such that the fat-soluble fatty acid or its derivative is 0.01 to 5 parts by weight, particularly 0.1 to 2 parts by weight, relative to 1 part by weight (dry weight) of the immobilized carrier. preferable. The contact temperature is preferably from 0 to 100C, particularly preferably from 20 to 60C. The contact time may be about 5 minutes to 5 hours. After this treatment, the immobilized carrier is recovered by filtration, but may be dried at this time. The drying temperature is preferably room temperature to 100 ° C., and drying under reduced pressure may be performed.
[0020]
Examples of the fat-soluble fatty acid used for the treatment with the immobilized carrier include a linear or branched, saturated or unsaturated fatty acid having 4 to 24 carbon atoms, which may be substituted with a hydroxyl group. Preferred fat-soluble fatty acids include fatty acids having 8 to 18 carbon atoms, for example, linear saturated fatty acids such as capric acid, lauric acid, and myristic acid, unsaturated fatty acids such as oleic acid and linoleic acid, and hydroxy fatty acids such as ricinoleic acid. And branched fatty acids such as isostearic acid. Examples of the fat-soluble fatty acid derivative include esters of a fatty acid having 8 to 18 carbon atoms and a compound having a hydroxyl group. Monohydric alcohol esters, polyhydric alcohol esters, phospholipids, and ethylene oxide are further added to these esters. Derivatives and the like are exemplified. Examples of the monohydric alcohol ester include methyl ester and ethyl ester, and examples of the polyhydric alcohol ester include monoglyceride, diglyceride, and derivatives thereof, and polyglycerin fatty acid ester, sorbitan fatty acid ester, and sucrose fatty acid ester.
It is desirable in the process that all of these fatty acids and their derivatives are liquid at room temperature. As these fat-soluble fatty acids or derivatives thereof, a mixture of the above-mentioned fatty acids, for example, naturally-occurring fatty acids such as soybean fatty acids can also be used.
[0021]
In the second step, the fatty acid to be reacted with the PUFA-containing partial glyceride is preferably a saturated or unsaturated fatty acid having 4 to 22 carbon atoms, such as butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, and capric acid. , Undecanoic acid, lauric acid, myristic acid, palmitic acid, zomaric acid, stearic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, arachidonic acid, gadrenic acid, arachinic acid, behenic acid, erucic acid, eicosapentaenoic acid, Docosahexaenoic acid or the like can be used. As the lower alcohol which forms an ester with the above fatty acid, a lower alcohol having 1 to 6 carbon atoms is preferable. Examples of the lower alcohol having 1 to 6 carbon atoms include methanol, ethanol, 1-propanol, 2-propanol, butanol, pentanol, and hexanol. These fatty acids or lower alkyl esters thereof may be used in combination of two or more. Also, a mixture of the above fatty acids, for example, a naturally occurring fatty acid such as soybean fatty acid can be used.
[0022]
In this reaction, the amount of the fatty acid or its ester used is R = [fatty acid group (mol) in fatty acid or its lower alkyl ester and PUFA-containing partial glyceride (monoglyceride + diglyceride)] / [PUFA-containing partial glyceride (monoglyceride + diglyceride) ), A range in which R is 1.5 to 2.6, particularly 1.7 to 2.4 is preferable.
[0023]
The reaction temperature varies depending on the melting points of the raw materials and products and the thermal stability of the enzyme, and is preferably from 20 to 70 ° C, particularly preferably from 30 to 50 ° C, in view of the reactivity and the stability of PUFA.
[0024]
According to the method of the present invention, the PUFA-containing diglyceride can be produced at a diglyceride concentration [diglyceride / total glyceride × 100] of 60% by weight or more, preferably 65% by weight or more.
[0025]
In order to further increase the diglyceride concentration of the obtained glyceride, the reaction is carried out while measuring the acid value (AV) of the reaction solution over time, and in order to achieve a diglyceride concentration of 60% by weight or more, the acid value must be 50R. The reaction is preferably terminated within a range satisfying -55>AV> 70R-150 (where AV> 0).
[0026]
By following these conditions, even when the conditions such as the raw material charging ratio, the reaction temperature, and the enzyme concentration are changed, a high-concentration diglyceride can be produced without being affected by the changes. That is, it is not preferable that the reaction is terminated when the acid value is out of the above range, since a sufficient diglyceride concentration cannot be obtained.
[0027]
【Example】
Production Example of Immobilized Enzyme 10 g of Duolite A-568 (Rohm & Hass) was stirred in 100 mL of a 0.1 mol / L aqueous sodium hydroxide solution for 1 hour, washed with 100 mL of distilled water for 1 hour, and then washed with 500 mM acetate buffer. The pH was equilibrated twice with 100 mL of the liquid (pH 5) for 2 hours, and then twice with 100 mL of 50 mM acetate buffer (pH 5) for 2 hours. The carrier was recovered by filtration, and ethanol replacement with 50 mL of ethanol was performed for 30 minutes. After filtration, 50 mL of ethanol containing 10 g of ricinoleic acid was added, and ricinoleic acid was adsorbed on the carrier for 30 minutes. The carrier was recovered by filtration, washed four times with 50 mL of 50 mM acetate buffer (pH 5), and after removing ethanol, the carrier was recovered by filtration. The carrier was contacted with an enzyme solution obtained by dissolving 20 g of a commercially available partial glyceride lipase (Lipase G "Amano" 50, Amano Enzyme) in 180 mL of 50 mM acetate buffer (pH 5) for 2 hours to perform immobilization. After filtration, the immobilized enzyme was recovered and washed with 50 mL of 50 mM acetate buffer (pH 5) to remove unimmobilized enzyme and protein. All of the above operations were performed at 20 ° C. The immobilization rate was determined from the difference between the remaining activity of the enzyme solution after immobilization and the activity of the enzyme solution before immobilization, and was 95%. Thereafter, 40 g of soybean fatty acid was added, and the mixture was dehydrated under reduced pressure at 40 ° C., and then separated by filtration from soybean fatty acid to obtain an immobilized enzyme. The immobilized enzyme thus obtained was washed with a substrate that actually undergoes a reaction before use.
[0028]
Example 1
100 g of PUFA-containing fats and oils (DHA-46G, Nippon Chemical Feed Co., DHA content 46% by weight, trans acid content 1.5% by weight) and 37.2 g of glycerin (fatty acid group / glyceryl group = 0.59 in molar ratio), 0.01 g of calcium hydroxide was mixed, and reacted at 170 ° C. for 4 hours while stirring under a nitrogen stream (glycerolysis reaction). After the completion of the reaction, excess glycerin was removed to obtain a glycerolysis reaction mixture. Then, 40 g of PUFA-containing fatty acid (DHA content: 44% by weight) [(fatty acid group in fatty acid, monoglyceride and diglyceride) / (molar ratio of glyceryl group in monoglyceride and diglyceride) R = 1.93 in 100 g of the reaction mixture. And 14 g (dry weight) of the washed immobilized lipase G were mixed, and the esterification reaction was carried out for 47 hours at 40 ° C. under reduced pressure while measuring the acid value of the reaction solution over time. At this time, the acid value of the reaction solution was 8.6. The reaction solution was analyzed by HPLC, and glyceride composition and unreacted substances were analyzed. The reaction solution was developed on a TLC plate (developing solvent: chloroform / acetone / methanol = 93.5 / 4.5 / 2.0 vol%), fractionated into glycerides, eluted with ethyl acetate, desolvated, and analyzed by GC. Was used to perform fatty acid composition analysis. Table 1 shows the results. The DG concentration was as high as 67.2% by weight, and the DHA content in DG was as high as 39% by weight. In addition, no increase from the raw material fat was observed while the trans acid content was kept at 1.5% by weight.
[0029]
Comparative Example 1
40 g of PUFA-containing fatty acid (DHA content: 44% by weight) 40 g [(fatty acid group in fatty acid, monoglyceride and diglyceride) / (molar ratio of (glyceryl group in monoglyceride and diglyceride)] in 100 g of the glycerolysis reaction mixture obtained in Example 1 R = 1.93] and 7 g of a commercially available immobilized enzyme, 1,3-position selective lipase: Lipozyme RM IM (Novozymes), and the acid value of the reaction solution was measured at 50 ° C. under reduced pressure with time. The esterification reaction was carried out for 48 hours while the measurement was carried out. At this time, the acid value of the reaction solution was 18.8.
The DG concentration was as low as 50.0% by weight, and DG could not be produced at a high concentration.
[0030]
Comparative Example 2
100 g of PUFA-containing fats and oils (DHA-46G, Nippon Chemical Feed Co., DHA content 46% by weight, trans acid content 1.5% by weight) and 37.2 g of glycerin (fatty acid group / glyceryl group = 0.59 in molar ratio), 0.01 g of calcium hydroxide was mixed, and reacted at 200 ° C. for 2 hours while stirring under a nitrogen stream (glycerolysis reaction). Thereafter, 42 g of PUFA-containing fatty acid (DHA content: 44% by weight) [(fatty acid group in fatty acid, monoglyceride and diglyceride) / (molar ratio of glyceryl group in monoglyceride and diglyceride) R = 1.88 to 100 g of the reaction mixture. And 7.1 g of a commercially available immobilized enzyme, 1,3-selective lipase: Lipozyme RM IM (Novozymes), and the acid value of the reaction solution is measured with time at 50 ° C. under reduced pressure. The esterification reaction was performed for 48 hours. At this time, the acid value of the reaction solution was 18.4.
The DG concentration was as low as 48.3% by weight, and DG could not be produced at a high concentration. In addition, the trans acid content increased compared with the raw material fats and oils, and increased to 2.1% by weight.
[0031]
Comparative Example 3
1,3-position selective lipase which is a commercially available immobilized enzyme in PUFA-containing fatty acid (DHA content 44% by weight) and glycerin 80g (molar ratio of fatty acid group / glyceryl group = 2.36): Lipozyme RM IM (Novozymes) 4) was added, and the esterification reaction was performed for 48 hours at 50 ° C. under reduced pressure while measuring the acid value of the reaction solution over time. At this time, the acid value of the reaction solution was 45.2.
The DG concentration was as low as 37.6% by weight, and DG could not be produced at a high concentration.
[0032]
[Table 1]

[0033]
【The invention's effect】
According to the production method of the present invention, a PUFA-containing diglyceride having a high ratio of DHA and the like and a high diglyceride concentration can be efficiently obtained.

Claims (5)

For highly unsaturated fatty acid-containing fats and oils, in the presence of a catalyst and in the absence of water, glycerol is allowed to act to carry out a glycerolysis reaction, and the resulting highly unsaturated fatty acid-containing partial glyceride and a fatty acid or a lower alkyl ester thereof. And a method for producing a highly unsaturated fatty acid-containing diglyceride which is reacted in the presence of an immobilized partial glyceride lipase. The production method according to claim 1, wherein the reaction temperature in the glycerolysis reaction is 120 ° C or more and less than 200 ° C. The production method according to claim 1 or 2, wherein a reaction molar ratio of total fatty acid groups to total glyceryl groups [total fatty acid groups (mol) / total glyceryl groups (mol)] in the glycerolysis reaction is 0.3 to 2.0. The method according to any one of claims 1 to 3, wherein the catalyst used in the glycerolysis reaction is selected from an alkali metal hydroxide, an alkaline earth metal hydroxide and an alkoxide having 1 to 3 carbon atoms. The method according to any one of claims 1 to 4, wherein the immobilized partial glyceride lipase is obtained by immobilizing a partial glyceride lipase on an immobilized carrier pretreated with a fat-soluble fatty acid or a derivative thereof.