JPS63105683A - Separation method for highly unsaturated fatty acid - Google Patents

Abstract

PURPOSE:To efficiently separate and obtain an unstable highly unsaturated fatty acid without denaturation thereof, by hydrolyzing a natural fat or oil with lipase and subjecting the hydrolyzed fat or oil to centrifugal liquid-liquid partition chromatography. CONSTITUTION:A natural fat or oil containing an 18-24C highly unsaturated fatty acid having 3-6 double bonds, e.g. eicosapentaenoic acid, docosahexaenoic acid, etc., as a fatty acid ingredient is hydrolyzed with lipase, preferable lipase produced by a lipase-producing microorganism. The resultant hydrolyzed solution is then subjected to centrifugal liquid-liquid partition chromatography, e.g. countercurrent partition chromatography in which one of the two-layer separated solutions is used as a stationary phase and the other as a mobile phase is continuously passed through the stationary phase while holding the stationary phase with centrifugal force and a sample injected into the mobile phase is continuously fractionated to separate and collect a fraction containing a free fatty acid and monoglyceride of the highly unsaturated fatty acid.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to eicosapentaenoic acid (hereinafter abbreviated as EPA)
, selective separation of PUFA from natural fats and oils (e.g., fish oil, whale oil, microbial-derived fats and oils) containing highly unsaturated fatty acids (hereinafter abbreviated as PUFA) such as docosahexaenoic acid (hereinafter abbreviated as DHA) as fatty acid components. It's about how to do it.

[Conventional technology]

EPA and DHA have an inhibitory effect on platelet aggregation, and research by Dr. Dyarberg et al. has suggested that they may serve as preventive agents for organ-related diseases such as cerebral thrombosis and myocardial infarction. EPA also has the ability to lower cholesterol in the blood, and its activity is said to be about four times that of linoleic acid, which is currently used as a cholesterol-reducing agent. As described above, PLIFA has attracted attention for its pharmacological effects.

Conventional PUFA separation methods include converting glycerides into esters of lower alcohols such as methanol and ethanol, distilling them, and then concentrating and separating them by urea inclusion treatment (for example, Japanese Patent Application Laid-open No. 8037-1983); A method of concentrating and separating using phase chromatography (for example, JP-A-58-88339, JP-A-58-109444) has been proposed.

On the other hand, a method has been proposed in which fish oil is hydrolyzed with lipase obtained from Candida cylindrasse to concentrate PUFA into glycerides and separate this from free fatty acids (hereinafter abbreviated as FFA) (JP-A-Sho). 58-165
No. 796).

[Problem that the invention seeks to solve]

However, due to its structure, PUFA is extremely unstable and easily oxidized to light, heat, oxygen, etc., and easily generates lipid peroxide. Lipid peroxide has a negative effect on the human body, causing diarrhea, vomiting, fever, and in the worst cases, queuing. Therefore P
When treating UFA, it is desirable to carry out the reaction at room temperature, normal pressure, and under neutral conditions.

However, in the conventional separation method using transesterification,
Since separation and concentration is performed after converting PUFA into a lower alcohol ester, there is a very high possibility that PUFA will be oxidized or denatured by heat, catalysts, alkalis, etc. during the esterification process.

In addition, the method of concentration separation using reversed phase chromatography requires the use of a large amount of packing material and solvent, has poor processing efficiency, and is not suitable for large-scale processing.

Furthermore, the conventional method using lipase as an application of enzymatic hydrolysis to fats and oils containing PUFA
This method takes advantage of the property that glycerides containing FFA are difficult to be hydrolyzed by lipase, and is a method in which normal fatty acid parts with 18 carbon atoms or less are decomposed by lipase to become fatty acids, and PUFA is separated from FFA as glyceride without being decomposed. However, there was a problem that the separation efficiency of PUFA was low and it was not possible to separate it at a high concentration.

The present invention is intended to solve the above-mentioned problems, and aims to propose a method for separating PUFA that can efficiently separate unstable PUFA to a high concentration without denaturing it.

[Means for solving problems]

The present invention decomposes natural fats and oils containing highly unsaturated fatty acids as fatty acid components using lipase, and then fractionates them using centrifugal liquid-liquid partition chromatography to separate both free fatty acids and monoglycerides of the highly unsaturated fatty acids. This is a method for separating highly unsaturated fatty acids.

In the present invention, PUFAs are not converted into lower alcohol esters, but are fractionated by centrifugal liquid-liquid partition chromatography as a mixture of various glycerides and FFAs by partial hydrolysis using lipase.
PUFA is separated by fractionating both fractions containing A and monoglyceride.

In the present invention, PUFA includes carbon atoms of 18 to 24,
There are long-chain PUFAs with 3 to 6 double bonds, examples of which include EPA and DHA, as well as γ-lylunic acid and arachidonic acid. The raw material oils and fats in the present invention are natural oils and fats containing these PUFAs as fatty acid components, and include fish oil, whale oil, evening primrose seed oil, pine seed oil, and oils and fats derived from microorganisms.

The lipase used in the present invention is preferably obtained from lipase-producing bacteria belonging to the genus Mucor, Chromobacterium, Pseudomonas, and Aspergillus.

Examples of the above-mentioned lipase-producing bacteria include Mucor minihai, Chromobacterium viscosum, Pseudomonas fluorescens, and Aspergillus niger. Of these, some lipases, such as those obtained from Mucor Minihai, can be used to completely remove fats and oils.
Although it is difficult to decompose it into FA, it is possible to decompose it into monoglycerides.

In order to decompose raw material fats and oils with lipase, water is required to develop its activity, and the amount of water is 3 times higher than natural fats and oils.
A suitable amount is 0 to 70% by weight, preferably about 50% by weight. The amount of lipase used is usually 1 g per 1 g of natural fat.
A suitable range is 0 to 10,000 units, preferably about 100 to 500 units.

The decomposition can be carried out by mixing the raw material oil, lipase and water, stirring under neutral conditions at a mild temperature and pressure suitable for enzymatic decomposition, and reacting for 24 to 72 hours.

In this reaction, raw fats and oils are hydrolyzed by lipase, and PUFA is decomposed from triglyceride to diglyceride to monoglyceride to FFA, but since PIJFA is bound to the β-position of glycerin, a large amount of PIJFA is present in monoglyceride as well. ing. On the other hand, according to centrifugal liquid-liquid partition chromatography, FFA and monoglyceride of PUFA can be converted into triglyceride, diglyceride,
Since it is possible to selectively separate PUFA from FFA and monoglycerides of other fatty acids, the decomposition of PUFA by lipase does not need to proceed completely to FFA, and if it is decomposed to monoglyceride, it is efficient.
PUFA can be separated.

In this way, lipase decomposes the raw material oil and produces P.
A mixture of UFA and other fatty acid tri-, di-, and monoglycerides and FFA was fractionated by centrifugal liquid-liquid partition chromatography, and PUFA monoglycerides and FFA were fractionated by centrifugal liquid-liquid partition chromatography.
A fraction containing FA is separated to separate PUFA.

Centrifugal liquid-liquid partition chromatography was published in Japanese Patent Publication No. 59-623.
No. 12, one of the two-layer separated liquid is held as a stationary phase by centrifugal force, while the other as a mobile phase is continuously passed through the stationary phase and injected into the mobile phase. This is countercurrent partition chromatography that continuously fractionates a sample.

In the present invention, the two phases differ in specific gravity and polarity and separate into two phases.
One of the seed solvents is used as a stationary phase and the other as a mobile phase, and the mobile phase is moved through the stationary phase by the action of centrifugal acceleration, and each component in the sample is chromatographed by multistage partition equilibrium using the difference in partition coefficient. fractionate. In this method, highly polar components in the sample are sequentially distributed into highly polar solvents, and less polar components are sequentially distributed into less polar solvents6.
The drawing is a system diagram of centrifugal liquid-liquid partition chromatography used in the present invention, and its principle is as follows.

=7- A large number of distribution pipes C (500 to 400
(1) are arranged parallel to the direction of centrifugal acceleration g and are connected to each other in series by a conduit T. Both ends of the conduit T are connected to rotation transfer liquid joints J7 and J installed at both ends of the centrifuge rotation shaft.
7 is connected. Recirculation liquid joint J, J2 is a 4-way valve v3.6-way valve (sample injector)v
2. Connected to the containers of stationary phase SP, mobile phase HP, and washing liquid wS via constant flow pump P and 4-way rotary valve v1, and also via 4-way valve 1, flow cell monitor M, and 3-way rotary valve v4 Connected to fraction collector F and waste liquid VT container. SL
is a sampling loop, and RC is a recorder.

Centrifugal liquid-liquid partition chromatography using this device is performed according to the following procedure. Specific gravity and polarity are different, 2
After mixing any solvent that separates into phases and allowing it to stand still, the mixture is separated into a heavy liquid phase and a light liquid phase and stored in a container. After filling distribution tube C with one of the liquid phases (heavy liquid phase in the drawing) as a stationary phase sp, the rotor R is rotated and while applying a constant centrifugal acceleration g, the other liquid phase (light liquid phase in the drawing) is filled. The liquid is continuously fed through the rotation transfer liquid joint J1 at one end of the rotating shaft as a moving phase. The mobile phase MP becomes minute droplets in the stationary phase SP under the action of the centrifugal acceleration g, passes sequentially through the distribution pipe C, and continuously flows out from the rotation transfer liquid joint J2 at the other end of the rotating shaft. The sample components introduced into a certain amount of mobile phase MP at the initial stage of liquid feeding through the sampling loop SL installed outside the centrifuge are separated into target components by centrifugal liquid-liquid partition chromatography during the above process, and are then separated into the desired components. It is fractionated by fraction collector F according to the amount.

When using a highly polar heavy liquid such as acetonitrile ethanol/water (90: IOV/V) mixed solvent as the stationary phase SP and a low polar light liquid such as n-hexane as the mobile phase, the polarity in the sample Those with lower chain length or longer chain length are sequentially flowed out together with the mobile phase. On the other hand, those with high polarity or short chain length will remain from the inflow side of the stationary phase, so if you reverse the flow of the mobile phase and push out the stationary phase from the inflow side, those with high polarity or short chain length will remain. Those with shorter chain lengths flow out sequentially.

The mobile phase that flows out in this way contains mainly triglycerides, diglycerides, and FF of less unsaturated fatty acids.
The stationary phase that flows out first upon inversion contains short-chain FFA and monoglyceride, and the stationary phase that flows out next contains long-chain, highly unsaturated FF.
A and monoglycerides. Therefore, by separating the final stationary phase fraction, PUFA can be separated with a high recovery rate.

〔Effect of the invention〕

As described above, according to the present invention, after decomposing natural fats and oils with lipase, both fractions of PUFA containing FFA and monoglyceride are separated by centrifugal liquid-liquid partition chromatography, so that unstable PUFA is denatured. Furthermore, since PLIFA can be separated in the form of FFA and monoglyceride without completely decomposing it into FFA, PUFA can be efficiently separated.

〔Example〕

The present invention will be explained below using examples. In each example, % indicates weight %.

Example 1 10g of fish oil and 4+J of purified water were placed in a screw tube with a capacity of 50 mρ, and 100 g of enzyme obtained from Mucor Minihai was added.
Unit was dissolved in purified water and added, and the reaction was carried out for about 48 hours by stirring at a speed of 500 revolutions per minute in a constant temperature bath at 50''C. Fractionation by chromatography was performed using a partition chromatograph cPc model LLN (manufactured by Miki Engineering ■) under the following conditions.

As a distribution liquid, n-hexane and acetonitrile were mixed and then allowed to stand to separate. 850 mfl of a heavy liquid was filled as a stationary phase, and a light liquid was used as a mobile phase. As a sample, a solution in which 10 g of the above fish oil decomposition product was dissolved in a mobile phase to give 50 mQ was injected, and then 1500 ml of mobile phase was injected to perform fractionation. At this time, the rotational speed was 1000 rprn, the liquid feeding rate was 10 n++Q/min, and the operating temperature was 20°C.

Mobile phase (light liquid) eluted above 1500+nIl
Concentrate to obtain fraction 1, then reverse the feeding direction and concentrate the first eluted stationary phase (heavy liquid) 100m+Q to obtain fraction 2, then concentrate the eluted stationary phase 80m+Q to obtain fraction It was set as 3.

After fraction 1, fraction 2, and fraction 3 were each subjected to solvent extraction with a methanol/chloroform-based solvent, esters were synthesized with methanol, and compositional analysis was performed by gas chromatography under the following measurement conditions. The results are used for EP from raw material oils and fats.
A. The recovery rate of DHA is shown in Table 1.

Fraction 1 mainly contains triglycerides, diglycerides, and FFAs of fatty acids with a low degree of unsaturation, fraction 2 mainly contains FFAs with short chain lengths and monoglycerides, and fraction 3 mainly contains FFAs of PUFA. and monoglycerides were mainly included. In addition, almost no change was observed in the peroxide value according to the standard oil and fat analysis test method established by the Japan Oil Chemists' Association.

Measurement conditions for gas chromatography Column: Cavillary chromatogram Salmon 3000A (manufactured by Shimadzu Corporation), (φ0.24
mm x 50m) Inlet, detector temperature: 250°C Column temperature: He; 25kPa, Air;
30 kPa, H,; 30 kPa detector: FID Example 2 Reaction and fractionation were performed using lipase obtained from Chromobacterium viscosum under the same conditions as in Example 1. The results are shown in Table 1.

Example 3 A reaction and fractionation were carried out under the conditions of Example 1 using lipase obtained from Pseudomonas fluorescens.

The results are shown in Table 1.

Table 1 From the above results, by separating fraction 3, PUF
It can be seen that A can be separated in high yield.

[Brief explanation of the drawing]

The figure is a system diagram of centrifugal liquid-liquid partition chromatography, where SP is the stationary phase, MP is the mobile phase, tys is the washing solution,
is a 4-way rotary valve, ■2 is a 6-way valve, and V3 is a 4-way valve.
■4 is a three-way rotary valve, SL is a sampling loop, P is a constant flow pump, J, J2 is a recirculation liquid joint, R is a rotor, C is a distribution pipe, T is a conduit,
M is a flow cell monitor, RC is a recorder, and F is a fraction collector.

Claims (4)

[Claims] (1) Natural oils and fats containing highly unsaturated fatty acids as fatty acid components are decomposed by lipase, and then fractionated by centrifugal liquid-liquid partition chromatography to separate fractions containing free fatty acids and monoglycerides of highly unsaturated fatty acids. A method for separating highly unsaturated fatty acids. (2) The separation method according to claim 1, wherein the highly unsaturated fatty acid has 18 to 24 carbon atoms and 3 to 6 double bonds. (3) The lipase is of the genus Mucor, Chromobacterium,
The isolation method according to claim 1 or 2, which is produced by a lipase-producing bacterium belonging to the genus Pseudomonas or the genus Aspergillus. (4) In centrifugal liquid-liquid partition chromatography, one of the two-phase separated liquids is held as a stationary phase by centrifugal force, while the other is continuously passed through the stationary phase as a mobile phase and injected into the mobile phase. The separation method according to any one of claims 1 to 3, wherein a separated sample is continuously fractionated.