JPH0142933B2 - - Google Patents

Description

[Detailed Description of the Invention] [Technical Field] The present invention provides γ-linolenic acid-containing natural oils and fats.
This invention relates to a method for concentrating and purifying linolenic acid as a fatty acid or lower alcohol ester. [Prior Art] γ-linolenic acid is contained in plant seed oils such as evening primrose seed oil, as well as in bacterial cell oils and fats. In this case, examples of the fungal oils include oils produced in the filamentous fungal cells of Isabelina, Vinacea, Lamaniana, Lamaniana anglispora, and Nana belonging to the genus Morteierella. By culturing such filamentous fungi in a medium using carbohydrates as a carbon source, it is possible to produce microbial cells with a high content of γ-linolenic acid-containing oil and fat at high density (Japanese Patent Laid-Open No. 1983-1993).
No. 205979). In addition, the γ-linolenic acid-containing fats and oils in the bacterial cells can be separated by the supercritical fluid extraction method (patent application No. 61-181481), solvent extraction method, etc., which was previously applied for. γ for
- The weight fraction of linolenic acid is about 5-7%,
Concentrations have been shown to be comparable to those in evening primrose seed oil. However, the concentration of γ-linolenic acid contained in these plant seed oils and bacterial cell oils is low, and it is necessary to concentrate and purify them. Regarding the concentration method, conventional methods include separating γ-linolenic acid using gooseberry fruit seed oil as a raw material and using reverse phase partition liquid chromatography using a gradient of a polar solvent mixture (Japanese Patent Application Laid-open No. 192828/1982); and γ-linolene obtained by hydrolyzing γ-linolenic acid-containing fats and oils and using silica gel-based or styrene-divinylbenzene copolymerized synthetic polymers with chemically bonded octadecyl groups as fatty acids or their derivatives by reverse-phase partition liquid chromatography. There is a method for concentrating acids (Japanese Patent Application Laid-open No. 192798/1983). However, none of these methods is yet satisfactory as a method for concentrating γ-linolenic acid. [Purpose] The present invention aims to improve
- It is an object of the present invention to provide a method for efficiently concentrating and purifying linolenic acid components. [Structure] According to the present invention, a fatty acid mixture obtained by hydrolyzing natural oils and fats containing γ-linolenic acid or a fatty acid lower alkyl ester mixture obtained by transesterifying with a lower alcohol can be used as is or by urea addition treatment. There is provided a method for concentrating and purifying γ-linolenic acid, which is characterized in that the γ-linolenic acid fraction is then fractionated by chromatography using a supercritical fluid as a mobile phase. In order to preferably carry out the method of the present invention, first of all, natural oils and fats such as vegetable oils or bacterial cell oils containing γ-linolenic acid are hydrolyzed using a conventional method to form fatty acid mixtures, methyl alcohol, ethyl alcohol, etc. A fatty acid lower alkyl ester mixture is obtained by carrying out a transesterification reaction with a lower alcohol having 1 to 6 carbon atoms. In this case, the target natural oil is a glyceride of fatty acids such as palmitic acid, oleic acid, linoleic acid, and γ-linolenic acid, so when this oil is hydrolyzed,
A fatty acid mixture containing each fatty acid is obtained.
Further, when a transesterification reaction is performed with a lower alcohol, an ester mixture containing lower alkyl esters corresponding to each fatty acid is obtained. When this fatty acid mixture or fatty acid lower alkyl ester mixture is subjected to chromatography using a supercritical fluid as a mobile phase (hereinafter referred to as supercritical fluid chromatography), γ-linolenic acid or its lower alkyl ester is separated from other fatty acids. Alternatively, since the retention time is different from that of other esters, it becomes possible to separate, concentrate, and purify the γ-linolenic acid fraction. In addition, when ultra-high purity γ-linolenic acid is required, the γ-linolenic acid content obtained by removing most of the saturated fatty acids or their esters is obtained using a conventional urea addition treatment method as a pretreatment. With increased concentrate, the column is less loaded and can be processed in one run.
A γ-linolenic acid fraction of 99.9% or more can be obtained. The fractionated γ-linolenic acid or ester can be trapped and desorbed into a product using an adsorbent such as activated carbon, or the supercritical fluid containing the γ-linolenic acid fraction can be depressurized and removed from the system. A product can be obtained by precipitating γ-linolenic acid or its ester. When treating fatty acid mixtures or fatty acid lower alkyl ester mixtures from natural oils and fats using supercritical fluid chromatography according to the present invention, the supercritical fluid includes carbon dioxide in a supercritical state;
In addition to chlorofluorocarbons, hydrocarbons such as methane and ethane are used, and their supercritical conditions are all known. For example, the supercritical conditions for carbon dioxide are: pressure: 72.4 Kg/cm ² or higher, temperature: 31.0°C. That's all.
In this case, sufficient separation performance can be obtained even if the supercritical fluid is used alone;
Separation performance can be improved by adding a hydrocarbon solvent such as hexane, heptane, or cyclohexane, and these may be used as a supercritical mixed fluid. This supercritical fluid is passed as a mobile phase through a column maintained under supercritical conditions. A fatty acid mixture or a fatty acid lower alkyl ester mixture, which is a raw material to be treated, is passed through the column in parallel with the direction of movement of the supercritical fluid. In this case, the fatty acid mixture or the fatty acid lower alkyl ester mixture may be dissolved alone in a supercritical fluid or in a liquid chromatograph using an organic solvent such as n-hexane, chloroform, acetone, lower alcohol, or cyclohexane. It can be fed to the column as a solution dissolved in the solvent used. In addition, as columns, silica gel-based columns with octadecyl groups,
A column packed with a styrene-divinylbenzene copolymer type polymeric filler or the like is used. The conditions for preparative separation using supercritical fluid chromatography include temperature of 35 to 100°C, preferably 40 to 60°C, and pressure of 80 to 300 Kg/cm ² , preferably 90 to 250 Kg/cm 2 .
^cm2 . The separated γ-linolenic acid fraction is preferably subjected to reduced pressure to precipitate γ-linolenic acid or its ester out of the system. Alternatively, it is preferable to adsorb the supercritical fluid containing the γ-linolenic acid fraction using a column packed with an adsorbent such as activated carbon. In this adsorbent-filled column, γ-linolenic acid or its ester is adsorbed under supercritical conditions of a supercritical fluid. After the fractionation is completed, the adsorbent-packed column is depressurized and only the supercritical fluid is separated from the column. The adsorbed γ-linolenic acid or its ester is desorbed from the adsorbent and recovered as a product. In this case, the desorption treatment is carried out by washing the adsorbent adsorbing γ-linolenic acid or its ester with a solvent such as n-hexane or chloroform, which is a conventional method. [Effect] In the present invention, the chromatogram of γ-linolenic acid is separated from the chromatogram of other fatty acids, and the chromatogram of γ-linolenic acid ester is separated from the chromatogram of other fatty acid esters. Therefore, a highly purified γ-linolenic acid fraction can be separated at a high yield. Furthermore, since a supercritical fluid is used as the mobile phase, there is no need for vacuum distillation to remove the organic solvent in the final step, unlike in conventional liquid chromatography-based fractionation methods and urea addition-based separation methods. Therefore, there is no decrease in yield due to thermal denaturation of the separated product. Furthermore, in addition to the advantages that the mobile phase used is inexpensive and harmless to the human body, the processing time required is from several minutes to several tens of minutes, which is significantly shorter than the several hours of conventional methods. is obtained. [Example] Next, the present invention will be explained in more detail with reference to Examples. Note that all percentages in the examples are based on weight. Example 1 Fats and oils extracted from filamentous fungi of the genus Morteierella were methyl esterified by a conventional transesterification method to obtain a fatty acid methyl mixture. The composition of this fatty acid methyl mixture was determined by gas chromatographic analysis to be 30.6% methyl palmitate, 0.5% methyl palmitoleate, 4.1% methyl stearate, 51.8% methyl oleate, 7.2% methyl linoleate,
It was found that methyl γ-linolenate was 5.3%. 14% hexane solution of this mixed fatty acid methyl
25μ was subjected to supercritical fluid chromatography,
A methyl γ-linolenic acid fraction was collected. The separation conditions for supercritical fluid chromatography at that time were: mobile phase: supercritical carbon dioxide, column packing material: 5 μm
ODS, column size: diameter 10mm x length 250mm,
Separation pressure: 120 to 140 Kg/cm ² , separation temperature: 40° C., and mobile phase flow rate: 3.0 Nl/min. After about 5 to 7 minutes under the above conditions, a peak of the methyl γ-linolenic acid fraction appears. In order to adsorb and separate this methyl γ-linolenic acid fraction, a trap filled with about 10 ml of 60 to 150 meshes of activated carbon was used. In the supercritical fluid chromatography used in this experiment, the throughput of the preparative column was small, so the above conditions
By repeating the fractionation 10 times, 14 of the mixed fatty acid methyl
Methyl γ-linolenate was adsorbed and trapped on activated carbon from a 250μ% hexane solution. As a result, the yield
Methyl γ-linolenic acid with a purity of 94.2% could be obtained at 90%. The composition of the separated ester is shown in Table 1. Example 2 The fatty acid methyl of Example 1 was subjected to urea addition treatment once by a conventional method to remove most of the saturated fatty acid methyl from the sample. The fatty acid methyl composition of the obtained concentrate was 2.5% methyl palmitoleate, 15.3% methyl oleate, 41.0% methyl linoleate,
The content of methyl γ-linolenate was 41.2%. 25μ of this 5% hexane solution of methyl mixed fatty acids was subjected to supercritical fluid chromatography under the same conditions as in Example 1, and fractionation was repeated five times.
When the γ-linolenic acid methyl fraction was collected from 125μ, it was found that the yield was 76.5% and the purity was over 99.99%.
Methyl linolenate was obtained. However, the yield at this time is based on the ester before the urea addition treatment.
The composition of the separated ester is shown in Table 1 as in Example 1.
It was shown to. Example 3 Fats and oils extracted from filamentous fungi of the genus Morteierella were hydrolyzed by a conventional method to obtain a fatty acid mixture. The composition of this fatty acid mixture was determined by gas chromatographic analysis to be 30.1% palmitic acid and 30.1% stearic acid.
5.4%, oleic acid 45.7%, linoleic acid 9.3%, γ
-Linolenic acid was 5.8%. This mixed fatty acid
25μ of a 10% hexane solution was subjected to supercritical fluid chromatography under the same conditions as in Example 1.
Repeat the fractionation 10 times to obtain γ from 250μ of the same solution.
- When the linolenic acid fraction was separated, γ-linolenic acid with a yield of 98% and a purity of 95.5% was obtained. The composition of the separated fatty acids is shown in Table 1. Example 4 The fatty acid mixture used in Example 3 was subjected to a single urea addition treatment in the same conventional manner as in Example 2 to remove most of the saturated fatty acids from the sample. The fatty acid composition of the obtained concentrate was 1.8% palmitoleic acid, 4.1% oleic acid, 45.9% linoleic acid, and 48.2% γ-linolenic acid. 25μ of a 10% hexane solution of this mixed fatty acid was subjected to supercritical chromatography under the same conditions as in Example 1, and fractionation was repeated 10 times to collect a 250μγ-linolenic acid fraction from the same solution. γ-linolenic acid with a purity of 99.9% or more was obtained at a yield of 75%. The yield at this time is based on the fatty acid before urea addition treatment. The composition of the separated fatty acids is shown in Table 1. Example 5 Evening primrose seed oil was prepared by a conventional transesterification method.
Methyl esterification was performed to obtain a fatty acid methyl mixture. The composition of this fatty acid methyl mixture is 6.5% methyl palmitate, 1.2% methyl stearate, 10.5% methyl oleate, and 74.5% methyl linoleate.
%, and methyl γ-linolenate was 7.6%. 25μ of this 10% hexane solution of methyl mixed fatty acids was subjected to supercritical chromatography under the same conditions as in Example 1, and fractionation was repeated 10 times.
When a methyl γ-linolenic acid fraction was collected from 250μ, methyl γ-linolenic acid with a yield of 90% and a purity of 93.7% was obtained. The composition of the separated ester is shown in Table 1. Note that the symbols shown in Table 1 have the following meanings. 16:0...Palmitic acid or its ester 16:1...Palmitoleic acid or its ester 18:0...Stearic acid or its ester 18:1...Oleic acid or its ester 18:2...Linoleic acid or its ester 18:3...γ-linolenic acid or its ester [Table]

Claims (1)

[Claims] 1. Chromatography using a supercritical fluid as a mobile phase of a fatty acid mixture obtained by hydrolyzing a natural oil containing γ-linolenic acid or a fatty acid lower alkyl ester mixture obtained by transesterifying with a lower alcohol. 1. A method for concentrating and purifying γ-linolenic acid, which comprises fractionating by graphie and separating a γ-linolenic acid fraction. 2 γ-linolenic acid obtained by subjecting a fatty acid mixture obtained by hydrolyzing a natural oil containing γ-linolenic acid or a fatty acid lower alkyl ester mixture obtained by transesterifying with a lower alcohol to urea addition treatment. A method for concentrating and purifying γ-linolenic acid, which comprises fractionating a concentrate by chromatography using a supercritical fluid as a mobile phase to separate a γ-linolenic acid fraction.