JP3813584B2 - Method for producing diglyceride - Google Patents

Description

【００３２】
【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]
BACKGROUND 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 oil and fat plasticity improving additives, foods, pharmaceuticals, cosmetics, and the like, and recently, foods that focus on the excellent physiological activity of diglycerides have attracted attention. On the other hand, bioactivity of polyunsaturated fatty acids (PUFA) such as arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid (DHA) is also attracting attention. In particular, DHA is effective in inhibiting platelet aggregation, lowering blood neutral fat, It is known to have an effect of lowering mid-cholesterol, an anticancer effect, an improvement of brain function, and the like. For this reason, research and development of the manufacturing method of fats and oils which contain PUFA in high concentration is energetically carried out.
[0003]
In general, 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 distribution, double bond addition, supercritical extraction, and combinations of these. A combined method is known. However, in these methods, the steps are complicated, inefficient, and expensive.
[0004]
Therefore, as a method for concentrating the PUFA fraction directly and in the form of triglyceride simply and efficiently, there is an enzymatic method in which triglyceride is hydrolyzed using lipase. In other words, since lipase generally has a weak effect on PUFA, it is possible to concentrate PUFA-containing triglycerides by selectively hydrolyzing ester bonds of constituent fatty acids other than PUFA in triglycerides and removing free acids. . For example, many methods for concentrating PUFA-containing triglycerides have been studied, such as a method of concentrating using a lipase derived from the genus Candida (see, for example, Patent Document 1).
[0005]
On the other hand, there are only a few examples of methods for producing PUFA-containing diglycerides. As an example, there is a method for producing a PUFA-containing glyceride in which PUFA or a lower alcohol ester thereof and glycerin are reacted using a heat-resistant lipase (see Patent Document 2). In this method, the reaction temperature is 50 to 70 ° C. and the enzyme The reaction is rather high, uses special enzymes, and cannot obtain diglycerides at high concentrations. In addition, as a so-called enzyme glycerolysis method in which fats and oils are reacted with glycerin in the presence of lipase, a method of obtaining monoglyceride by decomposing fats and oils in the presence of an alkaline agent using an alkaline lipase (see Patent Document 3), 30 ° C to 5 ° C A method of obtaining PUFA-containing partial glycerides by carrying out the reaction while gradually lowering the temperature is known (see Patent Document 4). These enzyme glycerolysis methods can perform a reaction in a shorter time than the esterification method using the above heat-resistant lipase, but these are not methods for obtaining diglycerides as main products, but high-concentration diglycerides. Can not be manufactured.
[0006]
[Patent Document 1]
JP 58-165796 [Patent Document 2]
JP 62-91188 A [Patent Document 3]
Japanese Patent Laid-Open No. 03-187385 [Patent Document 4]
JP 08-214892 A [0007]
[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.
[0008]
[Means for Solving the Problems]
The present inventor, after subjecting the PUFA-containing oil and fat to a glycerolysis reaction in the presence of lipase, by reacting the fatty acid or its ester in the presence of the immobilized partial glyceride lipase, in a mild condition and in a short time, It has been found that it is possible to produce high concentration PUFA-containing diglycerides.
[0009]
That is, the present invention is a glycerolysis reaction by reacting PUFA-containing fats and oils with glycerin in the presence of water and lipase, and the obtained PUFA-containing partial glycerides and fatty acids or lower alkyl esters thereof are immobilized partial glycerides. The present invention provides a method for producing a PUFA-containing diglyceride that is reacted in the presence of lipase.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
〔the first stage〕
In the enzyme glycerolysis, which is the first step of the method of the present invention, PUFA-containing fats and oils, lipase, glycerin and water are used as raw materials. The polyunsaturated fatty acid (PUFA) here 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 enriched by PUFA by selective hydrolysis using lipase or the like can also be used.
[0011]
The lipases include Candida genus, Mucor genus, Rhizopus genus, Aspergillus genus, Pseudomonas genus, Alcaligenes genus, Penicillium genus, Bacillus genus ) Genus, Geotrichum genus, Thermomyces genus, Fusarium genus, etc., preferably Candida genus, Pseudomonas genus, more specifically Candida rugosa, Pseudomonas fluorescence, Pseudomonas cepacia Lipases originating from etc. can be used. Examples of commercially available products include “Lipase AK“ Amano ”” and “Lipase PS“ Amano ”” (both Amano Enzyme). Moreover, the immobilized lipase which hold | maintained this lipase on various support | carriers (Celite, diatomaceous earth, a silica gel, an ion exchange resin etc.) can also be used. Many of these lipases or immobilized lipases can be easily obtained as commercial products. The amount of lipase used is preferably 0.1 to 30% by weight, particularly 0.5 to 10% by weight, based on the reaction raw material.
[0012]
The amount of water added in the enzyme glycerolysis reaction is preferably 0.2 to 10%, particularly preferably 0.5 to 5% of the weight of the PUFA-containing fat. The reaction molar ratio of the total fatty acid group to the total glyceryl group [total fatty acid group (mol) / total glyceryl group (mol)] is preferably 0.3 to 1.5, particularly preferably 0.5 to 1.0. If it is in the range of the said reaction molar ratio, a fatty acid and partial glyceride may exist in a reaction raw material other than PUFA containing fats and oils, glycerol, and water.
[0013]
The reaction temperature varies depending on the melting points of the raw materials and products and the thermal stability of the enzyme, but 20 to 70 ° C., particularly 30 to 50 ° C. is preferable from the viewpoint of reactivity and PUFA stability.
[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 (including triglyceride) obtained by simply removing excess glycerin from the product obtained in the first stage can be used as it is in the second stage.
[0015]
The partial glyceride lipase used in the second stage is a lipase that hydrolyzes monoglyceride and diglyceride partial glycerides but does not hydrolyze triglycerides. Examples of 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), monoglyceride lipase derived from Pseudomonas sp. LP7315 (Journal of Bioscience and Bioengineering, 91 (1), 27-32, 2001), lipase derived from Penicillium cyclopium (J. Biochem, 87 (1), 205) -211, 1980), Penicillium camembertii U-150-derived lipase (J. Fermentation and Bioengineering, 72 (3), 162-167, 1991), etc., among which Penicillium (Penicillium) genus Lipase is preferred. Examples of commercially available products include “Lipase G“ Amano ”50” (Amano Enzyme).
[0016]
The immobilized partial glyceride lipase used in the present invention is an inorganic carrier such as celite, diatomaceous earth, kaolinite, silica gel, molecular sieves, porous glass, activated carbon, calcium carbonate, ceramics; cellulose powder, polyvinyl Alcohol, polypropylene, chitosan, ion exchange resin, hydrophobic adsorption resin, chelate resin, synthetic adsorption resin, etc. are fixed to an immobilization carrier such as organic polymer, especially those fixed to ion exchange resin preferable.
[0017]
As the ion exchange resin, a porous anion exchange resin is preferable. The particle diameter of the resin is desirably 100 to 1000 μm, and the pore diameter is desirably 100 to 1500 mm. These pores give a large surface area to enzyme adsorption, and a larger adsorption amount can be obtained. Examples of the material for the resin include phenol formaldehyde, polystyrene, acrylamide, and divinylbenzene. In particular, phenol formaldehyde resin (trade name Duolite A-568) is desirable.
[0018]
The immobilization temperature of the partial glyceride lipase may be any temperature that does not cause enzyme inactivation, 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 pH 3-9. In particular, when using a lipase whose optimum pH is acidic, the pH should be 4 to 6 in order to obtain the maximum activity. Moreover, as a buffer solution used for the enzyme aqueous solution, a general acetate buffer solution, phosphate buffer solution, Tris-HCl buffer solution, or the like can be used. The concentration of the partial glyceride lipase in the enzyme aqueous solution is desirably a sufficient concentration that is not more than the solubility of the enzyme from the viewpoint of immobilization efficiency. If necessary, the insoluble part may be removed by centrifugation, and the supernatant may be used. Moreover, 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, with respect to 1 part by weight of the immobilized carrier.
[0019]
In the present invention, it is preferable to treat the immobilization carrier with a fat-soluble fatty acid or a derivative thereof as a pretreatment for immobilization in order to obtain an adsorption state that exhibits high activity. These may be used singly, but a further effect is exhibited by combining two or more. As a method for contacting these fat-soluble fatty acids or derivatives thereof and the immobilization carrier, these may be added as they are in water or an organic solvent, but in order to improve dispersibility, fat-soluble fatty acids or derivatives thereof are added to the organic solvent. Once dispersed / dissolved, it may be added to an immobilized carrier dispersed in water. Examples of the organic solvent include chloroform, hexane, ethanol, and the like. The ratio of the fat-soluble fatty acid or derivative thereof and the immobilized carrier is preferably 0.01 to 5 parts by weight, particularly preferably 0.1 to 2 parts by weight, based on 1 part by weight (dry weight) of the immobilized carrier. The contact temperature is preferably 0 to 100 ° C, particularly preferably 20 to 60 ° C. 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 may be dried under reduced pressure.
[0020]
Examples of the fat-soluble fatty acid used for the treatment of the immobilization carrier include 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, hydroxy fatty acids such as ricinoleic acid, Examples thereof include 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 ester, polyhydric alcohol ester, phospholipid, or ethylene oxide added to these esters. Examples include derivatives. Examples of monohydric alcohol esters include methyl esters and ethyl esters, and examples of polyhydric alcohol esters include monoglycerides, diglycerides, and derivatives thereof, polyglycerin fatty acid esters, sorbitan fatty acid esters, and sucrose fatty acid esters. These fatty acids and derivatives thereof are desirably liquid at room temperature in the process. As these fat-soluble fatty acids or derivatives thereof, a mixture of the above fatty acids, for example, naturally-derived fatty acids such as soybean fatty acid can be used.
[0021]
In the second stage, 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, capric acid. , Undecanoic acid, lauric acid, myristic acid, palmitic acid, zomarinic acid, stearic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, arachidonic acid, gadrenic acid, arachidic acid, behenic acid, erucic acid, eicosapentaenoic acid, Docosahexaenoic acid or the like can be used. Moreover, as a lower alcohol which forms the said fatty acid and ester, a C1-C6 lower alcohol is preferable. Examples of the lower alcohol having 1 to 3 carbon atoms include methanol, ethanol, 1-propanol, 2-propanol, butanol, pentanol, and hexanol. Two or more of these fatty acids or their lower alkyl esters can be used in combination. A mixture of the above fatty acids, for example, naturally derived fatty acids such as soybean fatty acid can also be used.
[0022]
In this reaction, the amount of 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) ) In the range where R is 1.5 to 2.6, particularly 1.7 to 2.4.
[0023]
The reaction temperature varies depending on the melting points of the raw materials and products and the thermal stability of the enzyme, but 20 to 70 ° C., particularly 30 to 50 ° C. is preferable from the viewpoint of reactivity and PUFA stability.
[0024]
According to the method of the present invention, a PUFA-containing diglyceride can be produced at a diglyceride concentration [diglyceride / total glyceride × 100] of 60% by weight or more.
[0025]
In order to further increase the diglyceride concentration of the resulting glyceride, the reaction is carried out while measuring the acid value (AV) of the reaction solution over time. In order to achieve a diglyceride concentration of 60% by weight or more, the acid value is 50R. The reaction is preferably terminated within a range satisfying −55>AV> 70R-150 (where AV> 0).
[0026]
By following these conditions, even when conditions such as the raw material charging ratio, reaction temperature, enzyme concentration, etc. change, a high concentration of diglyceride can be produced without affecting the change. That is, when the reaction is terminated when the acid value is outside the above range, a sufficient diglyceride concentration cannot be obtained, which is not preferable.
[0027]
【Example】
Examples of immobilized enzyme production
After stirring 10g of Duolite A-568 (Rohm & Hass) in 100mL of 0.1mol / L sodium hydroxide solution for 1 hour, it was washed with 100mL distilled water for 1 hour, and then with 100mL of 500mM acetate buffer (pH5). The pH was equilibrated for 2 hours and then with 100 mL of 50 mM acetate buffer (pH 5) twice for 2 hours each. The carrier was recovered by filtration, and ethanol substitution 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), ethanol was removed, and the carrier was recovered by filtration. Immobilization was carried out by contacting 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. After filtration, the immobilized enzyme was recovered and washed with 50 mL of 50 mM acetate buffer (pH 5) to remove non-immobilized enzyme and protein. All the above operations were performed at 20 ° C. The immobilization rate was found to be 95% from the difference between the residual activity of the enzyme solution after immobilization and the activity difference between the enzyme solution before immobilization. Thereafter, 40 g of soybean fatty acid was added and dehydration was performed under reduced pressure at 40 ° C., followed by filtration and separation from soybean fatty acid to obtain an immobilized enzyme. The immobilized enzyme thus obtained was washed with a substrate for actual reaction before use.
[0028]
Example 1
PUFA-containing fats and oils (DHA-46G, Nippon Chemical Feed Co., Ltd., DHA content 46% by weight) 100g and glycerin 36.4g (fatty acid group / glyceryl group = 0.6 in molar ratio), distilled water 1.7g (water amount 1.7% by weight (vs. Oils and fats)) were mixed, 4 g of lipase PS “Amano” was added, and a glycerolysis reaction was performed for 22 hours with stirring under a nitrogen stream at a temperature of 40 ° C. (first stage). After completion of the reaction, excess glycerin was removed to obtain a glycerolysis reaction mixture. Then, 10 g of PUFA-containing fatty acid (DHA content 44% by weight) [(fatty acid group in fatty acid, monoglyceride and diglyceride) / (glyceryl group in monoglyceride and diglyceride) R = 2.05] to 100 g of the reaction mixture The washed immobilized lipase G (11 g, dry weight) was mixed, and the esterification reaction was performed for 70 hours while measuring the acid value of the reaction solution over time at a temperature of 40 ° C. under reduced pressure (second stage). The acid value of the reaction solution at this time was 3.3. The reaction solution was analyzed by HPLC, and the glyceride composition and unreacted products were analyzed. In addition, the reaction solution is developed on a TLC plate (developing solvent: chloroform / acetone / methanol = 93.5 / 4.5 / 2.0 vol%), fractionated into each glyceride and eluted with ethyl acetate, and after solvent removal, fatty acid composition analysis is performed by GC. went. The results are shown in Table 1. The DG concentration was as high as 60.7 wt%, and the DHA content in DG was as high as 40 wt%.
[0029]
Comparative Example 1
PUFA-containing fatty acid (DHA content 44% by weight) and glycerin 80g (fatty acid group / glyceryl group = 2.36 in molar ratio), commercially available immobilized enzyme 1,3-position selective lipase: Lipozyme RM IM (Novozymes) 4g The esterification reaction was carried out for 48 hours while measuring the acid value of the reaction solution over time at 50 ° C. under reduced pressure. The acid value of the reaction solution at this time was 45.2.
The DG concentration was as low as 37.6% by weight, and DG could not be produced at a high concentration.
[0030]
Comparative Example 2
Mole ratio of PUFA-containing fatty acid (DHA content 44 wt%) 10 g [(fatty acid group in fatty acid, monoglyceride and diglyceride) / (glyceryl group in monoglyceride and diglyceride) to 100 g of the glycerolysis reaction mixture obtained in Example 1 R = 2.05] and a commercially available immobilized enzyme, 1,3-position selective lipase: 5.5 g of Lipozyme RM IM (Novozymes), and the acid value of the reaction solution over time at 50 ° C. under reduced pressure. While measuring, the esterification reaction was carried out for 70 hours. The acid value of the reaction solution at this time was 8.8.
The DG concentration was as low as 44.7% by weight, and DG could not be produced at a high concentration.
[0031]
[Table 1]

[0032]
【The invention's effect】
According to the production method of the present invention, a high concentration of PUFA-containing diglyceride can be obtained efficiently.

Claims (5)

Glycerol is allowed to act on highly unsaturated fatty acid-containing oils and fats in the presence of water and lipase to effect glycerolysis reaction. A method for producing a highly unsaturated fatty acid-containing diglyceride, which is reacted in the presence of glyceride lipase. The production method according to claim 1, wherein the amount of water added in the glycerolysis reaction is 0.2 to 10% of the weight of the highly unsaturated fatty acid-containing fat. The production method according to claim 1 or 2, wherein a reaction molar ratio [total fatty acid group (mol) / total glyceryl group (mol)] of all fatty acid groups and all glyceryl groups in the glycerolysis reaction is 0.3 to 1.5. The production method according to any one of claims 1 to 3, wherein the immobilized partial glyceride lipase is obtained by immobilizing the partial glyceride lipase on an immobilization carrier pretreated with a fat-soluble fatty acid or a derivative thereof. The production method according to any one of claims 1 to 4, wherein the polyunsaturated fatty acid-containing diglyceride is obtained at a diglyceride concentration [diglyceride / total glyceride x 100] of 60% by weight or more.