JP2004208539A - Method for producing diglyceride - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a diglyciride containing a highly unsaturated fatty acid, by which the diglyciride containing the highly unsaturated fatty acid in a high concentration can efficiently be produced. <P>SOLUTION: This method for producing the diglyciride containing the highly unsaturated fatty acid comprises reacting the highly unsaturated fatty acid or its lower alkyl ester with glycerol in the presence of an immobilized partial glyceride lipase, while removing the reaction produced water outside the reaction system. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００３０】
【発明の効果】
本発明の製造方法によれば、高濃度の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 at 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, there is an enzymatic method as a simple and highly efficient method for directly enriching the PUFA fraction in the form of triglycerides. In general, lipase has a weak effect on PUFA. Therefore, by utilizing this property, PUFA-containing triglyceride can be concentrated by selectively hydrolyzing and removing ester bonds of constituent fatty acids other than PUFA in triglyceride. For example, a method of concentrating using a lipase derived from the genus Candida is known (Patent Document 1).
Thus, many methods for concentrating PUFA-containing triglycerides have been studied.
[0005]
On the other hand, there are few examples of a method for producing a PUFA-containing diglyceride. An example of a method for producing PUFA-containing glyceride is a method in which PUFA or a lower alcohol ester thereof is reacted with glycerin using a thermostable lipase (Patent Document 2). In this method, the reaction temperature is 50 to 70 ° C. As a result, the enzyme reaction is somewhat expensive, a special enzyme is used, and diglyceride cannot be obtained at a high concentration. Also known is a method for producing a PUFA-containing fat or oil in which the total of monoglyceride and diglyceride is larger than triglyceride by adding an excess amount of glycerin to PUFA or an alcohol ester thereof in the presence of lipase to carry out the reaction. However, in this method, a large amount of unreacted glycerin remains, so that a removal step is required, and diglyceride cannot be obtained at a high concentration.
[0006]
Further, when synthesizing diglyceride by an esterification reaction, the optimal reaction end point for obtaining high diglyceride purity at a high reaction rate, depending on the charge ratio of the raw material fatty acid or its ester to glycerin, reaction temperature, enzyme concentration, etc. Is different. In particular, when the ratio of a fatty acid or an ester thereof to glycerin is increased to improve the reaction rate, there is a problem that if the reaction time is increased by increasing the reaction time, triglyceride is generated and the diglyceride purity is reduced.
[0007]
[Patent Document 1]
JP-A-58-165796 [Patent Document 2]
JP-A-62-91188 [Patent Document 3]
Japanese Patent Application Laid-Open No. 10-265795
[Problems to be solved by the invention]
Therefore, an object of the present invention is to provide a method for efficiently producing a high-concentration PUFA-containing diglyceride.
[0009]
Further, another object of the present invention is to cope with various fatty acid / glycerin ratios, reaction temperatures and enzyme concentrations. Particularly, even at a high fatty acid / glycerin ratio, a high reaction rate and a high concentration of PUFA-containing diglyceride can be obtained. It is to provide a manufacturing method.
[0010]
[Means for Solving the Problems]
The present inventors have adopted, as a lipase used in the esterification reaction, a partial glyceride lipase not targeted in any of Patent Literatures 1 to 3, and immobilized the same to remove water generated during the reaction from the reaction system. By reacting PUFA-containing fatty acid or its lower alkyl ester with glycerin while removing it, it has been found that the diglyceride production rate is dramatically increased, and that a high concentration of PUFA-containing diglyceride can be obtained efficiently. Further, the present inventors, when the acid value of the reaction solution is within a certain range in relation to the ratio of the raw material PUFA-containing fatty acid or its lower alkyl ester and glycerin, by terminating the reaction, It has been found that it is possible to produce high concentrations of diglycerides with the highest possible conversion.
[0011]
That is, the present invention provides a method for producing a PUFA-containing diglyceride in which PUFA or a lower alkyl ester thereof and glycerin are reacted in the presence of an immobilized partial glyceride lipase while removing reaction product water outside the reaction system. is there.
[0012]
BEST MODE FOR CARRYING OUT THE INVENTION
In the present invention, a polyunsaturated fatty acid (PUFA) 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. PUFAs derived from natural oils and fats such as fish oils such as sardines, mackerel and tuna, algae and fungi can also be used. As the lower alcohol forming an ester with PUFA, lower alcohols having 1 to 6 carbon atoms are preferable. Examples of the lower alcohol having 1 to 6 carbon atoms include methanol, ethanol, 1-propanol, 2-propanol, butanol, pentanol, and hexanol. These PUFAs or lower alkyl esters thereof may be used in combination of two or more.
[0013]
The partial glyceride lipase used in the present invention 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), Penicillium cyclopium-derived lipase, 87 (1), 87 (1), 87 (1), 87 (1), 87 (1), 87 (1), 87 (1). -211, 1980), Penicillium camemberti. U-0.99 lipase (J. Fermentation and Bioengineering, 72 (3), 162-167, 1991) and the like can be mentioned. Among them, those having an optimum temperature of 30 ° C. or more and less than 50 ° C., particularly 30 to 45 ° C. are preferable.
[0014]
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.
[0015]
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.
[0016]
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.
[0017]
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.
[0018]
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.
[0019]
In the reaction between PUFA or its lower alkyl ester and glycerin in the presence of the immobilized partial glyceride lipase, the reaction molar ratio R [R = PUFA or its lower alkyl ester (mol) / glycerin (mol)] is as follows: It is preferably from 1.5 to 2.6, and particularly preferably from 1.7 to 2.4.
[0020]
This reaction is carried out while removing the reaction water outside the reaction system. That is, the reaction is carried out under substantially anhydrous conditions except for the water contained in the lipase preparation, and the reaction product water generated by the esterification reaction is reduced in pressure; use of an absorbent such as zeolite and molecular sieves; drying in the reaction tank The inert gas is removed from the system by a method such as ventilation.
[0021]
The reaction temperature of the esterification reaction varies depending on the melting points of the raw materials and products, and the thermal stability of the enzyme, and is preferably 30 ° C. or more and less than 50 ° C., particularly preferably 30 to 45 ° C., from the viewpoints of reactivity and PUFA stability.
[0022]
According to the method of the present invention, diglycerides having a diglyceride purity [diglyceride / (diglyceride + triglyceride) × 100] of 80% by weight or more, more preferably 85% by weight or more, especially 90% by weight or more are converted to 60% by weight or more, preferably 65% by weight or more. It can be produced at a high reaction rate of at least 70% by weight, more preferably at least 70% by weight (total weight percentage of diglyceride and triglyceride in the reaction solution).
[0023]
In order to further increase the diglyceride purity of the obtained diglyceride, the reaction is carried out while measuring the acid value (AV) of the reaction solution over time to achieve a reaction rate of 60% by weight or more and a diglyceride concentration of 80% by weight or more. It is preferred that the reaction be terminated within the range where the acid value satisfies 50R-55>AV> 70R-150 (where AV> 0). Furthermore, in order to achieve a reaction rate of 70% by weight or more and a diglyceride purity of 85% by weight or more, 50R-70>AV> 70R-145 (where AV> 0), particularly a reaction rate of 80% by weight or more, In order to achieve a concentration of 90% by weight or more, it is preferable to terminate the reaction within a range satisfying 50R-80>AV> 70R-140 (where AV> 0).
[0024]
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 at the highest possible conversion without affecting the changes. That is, when the reaction is terminated at a time when the acid value is higher than the above range, the diglyceride purity is high, but the reaction rate is low, so that the productivity is low, and the reaction is performed after the acid value becomes lower than the above range. In the case of terminating the reaction, the ratio of triglyceride in the reaction solution is increased, and sufficient diglyceride purity cannot be obtained.
[0025]
【Example】
Example 1
10 g of Duolite A-568 (Rohm & Hass, average particle size: 480 μm) was stirred in 100 mL of a 0.1 mol / L aqueous sodium hydroxide solution for 1 hour. After stirring, the pH was equilibrated with 100 mL of a 500 mM acetate buffer (pH 5) for 2 hours. Thereafter, the pH was equilibrated twice for 2 hours with 100 mL of a 50 mM acetate buffer (pH 5). After that, filtration was performed to recover the carrier, and then ethanol replacement was performed with 50 mL of ethanol 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. Thereafter, the carrier was collected by filtration and washed four times with 50 mL of 50 mM acetate buffer (pH 5) to remove ethanol, and the carrier was recovered by filtration. Thereafter, 20 g of a commercially available partial glyceride lipase (Lipase G "Amano" 50, Amano Enzyme Co., Ltd.) was contacted with an enzyme solution dissolved in 180 mL of 50 mM acetate buffer (pH 5) for 2 hours for 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, which is a substrate for actually performing a reaction, was added, and dehydration under reduced pressure was performed at 40 ° C. Thereafter, the solution was separated from soybean fatty acid by filtration to obtain an immobilized enzyme.
The immobilized enzyme thus obtained was washed with a PUFA-containing fatty acid (DHA content: 44% by weight), which is a substrate for actually performing the reaction. 20 g (dry weight: 8 g) of the washed immobilized enzyme was weighed into a 200 mL four-necked flask. A PUFA-containing fatty acid (DHA content: 44% by weight) and glycerin (80 g (fatty acid / glycerin = 2.30 in molar ratio)) were added thereto, and the acid value of the reaction solution was measured with time at 40 ° C. under reduced pressure. The esterification reaction was performed for 45 hours. At this time, the acid value of the reaction solution was 27.8. 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% by volume), fractionated into glycerides, eluted with ethyl acetate, and after desolvation, Fatty acid composition analysis was performed by GC. Table 1 shows the results. The conversion was 74.2% by weight, and the DG concentration was as high as 85.3% by weight. The DHA content in DG was as high as 44% by weight.
[0026]
Example 2
20 g (dry weight: 8 g) of the washed immobilized enzyme obtained in Example 1 was weighed into a 200 mL four-necked flask. A PUFA-containing fatty acid (DHA content: 44% by weight) and glycerin (80 g (fatty acid / glycerin = 1.87 in molar ratio)) were added thereto, and the acid value of the reaction solution was measured with time at 40 ° C. under reduced pressure. The esterification reaction was performed for 165 hours. At this time, the acid value of the reaction solution was 3.6. The reaction mixture was analyzed in the same manner as in Example 1. As a result, the conversion was 83.8% by weight, and the DG concentration was as high as 88.3% by weight. Further, the DHA content in DG was as high as 37% by weight.
[0027]
Comparative Example 1
A commercially available immobilized enzyme, 1,3-position selective lipase: 4 g of Lipozyme RM IM (manufactured by Novozymes) was weighed into a 200 mL four-neck flask. A PUFA-containing fatty acid (DHA content: 44% by weight) and glycerin (80 g (fatty acid / glycerin = 2.36 in molar ratio)) were added thereto, and the acid value of the reaction solution was measured at 50 ° C. under reduced pressure with time. The esterification reaction was performed for 48 hours. At this time, the acid value of the reaction solution was 45.2. The reaction mixture was analyzed in the same manner as in Example 1. As a result, the conversion was 57.1% by weight, the DG concentration was as low as 65.8% by weight, and DG could not be produced at a high concentration.
[0028]
Comparative Example 2
20 g (dry weight: 8 g) of the washed immobilized enzyme obtained in Example 1 was weighed into a 200 mL four-necked flask. A PUFA-containing fatty acid (DHA content: 44% by weight) and glycerin (80 g (molar ratio fatty acid / glycerin = 2.66)) were added thereto, and the acid value of the reaction solution was measured with time at 40 ° C. under reduced pressure. The esterification reaction was performed for 18 hours. At this time, the acid value of the reaction solution was 30.6. The reaction mixture was analyzed in the same manner as in Example 1. As a result, the conversion was 77.2% by weight, the DG concentration was as low as 66.2% by weight, and DG could not be produced at a high concentration.
[0029]
[Table 1]

[0030]
【The invention's effect】
According to the production method of the present invention, a high concentration of PUFA-containing diglyceride can be efficiently obtained.

Claims (6)

A process for producing a highly unsaturated fatty acid-containing diglyceride, comprising reacting a highly unsaturated fatty acid or a lower alkyl ester thereof and glycerin with glycerin in the presence of an immobilized partial glyceride lipase while removing reaction water outside the reaction system. The method for producing a diglyceride containing highly unsaturated fatty acids according to claim 1, wherein the partial glyceride lipase has an optimum temperature of 30 ° C or more and less than 50 ° C. The reaction molar ratio R [R = fatty acid or lower alkyl ester (mol) / glycerin (mol)] of a highly unsaturated fatty acid or a lower alkyl ester thereof and glycerin is 1.5 to 2.6. 2. The production method according to 2. The method according to any one of claims 1 to 3, 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. A diglyceride having a diglyceride purity [diglyceride / (diglyceride + triglyceride) × 100] of 80% by weight or more is obtained at a reaction rate of 60% by weight or more (total weight percentage of diglyceride and triglyceride in a reaction solution). The method according to any one of claims 1 to 4. The method according to any one of claims 1 to 4, wherein the reaction is terminated when the acid value (AV) of the reaction solution satisfies 50R-55> AV> 70R-150 (where AV> 0).