JPS61192797A - Concentration of highly unsaturated acid - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention is directed to the use of a marine oil containing eicosapentaenoic acid (hereinafter referred to as EPA) and docosahexaenoic acid (hereinafter referred to as DHA) in the form of triglycerides. This invention relates to a method for concentrating unsaturated acids.

[Conventional technology]

The polyunsaturated fatty acids EPA and DHA contained in fish oil have the effect of inhibiting platelet aggregation, lowering the amount of cholesterol and triglycerides in plasma, and are used as preventive agents for cardiovascular problems such as cerebral thrombosis and myocardial infarction. Many possibilities have been reported.

When these fats and oils are ingested, the rate of substitution of arachidonic acid in platelet phospholipids with EPA and DMA clearly increases.

However, the amounts of EPA and DHA administered in these experiments were several grams or more per day, and as a result, food such as eating large amounts of canned fish or eating several green fish, such as sardines, at each meal for a long period of time was required. They have to make a living, and it is difficult to implement this in their daily diet.

For example, to ingest 4g of EPA from cod liver oil every day, 40+
off-Furthermore, in order to ingest ~15 g of EPAIO in mackerel, 750 g of fish is required, and in such a meal, prostaglandin 3 derived from EPA is detected in the urine.

For this reason, highly unsaturated fatty acids such as EPA and DH in fish oil
A is being used as a medicine and health food.

EPA and DNA have 511i and six double bonds in each molecule arranged in a cis-type, moreover, in a methylene-interlap tent pattern2, and chemical synthesis thereof is extremely difficult and has not been carried out at present. However, in the natural world, lipids from fish, shellfish, algae, crustaceans, and marine products are found in large amounts in the lipids of marine biological oils obtained from milk, etc., and especially in the fish oil of blue-green fish, there are 10
% or more, and because of the abundance of resources and the availability of raw materials, enrichment from fish oil, etc. is being considered in many cases.

Until now, there has been no physicochemical isolation method that can separate fats and oils containing a large amount of highly unsaturated fatty acids of interest from complex systems such as fish oil in a single step, so molecular distillation methods based on boiling point differences, wintering based on melting point differences, and solubility methods have not been available. Attempts have been made to concentrate the desired glycerides by combining methods such as dissolution fractional crystallization based on differential polarity, liquid chromatography based on analytical polarity, and solid fat fractionation at ultra-low temperatures.

On the other hand, fats and oils are decomposed into fatty acids or their derivatives, and then highly unsaturated fatty acids are concentrated through chemical and physical treatments.
A method for separating and purifying highly unsaturated fatty acids by reverse-phase partition chromatography has been proposed (Japanese Patent Application Laid-Open No. 109-1989).
No. 444).

[Problem that the invention seeks to solve]

However, among the conventional methods mentioned above, the single molecule distillation method generates artifacts due to thermal denaturation of highly unsaturated fatty acids.
Wintering and dissolution fractional crystallization methods can be used to separate similar continuous phases such as fish oil glycerides without significant changes in solid-liquid composition, and analytical liquid chromatography methods require very small throughputs. . Although it is possible to concentrate highly unsaturated fatty acids in the solid fat fractionation method at ultra-low temperatures, it requires filtration in the presence of a solvent at around -70°C, and it is difficult to control the temperature and recover the mother liquor at that temperature. Yield is low.

In the case of the method of decomposing fats and oils into fatty acids and their derivatives, and then concentrating highly unsaturated fatty acids through chemical and physical treatments, resynthesizing these fatty acids is unsuitable for human consumption and cannot be consumed as food for a long period of time. It is clear that concentrated fish oil that remains in the form of triglycerides is better for the human body. In addition, in order to separate highly unsaturated fatty acids using reversed-phase partition chromatography in this method, the components of the effluent must be constantly monitored, and when the desired polyunsaturated fatty acids flow out, only that portion must be collected. There were problems such as not being able to do so.

The present invention is intended to solve the above-mentioned conventional problems.The present invention is to fractionate marine biological oils by reverse phase partition chromatography in the form of triglycerides without decomposing them. The purpose of the present invention is to provide a method for concentrating highly unsaturated fatty acids, which can efficiently concentrate highly unsaturated fatty acids in an edible form with simple operations by separating a fraction of .

[Means for solving problems]

The present invention involves fractionating marine biological oils containing eicosapentaenoic acid and docosahexaenoic acid as fatty acid components by reverse phase partition chromatography in the form of triglycerides without decomposing them, and extracting an initial fraction containing a high concentration of polyunsaturated acids. This is a method for concentrating highly unsaturated acids, which is characterized by fractionating.

Gas chromatography with a set of capillaries (below).

When the fatty acids constituting the triglycerides of marine biological oils were measured by GC, 60 to 70 types of fatty acids ranging from lauric acid to tetracosenoic acid were detected. In addition, when we measured the triglyceride composition by heating GG using a short non-polar column and high performance liquid chromatography (hereinafter referred to as HPLC) using a reversed phase partition column, we found that the carbon number distribution spanned a very wide range and that the carbon number distribution spread over a very wide range in parallel. The degree of unsaturation distribution was observed, and it was found that the highly unsaturated fatty acids in the marine biological oil were fairly evenly distributed in the glycerides. Furthermore, from the above studies, it was found that there are very few monoglycerides and diglycerides in marine biological oils.

Therefore, in marine biological oils, highly unsaturated fatty acids of tetraenoic acid or higher form triglycerides together with saturated acids and monounsaturated acids, but each fatty acid is evenly distributed in both the number of carbon atoms and the degree of unsaturation in the triglycerides. It is presumed that this exists as follows.

By the way, regarding the flow order in the analysis of triglycerides by reverse-phase partition HPLC, the number of carbon atoms and the log retention capacity are in a linear relationship, and the number of unsaturated bonds is calculated by the number of carbon equivalents, that is, (number of carbons - 2 × unsaturated It is known that there is a linear relationship between the number of devices) and the log storage capacity. For example, trilaurin (36 carbon atoms) and trilylune (54 carbon atoms)
2XX, which has 9 unsaturated units and 36 carbon equivalents, has a very close retention capacity.

Therefore, in the case of marine biological oils in which both the number of carbon atoms and the degree of unsaturation of each fatty acid are evenly distributed in the triglycerides, the flow order of triglycerides by reversed phase partition chromatography is that the low chain length and high unsaturation categories are in the first half, and the high chain length and high unsaturation categories are in the first half. Since the long and low unsaturation fractions flow out in the latter half, if the first half is fractionated, EPA and DHA
It is possible to separate a fraction enriched with highly unsaturated fatty acids such as

Therefore, in the present invention, marine biological oil containing EPA and DMA is fractionated in the form of triglycerides by reverse phase partition chromatography without decomposition, and the initial (first half) fraction containing a high concentration of polyunsaturated fatty acids is extracted. By fractionating, highly unsaturated fatty acids are efficiently concentrated and concentrated oil suitable for consumption is obtained.

In the present invention, the target of processing is seafood.

Algae, crustaceans, and fisheries are aquatic biological oils obtained from aquatic organisms such as milk. In the present invention, these marine biological oils are fractionated in the form of triglycerides without decomposition, but pretreatment such as concentration and purification by conventional methods may be performed.

The reversed phase partition chromatography used for fractionation is preferably one for preparative separation, particularly one for high pressure, high speed, and large volume fractionation.

What columns are used for reversed phase partition chromatography?

Columns that are generally used for reversed-phase partition chromatography can be used, but columns packed with silica gel-based or synthetic polymer-based carriers for reversed-phase partition chromatography can be used, and in particular, silica gel-based or styrene-based columns with chemically bonded octadecyl groups can be used. A chromatography column filled with a slurry of a divinylbenzene copolymerized synthetic polymer support for reversed phase partition chromatography is preferred.

The eluent used for reversed phase partition chromatography can be one that is generally used for reversed phase partition chromatography, especially an eluent in which unsaturated acid triglyceride flows out in a state separated from other components. Can be used.

Such eluents include aliphatic ketones, lower alcohols, acetonitrile, dichloromethane, and tetrahydrofuran.

Some include combinations of n-hexane, water, etc.

Preferred eluents include a system consisting of 0-10% by volume of water and 90-100% by volume of aliphatic ketones, as well as 70-90% by volume of acetonitrile, 7-20% by volume of isopropyl alcohol, and 3-15% by volume of n-hexane. There are systems consisting of the following, and each component can be replaced with another component.

The fractionation method is by reverse phase partition chromatography.

Marine biological oils such as fish oil remain in the form of triglycerides without being broken down, benzene and chloroform.

Dissolve in a suitable solvent such as acetone or n-hexane and inject into a column for reversed phase partition chromatography.

Next, an eluent for reverse phase partition chromatography is passed through the column to perform fractionation.

With such reversed-phase partition chromatography, mono- and diglycerides flow out as the leading components, but these are in trace amounts and do not need to be discarded.Then, triglycerides flow out, but highly unsaturated acids such as EPA and DHA Leaked early.

Most of the highly unsaturated acids flow out into the first half outflow section.

Depending on the composition of the feedstock oil, the unsaturated acid may start flowing out midway through, but the effluent before that can be collected as is.

In this way, by starting fractionation immediately after the start of outflow and finishing the fractionation when the first half of the oil flows out, where most of the highly unsaturated acids flow out, concentrated oil containing a high concentration of highly unsaturated acids can be obtained. The end point of the fractionation is the point at which the EPA concentration of the total fractionated concentrated oil becomes 18% by weight or more, and the highly unsaturated acid content can be adjusted by the length of the fractionation time. The end point of fractionation may be determined by the amount of effluent instead of the length of fractionation time, and in some cases, it may be determined by the refractive index response, etc. By removing the solvent from the fractionated effluent, A concentrated oil enriched in highly unsaturated acids can be obtained.

Below, we will explain the experimental results using reverse phase partition HPLC.
When glycerides from marine biological oils were separated and concentrated using a commercially available silica column with chemically bonded analytical octadecyl groups and a high-porous polymer gel column made from styrene-divinylbenzene copolymer, both After a small amount of mono- and diglyceride flows out as the leading component, a peak that is thought to be triglyceride appears from before the outflow position of triglyceride (9 unsaturated bonds), which was analyzed under the same conditions to estimate the degree of unsaturation. Initially, several dozen peaks appeared over a very wide range, and the number of peaks changed depending on the flow rate and eluent composition.
It could not be identified at all from the triglycerides composed of ~18 groups. However, the eluent from the time the sample solvent elutes in the analytical column until the final peak has finished flowing out is continuously fractionated in fixed amounts, and the fractionated triglyceride is converted into fatty acid methyl and then subjected to gas chromatography. As a result of the measurement, it was confirmed that highly unsaturated acids were highly concentrated in the first outflow section, and from the weight yield point of view, it was confirmed that highly unsaturated acids could be sufficiently recovered from the first half outflow section. Understood.

Especially regarding EPA, 30% within a certain range.
The yield can be increased by widening the fractionation range before and after the above-mentioned fraction, but since the degree of increase in yield is correlated with the decrease in the EPA content in the recovered oil, The fractionation range can be set according to the target setting value, and an arbitrary content can be obtained in consideration of economic efficiency.

In the above operation, there is no need to decompose the feedstock oil, and the highly unsaturated acids flow out at the beginning, so it is sufficient to start the fractionation at the same time as the fractionation begins, and there is no need to constantly monitor the components that flow out. In addition, since the end point of fractionation can be easily determined, the operation is extremely easy. The concentrated oil obtained is in the form of triglycerides and does not require resynthesis, making it suitable for human consumption.

〔Effect of the invention〕

According to the present invention, marine biological oils are fractionated in the form of triglycerides by reverse-phase partition chromatography without being decomposed, and the initial fractions are collected. Highly unsaturated acids can be efficiently concentrated in a suitable form.

〔Example〕

Examples of the present invention will be described below.

Example 1 Sardine oil 500% was obtained from sardines using the spotting method under a nitrogen stream.
I got g. The standard number for this oil is No. 5 according to the Gardner method established by the Japan Oil Chemists' Association, and the peroxide value is 3.2, the iodine value is 185, and the saponification value is 193. The base point was -9°C. After partially decomposing this fat and oil, we heated it under reflux with a boron trifluoride methanol solution, converted all the fatty acids into methyl esters through a transesterification reaction, and then analyzed the fatty acid composition using gas chromatography.As a result, the content of EPA was determined. was 14%, and the DMA content was 8%.

12.0 g of this sardine oil was dissolved in n-hexane, and a column (inner diameter x length 1. 91an x 50am, filling volume 157aJ, filling amount 81.6g). As an eluent, acetone/l water (96/4 vol/vol) was flowed for 2Q at a flow rate of 1.0 mQ/win after being fractionated into each fraction of 20 to 70+sfl. Furthermore, acetone IQ and methanol IQ were flowed to elute residual fats and oils in the column. The solute concentration in the eluent was monitored using a refractive index detector for liquid chromatography (Elma Optical Co., Ltd. ml) by flowing a portion of the eluent into a bypass. the result,
The solute started to appear in the eluent from the time when the eluent had flowed 5001 times. After removing the solvent from all subsequent fractions, a portion thereof was collected and converted into methyl ester in the same manner as described above, and then the fatty acid composition was measured by gas chromatography.

EPA determined from the relationship between the fraction number eluted by the eluent and the solute concentration, and the solute weight and fatty acid composition
Figure 1 shows the relationship between the elution weights of . The EPA concentration in the solute was over 30% in the 700-8001 eluate portion of the eluent, and the weight yield was 6%.

Also, the front and rear sections near this outflow part, that is, the eluent
0 to 900 companies, EPA content is 25.1% for the entire elution group
The yield per weight was 34.2%. The analytical values and physical property values of highly unsaturated acid-enriched fish oil in the range of arrow A in FIG. 1 are as follows.

Analysis value: EPA 25.1%, DHA 13.4%
Physical properties: pale yellow liquid, JOL-4901-saponification value 1
87°Iodine value 254. Viscosity 36.6eP (30°C).

Base point -10℃, specific gravity d180.9340, peroxide value 4
．． 2. Amount of unsaponifiable matter 1.6% Example 2 500 g of mackerel oil obtained from Japanese mackerel by point method under nitrogen flow
I got it. The standard number for this oil is No. 5 according to the Gardner method established by the Japan Oil Chemists' Association, the peroxide value is 2.8, and the iodine value is 159. The saponification value was 192, and the base point was -4°C. After saponifying and decomposing a portion of this fat, we heated it under reflux with a boron trifluoride methanol solution, converted all fatty acids into methyl esters through a transesterification reaction, and then analyzed the fatty acid composition using gas chromatography. As a result, the content of EPA was determined. The content of DHA was 9.5%, and the content of DHA was 11.5%.

This mackerel oil12. Fully porous spherical YMC-GEL dissolved in n-hexane and chemically bonded by reacting octadecyldimethylchlorosilane and silica gel.
005-30150 (manufactured by Yamamura Kagaku Kenkyushin Co., Ltd.) was injected into a column (inner diameter x length 1.9 cm x 50 mm, packing volume 157 aJ, packing amount 93.3 g). Acetonitrile/isopropyl alcohol/n- as eluent
Hexane (12/3/1 vol/vol/vol) at a flow rate of 1. Flowed with OmR/win, 25% of the initial outflow part in the entire section from after the solvent peak outflow to the outflow of all fractions,
The eluent corresponding to 25% of the eluent that flowed out thereafter was separated.

Furthermore, in order to elute the remaining fats and oils in the column, acetone IQ
, methanol H1 was flowed.

The solute concentration in the eluent was monitored using a refractive index detector for liquid chromatography (Elma Optical Co., Ltd. II) by flowing a portion of the eluent into a bypass. After removing the solvent from the fractions from the first outflow section and the subsequent first half outflow section, a portion thereof was collected and converted into methyl ester, and then the fatty acid composition was measured by gas chromatography. FIG. 2 shows the relationship between the concentration of solute eluted by the eluent and the elution time. The weight yield of the first outflow part (arrow B range) is 27.5%, the EPA content is 25.1%, and the next first half outflow part (arrow C range)
The weight yield was 8.7% and the EPA content was 21.9%. The analytical values and physical property values of the highly unsaturated acid-enriched fish oil in the range of arrow B in the first outflow section of FIG. 2 are as follows.

Analysis value: EPA 25.1%, DHA 10.7%
Physical properties: pale yellow liquid, n 601-4987, saponification value 189°, iodine value 239. Viscosity 35.9cP (30℃
), base point -8℃, specific gravity d i80.9340, peroxide value 3.8, amount of unsaponifiables 1.8%

[Brief explanation of the drawing]

Figure 1 is the chromatogram of Example 1, Figure 2 is Example 2.
This is the chromatogram. Agent: Patent Attorney Yanagihara Seihanpo, SaiΔjo0 ()

Claims (4)

[Claims] (1) Fractionate marine biological oil containing eicosapentaenoic acid and docosahexaenoic acid as fatty acid components by reverse phase partition chromatography in the form of triglycerides without decomposing them, and collect the initial fraction containing a high concentration of polyunsaturated acids. A method for concentrating highly unsaturated acids, characterized by fractionation. (2) A patent claim in which the reversed-phase partition chromatography is performed using a column packed with a silica gel-based carrier or a styrene-divinylbenzene copolymer type synthetic polymer carrier for reversed-phase partition chromatography to which octadecyl groups are chemically bonded. The method described in item 1. (3) The reversed phase partition chromatography is carried out using an eluent selected from aliphatic ketones, lower alcohols, acetonitrile, dichloromethane, tetrahydrofuran, n-hexane and water. Method. (4) The method according to any one of claims 1 to 3, wherein the fraction is collected immediately after the start of outflow, and the highly unsaturated acid content is adjusted depending on the length of the fractionation time. the method of.