JPH0787793B2 - Method for separating highly unsaturated fatty acids - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to eicosapentaenoic acid (hereinafter abbreviated as EPA),
Method for selectively separating PUFA from natural fats and oils (for example, fish oil, whale oil, fats and oils derived from microorganisms) containing highly unsaturated fatty acid (hereinafter, abbreviated as PUFA) such as docosahexaenoic acid (hereinafter, abbreviated as DHA) as a fatty acid component It is about.

[Prior art] EPA and DHA have an inhibitory effect on platelet aggregation, and may have potential as preventive agents for cardiovascular diseases such as cerebral thrombosis and myocardial infarction.
Suggested by research by Dr. Berg. Also EP
A has a function of lowering cholesterol in blood, and its activity is said to be about four times that of linoleic acid currently used as a cholesterol-removing agent. As described above, attention has been paid to the pharmacological action of PUFA.

As a conventional method for separating PUFA, a method in which glyceride is converted to an ester of a lower alcohol such as methanol or ethanol, distilled, and then concentrated by urea inclusion treatment (for example, JP-A-58-8037), reverse method A method of concentrating and separating by using phase chromatography (for example, JP-A-58-883).
39, JP-A-58-109444) and the like have been proposed.

On the other hand, a method has been proposed in which fish oil is hydrolyzed with a lipase obtained from Candida cylindrasse to concentrate PUFA on the glyceride side, and this is separated from free fatty acids (hereinafter, abbreviated as FFA) (Japanese Patent Laid-Open Publication No. S60-187). 58-165796).

[Problems to be solved by the invention]

By the way, because of its structure, PUFA is extremely unstable with respect to light, heat, oxygen, etc., and is easily oxidized, and easily produces lipid peroxide. Lipid peroxide adversely affects the human body, causing diarrhea, vomiting,
Fever and, in the worst case, death can occur. Therefore, when treating PUFA, reaction under normal temperature, normal pressure and neutral conditions is desirable.

However, in the conventional transesterification separation method,
Since PUFA is converted into esters of lower alcohols and then separated and concentrated, there is a high possibility that PUFA will be oxidized or modified by heat, catalyst, alkali, etc. in the process of esterification.

Further, the method of concentrating and separating by using reverse phase chromatography is not suitable for a large amount of treatment because it requires the use of a large amount of filler and solvent and the treatment efficiency is poor.

Furthermore, the method of using lipase as an application of enzymatic hydrolysis to fats and oils containing conventional PUFAs utilizes the property that glycerides containing PUFAs are less likely to be hydrolyzed by lipases, and has a carbon number of 18 or less. This is a method in which the fatty acid part is decomposed into a fatty acid by lipase and PUFA is separated from FFA as a glyceride without decomposition, but there is a problem that the separation efficiency of PUFA is low and it cannot be separated at a high concentration.

The present invention is intended to solve the above problems and
The purpose of the present invention is to propose a method for separating PUFA that can be efficiently separated into a high concentration without denaturing PUFA.

[Means for solving problems]

The present invention decomposes natural fats and oils containing polyunsaturated fatty acids as a fatty acid component with lipase to produce a mixture containing monoglyceride and free fatty acid, and fractionating the mixture by centrifugal liquid-liquid partition chromatography to obtain a mixture of highly unsaturated fatty acids. A method for separating highly unsaturated fatty acids, which comprises collecting a fraction containing saturated free fatty acids and monoglycerides.

In the present invention, PUFA is not converted to an ester of a lower alcohol, but a mixture of various glycerides and FFA by partial hydrolysis with lipase is fractionated by centrifugal liquid-partition chromatography to obtain a fraction containing PUFA FFA and monoglyceride. PUFA is separated by collecting the fractions.

In the present invention, the PUFA includes a long-chain PUFA having 18 to 24 carbon atoms and 3 to 6 double bonds, examples of which include the EPA,
In addition to DHA, γ-linolenic acid, arachidonic acid, etc. may be mentioned. The raw material oil and fat in the present invention is a natural oil and fat containing these PUFAs as a fatty acid component, and examples thereof include fish oil, whale oil, evening primrose seed oil, pine nut oil, and microorganism-derived oil and fat.

The lipase used in the present invention is preferably obtained from a lipase-producing bacterium belonging to the genera Mucor, Chromobacterium, Pseudomonas and Aspergillus.
Examples of the lipase-producing bacterium include Mucor miehai, Chromobacterium viscosum, Pseudomonas fluorescens, Aspergillus niger and the like. Some of these lipases, such as those obtained from Mucor Miehai, completely removed the fat and oil.
It is difficult to decompose to A, but it is possible to decompose to monoglyceride.

In order to decompose the raw material fats and oils with lipase, water is necessary to express its activity, and the amount of water is larger than that of natural fats and oils.
30 to 70% by weight, preferably about 50% by weight is suitable.
The amount of lipase used is usually 10 to 1000 per 1 g of natural fats and oils.
0 unit, preferably about 100 to 500 units is suitable.

As the method of decomposition, the above-mentioned raw material fats and oils, lipase and water are mixed and stirred under a neutral condition at a moderate temperature and pressure suitable for enzymatic decomposition, and the reaction can be carried out for 24 to 72 hours.

In this reaction, the raw fats and oils are hydrolyzed by lipase, and PUFA is decomposed from triglyceride to diglyceride and monoglyceride into FFA. ing. On the other hand, according to centrifugal liquid-liquid partition chromatography, FFA of PUFA and monoglyceride were compared with those of triglyceride, diglyceride, and other fatty acids.
Since it can be selectively separated from monoglyceride, it is not necessary that the decomposition of PUFA by lipase completely progresses to FFA, and PUFA can be separated efficiently if monoglyceride is decomposed.

In this way, PU produced by decomposing raw fats and oils with lipase
FA and other fatty acids tri-, di-, monoglycerides and
The mixture of FFAs is fractionated by centrifugal separation-partition chromatography, and the fractions containing PUFA monoglyceride and FFA are fractionated to separate PUFAs.

Centrifugal liquid-partition chromatography is disclosed in Japanese Patent Laid-Open No. 59-62312, in which one of the two-layer separation liquid is retained as a stationary phase by centrifugal force while the other is continuously immobilized as a mobile phase. Countercurrent partition chromatography, in which the sample injected through the mobile phase is continuously fractionated.

In the present invention, the specific gravity and the polarity are different and the two phases are separated.
One of the seed solvents is the stationary phase, the other is the mobile phase, 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 a sample are sequentially distributed to a highly polar solvent, and low polar components are sequentially distributed to a less polar solvent.

The drawing is a systematic diagram of centrifugal liquid-partition chromatography used in the present invention, and the principle thereof is as follows.

Multiple distribution pipes C (500 to 4000 pipes) on the centrifuge rotor R
Are arranged parallel to the direction of the centrifugal acceleration g, and are connected in series with each other by a conduit T. Both ends of the conduit T are connected to retransfer liquid joints J ₁ and J ₂ installed at both ends of the rotary shaft of the centrifuge. Transfer liquid joint J ₁ , J ₂ is a 4-way valve V ₃ ,
6-way valve (sample injector) V ₂ , constant flow pump P
And a four-way valve V ₃ , connected to the stationary phase SP, mobile phase MP, and washing liquid WS via a four-way rotary valve V ₁ .
It is connected to the fraction collector F and the waste liquid WT container via a flow cell monitor M and a three-way rotary valve V ₄ . SL is a sampling loop and RC is a recorder.

Centrifugal liquid distribution chromatography using this apparatus is performed in the following procedure. Different in specific gravity and polarity 2
After mixing an arbitrary solvent that separates into phases and allowing it to stand, it is separated into a heavy liquid phase and a light liquid phase and stored in a container. After filling one of the liquid phases (heavy liquid phase in the drawing) into the distribution pipe C as the stationary phase SP, the rotor R is rotated to give a constant centrifugal acceleration g, while the other liquid phase (light liquid in the drawing). Phase) as the mobile phase MP, and continuously feeds it through the transfer fluid joint J ₁ at one end of the rotary shaft. The mobile phase MP becomes fine droplets in SP in the stationary phase by the action of the centrifugal acceleration g, successively passes through the distribution pipe C, and continuously flows out from the retransfer liquid joint J _{2 at the} other end of the rotating shaft. From the sampling loop SL installed outside the centrifuge, a fixed amount of the sample components introduced into the mobile phase MP in the initial stage of the liquid separation are separated into the target components by chromatography in the centrifugal liquid distribution during the above process. Fractionate with Fraction Collector F as needed.

As the stationary phase SP, use a heavy liquid with high polarity, such as acetonitrile ethanol / water (90: 10V / V) mixed solvent, and use it as a mobile phase.
When a light liquid having a low polarity, such as n-hexane, is used as the MP, the liquid having a low polarity or a long chain length in the sample is sequentially discharged together with the mobile phase. On the other hand, since the ones with high polarity or those with a short chain length will sequentially remain from the inflow side of the stationary phase, if the mobile phase solution is reversed and the stationary phase is extruded from the inflow side, the one with high polarity or Those with a short chain length flow out sequentially.

In the mobile phase thus discharged, triglycerides, diglycerides, and FFA of less unsaturated fatty acids are mainly used.
The first stationary phase flowing out by inversion contains short chain FFAs and monoglycerides, and the subsequent stationary phase containing long chain lengths and highly unsaturated FFAs and monoglycerides. . Therefore, PUFA can be separated at a high recovery rate by collecting the final stationary phase fraction.

〔The invention's effect〕

As described above, according to the present invention, natural fats and oils are decomposed with lipase to produce a mixture containing monoglyceride and free fatty acid, and the mixture is subjected to centrifugal liquid-partition chromatography to separate a fraction containing PUFA FFA and monoglyceride. Since it is possible to separate the unstable PUFA without denaturing it, and because it can be separated in a high yield in the form of FFA and monoglyceride without completely decomposing PUFA into FFA, PUFA can be separated. Can be efficiently separated.

〔Example〕

Hereinafter, the present invention will be described with reference to examples. In each example,% indicates% by weight.

Example 1 10 g of fish oil and 4 ml of purified water were put into a screw tube having a capacity of 50 ml, 100 units of enzyme obtained from Mucor Miehai was dissolved in purified water and added, and the mixture was rotated at a speed of 500 rpm in a constant temperature bath at 50 ° C. The mixture was stirred and reacted for about 48 hours. The decomposed product is a centrifugal liquid-liquid partition chromatograph CPC having the structure shown in FIG.
Fractionation by chromatography was performed using a model LLN (manufactured by Miki Engineering Co., Ltd.) under the following conditions.

Mix n-hexane and acetonitrile as a distribution liquid, and leave it to stand to separate 850 ml of the heavy liquid as a stationary phase.
The light liquid was used as the mobile phase. As a sample, 5 g of the above-mentioned fish oil decomposed product 10 g
The solution dissolved in the mobile phase was injected to 0 ml, and then 1500 ml of the mobile phase was injected for fractionation. At this time, the rotation speed is 1000 rpm, the liquid feeding speed is 10 ml / min, and the operating temperature is 20 ° C. 1500 ml of the mobile phase (light solution) eluted above was concentrated to fraction 1, and 100 ml of the stationary phase (heavy liquid) eluted first by reversing the flow direction was concentrated to fraction 2 and then eluted. 800 ml of stationary phase was concentrated to obtain fraction 3.

Fraction 1, Fraction 2, and Fraction 3 were subjected to solvent extraction with a methanol / chloroform solvent, respectively, and then ester was synthesized with methanol, and the composition was analyzed by gas chromatography under the following measurement conditions. The result is EPA from the raw oil and fat,
The recovery rate of DHA is shown in Table 1.

Fraction 1 mainly contains triglyceride, diglyceride and FFA of low unsaturation fatty acid, Fraction 2 mainly contains short chain FFA and monoglyceride, and Fraction 3 contains PUFA FFA. And monoglycerides were mainly included. Moreover, the peroxide value by the standard oil and fat analysis test method established by the Japan Oil Chemistry Association showed almost no change.

Gas chromatography measurement conditions Column: Capillary chromatogram Salmon 3000A (manufactured by Shimadzu), (φ0.24mm × 50m) Injection port, detector temperature: 250 ° C Column temperature: He; 25kPa, Air; 30kPa, H ₂ ; 30kPa Detector: FID Example 2 The reaction and fractionation were carried out using the lipase obtained from Chromobacterium viscosum under the same conditions as in Example 1. The results are shown in Table 1.

Example 3 Under the conditions of Example 1, the reaction and fractionation were performed using the lipase obtained from Pseudomonas fluorescens.
The results are shown in Table 1.

From the above results, it is understood that PUFA can be separated in a high yield by collecting fraction 3.

Comparative Example 1 The degradation product of Mucor miehai obtained in Example 1 was fractionated by column chromatography. 7% hydrous Florisil
800 g was suspended in hexane and filled in a glass tube (10 × 20 cm). 10 g of the decomposed product was dissolved in hexane and packed in a column. Hexane 500 ml, hexane / ether (90:10)
500 ml, hexane / ether (70:80) 500 ml, hexane / ether (50:50) 500 ml, methanol / ether (1
(0:90) Fractionated with 500 ml. Elute 1000 ml, then 500 ml
Each was fractionated into fractions 1, 2, and 3. The fatty acid composition of each was analyzed and shown in Table 2.

From the above results, it can be seen that PUFA cannot be separated in a high yield even by using a separation means other than centrifugal liquid-liquid partition chromatography after the decomposition with lipase.

[Brief description of drawings]

The drawing is a system diagram of centrifugal liquid distribution chromatography. SP is stationary phase, MP is mobile phase, WS is washing solution, V ₁ is 4-way rotary valve, V ₂ is 6-way valve, V ₃ is 4-way valve, V ₄
Is a 3-way rotary valve, SL is a sampling loop, P
Is a constant flow pump, J ₁ and J ₂ are recirculating fluid joints, 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.

Front page continuation (72) Inventor Yoshiaki Nunogaki Uguisudai 43, Nagaokakyo, Kyoto (56) References JP-A-51-80305 (JP, A) JP-A-59-62312 (JP, A)

Claims (4)

[Claims] 1. A natural fat or oil containing a polyunsaturated fatty acid as a fatty acid component is decomposed with a lipase to produce a mixture containing a monoglyceride and a free fatty acid, and the mixture is fractionated by centrifugal liquid-liquid partition chromatography. A method for separating highly unsaturated fatty acids, which comprises collecting a fraction of unsaturated fatty acids containing free fatty acids and monoglycerides. 2. The separation method according to claim 1, wherein the polyunsaturated fatty acid has 18 to 24 carbon atoms and 3 to 6 double bonds. 3. The method according to claim 1 or 2, wherein the lipase is produced by a lipase-producing bacterium belonging to the genus Mucor, Chromobacterium, Pseudomonas, or Aspergillus. 4. Centrifugal liquid distribution chromatography holds one of the two-phase separation liquid as a stationary phase by centrifugal force, while the other one is continuously passed through the stationary phase as a mobile phase to obtain a mobile phase. The separation method according to any one of claims 1 to 3, wherein the injected sample is continuously fractionated.