JPH0219338A - Extraction and purification of poly (unsaturated fatty acid) from natural source - Google Patents

Abstract

PURPOSE: To obtain the subject substantially pure compd. without substantially generating cis-trans transformation by treating an extract containing a mixture of poly- unsaturated free fatty acids or esters of them from marine animal oil with urea and subjecting the treated extract to low temp. fractional crystallization.

CONSTITUTION: A mixture of poly-usaturated free fatty acids or methyl or ethyl esters of them is extracted from marine animal oil and urea of which the amt. is sufficient to form a coordinated complex along with substantiall all of fatty acids (esters) capable of complexing free fatty acids and esters is mixed with a solvent of which the amt. is sufficient to dissolve urea to remove saturated fatty acids as a precipitate. After urea and solvent are removed from a filtrate, the precipitate is again dissolved in the solvent and the soln. is gradually cooled to form a precipitate to remove the same and, after the solvent is removed from the remaining filtrate in an amt. sufficient to increase the concn. of the soln., the soln. is cooled to remove a second precipitate and, further, the aforementioned operation is applied to the remaining filtrate to remove a formed separated liquid phase or solid phase to obtain a residual liquid phase containing substantially pure EPA and DHA.

COPYRIGHT: (C)1990,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the production of polyunsaturated fatty acids from natural polyunsaturated fatty acid sources.
The present invention relates to a method for separating substantially pure EI'A and DHA from oils of natural origin, such as marine animal oils; It relates to a method that uses only materials generally recognized as safe and only mild reaction conditions, and the resulting substances have cis-trans rearrangements.
n) is substantially 100% pure and can be used in food and medicine.

Various fats and fatty oils are composed of triglycerides of fatty acids. Many fatty acids, especially polyunsaturated fatty acids, are
They are difficult to synthesize and are therefore only obtained by extraction from natural fats or oils where these fatty acids naturally occur. Many of these unsaturated fatty acids have therapeutic potential.
tic potential). Others are believed to have favorable effects.

Also, fatty acids with therapeutic potential must be in the form of specific cis-trans isomers.

There has long been a need for a method of obtaining such unsaturated fatty acids in large quantities in pure form and without cis-trans conversion.

Two particular polyunsaturated fatty acids that have therapeutic effects and have been difficult to obtain in pure form and in large amounts are: (of all 2) -5,
8,11,14,17-eicosapentaenoic acid ((al
l-z)-5,8,1! , 14.17-eicosa
penta-enoic acid), hereinafter referred to as EP
A, and (all Z) -4,7,1,0,1
3,16,19-docosahexaenoic acid ((all-z)
-4,7,10,13,16,19-docosaha
-xaenoic acid), hereinafter referred to as DHA. EPA and DHA are both known as precursors in the biosynthesis of prostaglandin PGE.

British Patents Nos. 1,604,554 and 2,033
, 745, EPA in the treatment and prevention of thrombotic disorders.
Discloses the effects of British Patent Publication No. 2,148,713 to the present inventor also discloses the use of EPA and/or DHA in combination with other specific fatty acids to reduce serum cholesterol and triglyceride levels. are doing.

Traditionally, it has been very difficult to obtain pure EPA and DHA. The reason is that the main source of these fatty acids is
This is because they are oils from marine animals such as mackerel, sardines, or cod, vegetable oils such as linseed oil, or derivatives of these oils and fats such as triglycerides. Unfortunately, fatty acids other than these are always present in large amounts. Because EPA and DHA are known to have medicinal effects, large amounts of highly pure EPA and DHA are required for clinical research.

Conventional methods for extracting EPA, DHA and other useful higher unsaturated fatty acids from their triglycerides include:
The production of highly pure fatty acids has not been satisfactory. Purity used here
The term refers to groups of different chain lengths and also unsaturated moieties (un
It is used not only to separate all other fatty acids that differ in the number and position of saturation, but also to mean purity in terms of a specific cis-trans structure. Not only do conventional methods not provide sufficient purity, but in many cases conventional methods require extreme physical and chemical conditions, resulting in fatty acid degradation.
radiation), peroxide formation, and/or conversion of at least some cis-bonds to the trans-form. Furthermore, the United States Food and Drug Administration (FD)
A) those generally recognized as safe (Gen
Erally Recognized As 5afe
) (GRAS) contains unrestored materials (1
material) is 1. It is used in many conventional methods. In order for the final product to be used in food or medicine, non-GRAS substances must be present in the final product.
It is important that there are no tances.

One conventional method for purifying EPA is described in U.S. Patent No. 4.37.
No. 7.526. In this patent, a fatty acid mixture containing EPA is treated with urea to remove saturated and relatively unsaturated fatty acids. The resulting solution is then prepared.

In order to further increase the yield of EPA, a fractional distillation process is performed.

However, to carry out fractional distillation, at least 180
°C for at least 40 minutes (over a
period of) is required. The highest purity obtainable by this method, as shown in all examples of this patent, is 92.9%. Furthermore,
It has also been discovered that the substantial amount of EPA produced by this method, in some cases reaching as high as 20%, has some degree of cis-trans conversion. .

The trans-form of EPA is completely harmful in any amount for use in food or medicine.

Abu-Nasr, A. N., et al., J. Am, O.
il Chemist.

Soc., 31.41-45 (1945) discloses that methyl Eicosapentaenoate (methyleico
sapentaenoate) and ethyl docosahexaenoate
oate). This technique does not provide sufficiently high purity, and chromatographic separation requires large amounts of solvent, which is undesirable.

Te5hinia, S, et al., Bulletin of
the Japanese Society of 5
scientific Fisheries, 44(8
), 927 (1978) describes a method for isolating EPA and DHA using squid liver oil as a raw material, saponifying it with ethanolic potassium hydroxide, extracting fatty acids with ether, and methylating it. has been done. Crude fatty acid methyl ester is silica gel 60
EPA is purified from DHA by column chromatography on (Silica Gel 60) and then column chromatography on a mixture of silver nitrate and silica gel. The problem with this technology is that trace amounts of silver often remain in the final product;
This is extremely harmful in food or medicine for human consumption. Furthermore, very large amounts of solvent are required to perform column chromatography.

Disclosures regarding the use of column chromatography to separate and purify EPA to some extent are described above as well as in Japanese Jikai No. 56-115736 and USSR Patent No. 973,128.

Another conventional method for obtaining high purity EPA is disclosed in British Patent Publication No. 2,148,713. This publication describes the following method: iodizing the double bonds of unsaturated fatty acids in a fatty acid mixture, then saponifying the iodinated oil, extracting the fatty acids from the saponified mixture, and A method is described for methylating the fatty acids obtained, separating the fatty acids by column chromatography, and then deiodinating the desired fraction.

This method enables excellent resolution of fatty acids in the final (eventual) column chromatography,
The fatty acids can then be protected from oxidation during the treatment period. Using this method to separate EPA from natural EPA sources such as cod liver oil, yields of 9.
0% or more (over 90%) and purity 96-100%
is obtained. However, it has been found that a substantial amount of cis-trans conversion occurs during the steps of this process, and that the resulting 96-bEPA is not always the same. Furthermore, iodine is GRA
It is not included in the S material list.

Various separation techniques have been used for fatty acids in general. These known techniques include separation by urea complexes and separation by cryogenic fractional crystallization.

Separation techniques using urea include the formation of crystalline clathrates. This is also called an adduct or a complex (coIlρlex), which is formed between urea and various linear organic compounds. An inclusion compound is a combination of two or more compounds, one of which is included in the crystal skeleton structure of the other compound. Each component of an clathrate compound can exist separately, but the chemical bonding method is unclear.

Each of these components has a secondary valence force (secondary valence force).
They are mutually held together by force (alence force) and hydrogen bonds. However, clathrates are different from normal hydrogen-bonding systems. The reason is that in the former case, "host"
” and the size and shape of the “guest” molecules are of critical importance, whereas in the latter they play little or no role.

It is known that the higher the degree of saturation of long chain fatty acids, the easier it is to form urea complexes. Therefore, by treatment with urea, it will be possible to separate saturated compounds and most heptano-unsaturated compounds from polyunsaturated compounds. This urea complex separation technique is detailed in the following literature: Production and Uses J.
are S, Markley, part 3.2309
~2358 pages, Interscience Publishers
).

Swern, D., New York, 1964.
“Separation technology: E, urea complex (Techn
Iques of Separation: E, U
For the purposes of this section, all contents described on these pages will be treated as references.

Low-temperature fractional distillation has been used for the separation of fatty acids and monoesters, as well as for the separation of glycerides and other lipid substances in natural fats and oils. This technique involves dissolving fatty acids in a solvent and then lowering the temperature to crystallize the various fatty acids from the solvent.

Often sub-zero ('C) temperatures are used. However, this technology has many limitations. When separating a mixture of a large number of fatty acids, it is difficult to obtain high purity, and there is also the problem of mutual solubility of various acids.

This technique and the limitations of low temperature crystals are discussed in the Markley book cited above, pages 2081-2123.

Markley, K. S., “Separation Techniques: A.
Distillation, salt freshness, low temperature crystallization (Techniques o
f5eparation: A.

Distjllation, Sal, t 5olub
ility, Low-Tempera-ture C
rystalization)”.

Here, all contents described in these sections will be treated as reference documents.

Pr1vett, 0.5. et al., J., Am., Oil
Chemist, Soc.

36, 443-449 (1959) describes a technique involving the combination of low temperature crystals and urea complexes. For the analysis of pig liver lipids, two fractions were first obtained using cryofractionation, and the filtrate was subjected to fractionation from methanol via the urea clathrate. Each fraction and filtrate was individually esterified and distilled, and the various distillates were analyzed. Swern,
D, et al., J. Am, Oil Cheo+ist.

Hachihong, 29.614-615 (1952), precipitates a urea complex from olive oil to separate saturated compounds and unsaturated compounds, and then subjects the acid or ester isolated from the urea complex to a low-temperature crystallization process.
It is disclosed that oleic acid is produced with a purity of 97 to 99% by fractional distillation.

Swern, D. et al., J. Am., Oil Che.
mist, Sac,, 29°431-434 (19
52) and US Pat. No. 2,838,480, the saturated acid is first separated by crystallization from 90% methanol at 0° C. and then urea is added to the filtrate to precipitate the oleic acid adduct at room temperature. Possibly tallow, grease or red oil (r.
ed oil).

However, it is possible to substantially remove EPA and D11A from marine animal oils using only materials that are generally recognized as safe and can be used in industrial processes, and without causing cis-trans conversion. 100 to
% purity, there is no prior art that suggests this. There has also been a long-standing need in the art for methods of separating certain other polyunsaturated fatty acids from natural sources with very high purity.

Therefore, it is an object of the present invention to solve the problems of the prior art.

A further object of the present invention is to provide a method for separating and purifying desired polyunsaturated fatty acids such as EPA, DHA and their esters without degradation.

Another object of the invention is that from marine animal oil or other natural sources,
A method for extracting mixtures of EPA and DNA or other desired polyunsaturated fatty acids at temperatures readily obtainable under commercial conditions without degradation or cis-trans conversion. Moreover, the objective is to provide a method for extraction with high yield and high purity.

These and other objects are achieved in accordance with the present invention by performing the following operations: First,
Hydrolyze oil-derived triglycerides under mild conditions, such as using the enzyme lipase; separate unsaponifiables by washing with an organic solvent; and separate urea from saturated and mono-unsaturated fatty acids by treatment with urea. to form a complex, thereby removing these saturated and mono-unsaturated fatty acids; dissolve the residue in an organic solvent, preferably acetone; cool slowly; and as the solidified material forms, The above objective is achieved by removing the fraction. In a preferred embodiment, after forming and removing each precipitate,
By repeating the steps of evaporating and then cooling the solvent portion to increase the fatty acid concentration in the solution, the cryogenic treatment required to completely precipitate the pure fatty acids is avoided. In this way, virtually the same yield and purity can be obtained without the use of cryogenic temperatures, which are less desirable in industrial processes.

Furthermore, this technique can also be successfully used to separate and purify other polyunsaturated fatty acids from natural sources, such as separating cis-linoleic acid and α-linolenic acid from flaxseed oil. can.

Preferred embodiments of the present invention provide substantially 100% pure EPA and D without substantially cis-trans conversion.
Although directed to the separation of 11A, the method of the present invention can also be applied to separate and purify other highly unsaturated fatty acids from natural sources. Although much of the description that follows is directed to the isolation of pure EPA and D11A from marine animal oils, the isolation of other highly polyunsaturated fatty acids from other natural sources Also.

It should be fully understood that the same technology can be used.

The essential features of the present invention are the avoidance of all extreme conditions throughout the process and the combination of urea treatment with cryogenic fractionation crystallization, which unexpectedly results in substantially This method produces substantially pure EPA and DNA without any cis-trans conversion.

The oil from which EPA and D11A are separated according to the present invention is preferably as fresh as possible so that the separation occurs before any substantial denaturation of the fatty acids occurs. Natural fats and oils containing high levels of EPA and D11A and suitable for use in the present invention include, for example: mackerel, sardines, barracuda (n+ac
kerel pike), herring and other bluish fish; cod liver oil; and marine zooplankton oils such as krill and various shrimp-like copepods.

However, in the present invention, any other EPA
It should be understood that and DHAI can also be used. Preferably, the fish source is obtained from an environment that is as cold as possible.

Δ5-desaturase, the enzyme that catalyzes the conversion of eicosate 1-laenoic acid to EPA
turase), optimal M inactivation occurs at 90°C. Therefore, fish from cold environments have higher EPA content than warm ice fish.

Additionally, if the fish are kept in a controlled environment, they can produce even more
It even becomes possible to increase the PA yield. α-Lilunic acid (
The best EPA levels will be obtained if the fish are fed a diet rich in a-11 nolenic acid and maintained in salt water at 9°C.

Natural fats and oils can be saponified or alcohololyzed.
lysis) to convert triglycerides into free fatty acids or esters of fatty acids. however,
High temperatures and strongly basic reagents can cause peroxidation and cis-trans conversion, and methods must be chosen to avoid these.

A preferred hydrolysis method involves using the enzyme lipase at a temperature of 35-40°C.
and enzymatic hydrolysis used in p116-7.

Lipase must be activated using trace amounts of cysteine or ascorbic acid according to conventional methods. Another advantage of using lipases for saponification is that, because of their stereospecificity, lipase enzymes do not clsave trans-fatty acids that can be naturally produced from triglycerides. It is a point.

Therefore, even if there is trans-EPA or DI in the starting material! Even if A were present, they would be removed together with the non-saponifiable substances and would no longer be present in the final product.

Alternative methods for hydrolyzing natural fats and oils include partially hydrolyzing these fats and oils using lipases or strong bases. When using lipase, rather than carrying out hydrolysis for 6 hours as usual, 1172-2
By time hydrolysis, the EPA source can be further enriched. This is because lipase preferentially removes the first and third branches of the processed triglycerides. It is already known that in natural triglycerides, there are far more saturated chains in the outer branch than in the middle branch. Therefore, if the amount of hydrolysis is limited, , a substantial amount of further saturated acid can be automatically removed.

Potassium hydroxide can also be used for partial hydrolysis of natural fats and oils. The oil source is potassium hydroxide, about 15~
It is treated for 20 minutes to partially hydrolyze triglycerides. As in the case of lipases, this partial hydrolysis produces more enriched E from triglycerides.
A PA source is obtained.

The reason is that the first and third branched portions of the triglyceride are preferentially affected by the base.

After partial hydrolysis, sulfuric acid or a strong mineral acid such as nitric acid or hydrochloric acid is added to the ice melting mixture to separate the fatty acid mixture. The fatty acid mixture floats to the top and the bottom aqueous phase is discarded.

This hydrolysis step is also useful to enhance the separation of other polyunsaturates from triglycerides, regardless of their source.

Free fatty acids can also be separated from their natural sources by transesterification methods.

A fatty acid containing material (marine animal oil, linseed oil, soybean oil, etc.) is refluxed with dry ethanol or dry methanol and traces of metallic sodium. This produces methyl or ethyl esters of free fatty acids, respectively, which are liberated from triglyceride molecules. This method involves substantially milder conditions than alkaline hydrolysis and prevents blackening of the reaction mixture due to harsh conditions. These esters can be freely converted to free fatty acids during any part of the extraction process by standard hydrolysis techniques. Depending on the purpose, it may be preferable to use these esters directly without conversion to the free acid.

In the next step, unsaponifiable substances such as cholesterol, vitamins A and D, and hydrocarbons are removed by washing with an organic solvent. All organic solvents can be used for this purpose, such as petroleum ether, methylene chloride, ethyl ether.

After removing the organic phase, the aqueous phase is acidified. Although any acid can be used for this acidification step.

Pharmaceutically acceptable acids are preferred. This treatment will result in the separation of free fatty acids into the separate organic phase. The aqueous phase is then discarded. Adding small amounts of sodium chloride or other salts enhances this separation.

The fatty acid mixture is then treated with urea to remove saturated and mono-unsaturated fatty acids. In this urea treatment, urea is added to a polar organic solvent in which both urea and fatty acids can be easily dissolved. Examples of workable solvents include methanol, ethanol, isopropanol, petroleum ether, benzene, trichlorethylene, and methyl isobutyl ketone. It is preferable to use ethanol, since maternal problems can be avoided. If necessary, dissolve urea in the solvent while heating.
A urea solution containing usually 10-20% urea is obtained. Mix the urea solution and fatty acid mixture. When adding the fatty acid mixture to the urea solution, it is preferred to first dilute the fatty acid mixture separately with an organic solvent to provide some protection of the fatty acids from the high temperatures of the urea solution. Free fatty acids may be dissolved in petroleum ether or polar solvents such as acetone, ethanol or methanol.

The amount of urea solution is adjusted to at least 0.5 parts by weight, preferably from 1 to 4 parts by weight, per part by weight of the fatty acid mixture.

The urea solution is homogeneously mixed with the fatty acids.

Therefore, the urea is preferably precipitated by cooling the urea-treated solution. At this point, the saturated and unsaturated fatty acids in the fatty acid mixture will form a complex with the urea crystals and crystallize out. If desired, a cooling process may be carried out by storing the solution for an extended period of time. Alternatively, the solution may be forcibly cooled using, for example, a water bath. Good results will be obtained if the solution is cooled to at most 50°C, preferably 30-40°C. If you want to further increase the urea removal rate, store the solution at about -10°C in the refrigerator.
It should be cooled to

The complex between urea and saturated and mono-unsaturated fatty acids is then filtered off or removed by other methods.

The filtrate thus obtained is concentrated, for example in an evaporator, to remove most of the solvent, and then all remaining urea is washed from the fatty acid mixture using 5% hydrochloric acid.

Alternatively, the solvent may be removed by performing water extraction using 10 parts of water per 1 part of the solvent.

The fatty acid mixture thus remaining is a substantially pure conjugate of highly unsaturated fatty acids. The following was discovered: First, fatty acids were dissolved in an organic solvent such as acetone2.
It has been found that by then slowly cooling the individual fatty acids of interest until they solidify from solution, it is possible to completely separate the individual fatty acids in a very simple and accurate manner. As the solution is slowly cooled, various fatty acids precipitate out, depending on their individual solubility in the solution. Each of these fatty acids precipitates and is separated from the solution. For various fatty acids.

Depending on the concentration of fatty acids in solution, there are specific points at which they precipitate out of solution. For example, D.N.
A was found to precipitate from a 10% acetone solution at about -38 to -40°C. EPA precipitates at about -60°C. Most of the other fatty acids are above -30℃ (
precipitation at temperatures above).

The solution is frozen carbon dioxide (dry ice) in acetone.
Cool in bath. The precipitate formed at -38 to -40°C is
Sintered glass (sintered glass) without substantially increasing the temperature
This is separated by filtration through a red glass) or Buchner funnel. Analysis of the resulting crystals revealed that they were essentially pure DHA; NMR analysis revealed that cis-trans conversion (c
oversoin) was not observed.

The material that precipitated at about -60°C was found to be essentially 100% pure EPA with no cis-trans conversion.

To avoid the extremely low temperatures required to separate EPA and DHA separately, the supernatant is reduced after each crystallization to reduce the solubility of the fatty acids and produce substantially pure A mixture of NA and EPA can be obtained.

Also, a combination of low temperature treatment and solvent removal treatment can be used.

The following table shows the difference between Irk-Othmer, lj, Enc clo edia of Che+5ic
al Technolo, 3rd edition, Volume 4, 827
Page (1978), which shows the differences in solubility of various fatty acids at various temperatures in acetone, toluene, and n-hebutane.

Solubility of fatty acids at various temperatures in organic solvents 18:2 (9c, 12c) -50162018:O 18:1 (9t) 18:1 (9c) 1.60 0.66 0.27 0. 27 0.54 0.11 0.023 0.005 5.2 1.68 0.53 0.17 0.26 0.092 4.10 1.20 0.35 1.41 0.36 0.086 0 .018 0.390 0.080 o, ois 0.003 3.12 0.06 0.28 0.86 0.20 0.056 0.30 0.08 0.02 0.005 0.080 0.018 0.004 2.25 0.66 0.19 0.05 0, 19 0.06 0.019 0.98 0.20 0.042 From the above table, it can be seen that by decreasing the temperature by 10%, all solvent It can be seen that the solubility factors of all fatty acids are reduced by about 3 to 7. This solubility factor (s
It has been found that the desired fatty acids can be extracted by the following procedure: by reducing the amount of solvent and reducing the concentration of the solution by a factor of about 2 to 9.
about 2 to 9) raising (i.e. reducing the infusion to 1/2 to 1/9 of its original volume) 1 then a temperature substantially higher than that required without reducing the volume of the solvent; It was discovered that the desired fatty acids could be extracted by cooling the mixture.

This solvent reduction technique has a number of advantages.

In particular, it has the advantage that the required temperature can be obtained by many commercially available freezers, and the processing method does not require any special techniques or special equipment.

This solvent reduction method for low-temperature fractionated crystallization is
), can be completed by using a more highly unsaturated fatty acid conjugate (coa+bination) obtained as a result of the urea treatment step described above. The conjugate of highly unsaturated fatty acids obtained through the urea treatment process is
Dissolve it in the same type of organic solvent used for the temperature reduction method: for example, dissolve it in acetone or petroleum ether.
It can be placed in a 0% solution. When cooled overnight to about -20°C, the remaining saturated and less unsaturated fatty acids solidify from solution. Therefore,
Separate the precipitate and discard.

The volume of the solution is then reduced and its concentration increased by evaporating or distilling the solvent to a predetermined amount of the original volume (eg, 1/3 of the original volume). When the volume-reduced solution thus obtained is cooled again to about -20 DEG C., new crystals of mono-unsaturated fatty acids appear, which are again filtered off. So, once again, the volume of this filtrate is about 1 of the original volume.
/2~1/9 factor reduction
ed by a factor ofabout on
e half to one n1nth), again -2
Cool to 0°C overnight. Here, di-unsaturated fatty acids (di
-unsaturated fatty acid) will solidify from solution. These crystals are filtered off again and discarded. If a temperature low enough to form and precipitate unsaturated fatty acid crystals cannot be obtained,
The fatty acids will form a separate aqueous phase, which can be removed in a separatory funnel.

To ensure that all undesired fatty acids crystallize from solution, the filtrate may be further cooled to -30°C. If no separated phases or crystals appear, the purification process is complete.

Therefore, the solvent may be evaporated.

The remaining liquid consists of a substantially pure EPA and D11A combination. If desired, these two components may be separated by cooling to -38 to -40°C to precipitate the DHA. However, most practically, EPA and D
Since the conjugate with HA is at least the same as the individual components, there is no need to separate these secondary components.

The fact that such high purity could be obtained by the method of the present invention was truly unexpected, especially considering that the fractional crystallization method cannot be carried out without first undergoing urea treatment. This is especially beyond expectations.

After hydrolysis and removal of the organic solvent that dissolves the unsaponifiables, the resulting total fatty acid mixture is cooled and a mixture of fatty acids precipitates out of solution as a mixture. Quite unexpectedly it was discovered that: the difference in precipitation points between EPA and D11A and the highest precipitation point only if the saturated and mono-unsaturated fatty acids were first removed by the urea method. The difference in precipitation point between the fatty acids and the similar fatty acids is sufficiently large that they precipitate as pure EPA or DHA rather than as a mixture of fatty acids. Due to the large number and diversity of fatty acids found in marine animal and vegetable oils, it has been extremely difficult to separate pure EPA and DHA from them. This point is explained in the various documents cited in the Background of the Invention section above. It is only through the organic combination of a specific process of urea treatment followed by low-temperature crystallization, as well as the specific treatment sequence, that 100% pure EPA and DHA can be obtained without any cis-trans rearrangement. You can get it.

This is not something that is obvious from common knowledge in the prior art.

Although the method of the present invention has been described using acetone as the organic solvent, from which fatty acids are precipitated by cooling or slow cooling while increasing the concentration of the solution,
The following points should be understood: That is, the relative precipitation points of EPA and DMA
Other organic solvents other than acetone can also be used for the above purpose, as long as the tation points are sufficiently separated from each other and also from other fatty acids in the mixture that a pure precipitate can be separated. So all can be used. It is believed that: That is, the temperature at which various fatty acids precipitate in most solvents depends on the concentration of fatty acids in the solution.

It is independent of the particular solvent used. acetone,
10 of methylene chloride, cyclohexane and petroleum ether
The precipitation points of DNA from % solutions are all about -38 to -
The temperature is 40°C. Similarly, the precipitation point of EPA from 10% solutions in acetone, cyclohexane, and petroleum ether is from about -60<0>C to about -30<0>C, depending on the concentration of DNA in these solutions. Whether a given solvent is workable or unworkable for this purpose can easily be determined by testing in accordance with conventional methods. for example,
It has been found that while toluene and heptane may be viable, they are relatively toxic and are therefore not preferred. The optimal organic solvent will be one that is non-toxic and generally recognized as safe, or one that can be completely removed without substantial treatment. Examples of suitable solvents other than acetone include cyclohexane, petroleum ether and methylene chloride. Also, depending on the concentration of the solute, the solvent remains in a liquid state over the entire precipitation temperature range, even at temperatures of 1, for example at least -70°C.

is necessary.

In implementing the capacity reduction embodiment of the present invention.

The exact temperature at which the solution is cooled and the exact volume reduction will vary depending on the particular fatty acids present in the conjugate. These parameters can be determined empirically by one skilled in the art without undue experimentation. The solution can be cooled to a temperature slightly below the precipitation initiation temperature and maintained at that temperature until precipitation is complete. Therefore, a capacity reduction process is performed. According to a rough but practical rule of thumb, in each step the capacity should be reduced to 173 of the capacity in the first step. However, if the conjugates of fatty acids precipitate together, depending on the particular fatty acid in the conjugate, the reduction in capacity will prove to be too great. On the other hand, if precipitation does not occur at a temperature suitable for reducing the volume of the solution, it will be an indication that the volume reduction is not sufficient. Routine experimentation will determine the amount of reduction in volume at which precipitation of the next fraction will occur at the desired temperature.

The invention is illustrated by the following examples, but the invention is not limited to these examples: 500 cc of cod liver oil was vigorously stirred in 2Q of distilled water. 50 g of sodium salt of EPA was added as an emulsifier. The pH was maintained at 7 during one reaction using phosphate buffer and ascorbic acid. 1 g of porcine pancreatic lipase was added to the emulsion and the emulsion was maintained at 40° C. with stirring for 6 hours.

Add 100cc of petroleum ether to remove cholesterol,
Unsaponifiables such as vitamin D, vitamin A and other impurities were removed. After thorough mixing, the petroleum ether phase was separated from the aqueous phase and the petroleum ether phase was removed using a separatory funnel.

The aqueous phase consists of 5%) 12So4 solution and 50 g of sodium chloride.
The organic phase was salted out. The organic phase separated from the aqueous phase contained the free acid and was separated using a separatory funnel and dried using sodium sulfate. Sodium sulfate was filtered off. At this point, the organic phase was approximately 450 cc and contained free fatty acids. Then add 200 c of absolute ethanol to this organic phase.
c mixed. 800 g of urea was dissolved in absolute ethanol 2Q with continuous stirring and heating to 70°C.

Therefore, this urea-alcohol solution was mixed with an ethanol solution of free fatty acids. Immediately, a complex of urea and saturated and mono-unsaturated fatty acids formed and precipitated. The liquid phase was decanted and the ethanol removed using an evaporator, which was recovered for reuse. Urea remaining in the liquid phase was removed by washing with a 5% hydrochloric acid solution and then with distilled water. The organic phase thus obtained was analyzed by gas chromatography. The results of the analysis were as follows: Tane-ban Ichiba percent 16: 1 4.818:
1 1.518: 2
1.220: 5
5722: 6 1822:
1 17.5 This organic phase was then dissolved in acetone to give an approximately 10% solution. This solution was placed in a commercially available freezer and the temperature of the solution was slowly lowered.

Initial precipitation occurred at about 0 to -2°C. The precipitate was filtered off and found to be essentially pure 24:1 fatty acid. A subsequent precipitate occurred at about -5 to -8°C and was collected by filtration. This precipitate was found to be a mixture of 17:1 and 16:1 fatty acids.

The next precipitation occurs at about -12°C, which is substantially 18:2
It turned out to be a fatty acid.

The residual solution was then placed in a dry ice-acetone bath for further cooling. Another precipitation occurred between -38 and -40°C.

without substantially increasing the temperature using a Buchner funnel.
This precipitate was removed and analyzed by gas chromatography. This product was found to be 100% D11A. The accuracy of the gas chromatography device used was o,
oot%, and no impurities were observed. This sample was also analyzed by NMR, and it was found that no cis-trans conversion occurred. The product was 100% all-cis D11A. As a result of NMR studies, no peaks corresponding to the trans-configuration were observed.

As the temperature of the residual solution continued to decrease to about 60 DEG C., another precipitation occurred. Separated in the same manner as described above for DHA, this precipitate was 100% pure [EPA
It turned out to be. Results of NMR study.

Indeed, no trans-configuration was observed.The yield of EPA was approximately 15% compared to the starting material, cod liver oil.
%, and the DNA yield is approximately 9% of the original cod liver oil.
Met.

Run 17 - For comparison, the same process as described above was repeated except for the urea treatment. By all the same methods as in Example 1 above, 500 cc of cod liver oil was treated with lipase and non-saponifiable
erial) was removed, the aqueous phase was then acidified and the organic phase was salted out and collected. Since this organic phase contains free fatty acids, the organic phase is then dissolved in acetone and
A 0% solution was prepared. This solution was placed in a commercially available freezer and the temperature of the solution was slowly lowered. Precipitation started at about +20°C. This precipitation occurred continuously until the temperature dropped to about -15°C. The precipitate was filtered off and found to be a mixture of fatty acids. The filtrate was further cooled,
No further precipitation occurred up to -70°C. No residue remained even after the acetone was evaporated. Thus, resolution of fatty acids is not possible without first being treated with urea.

Treatment of this precipitate with urea should yield substantially the same results as the initial urea treatment described in Example 1, but with the exception of resolving pure EPA or DHA.
lve). This will only slightly separate the saturated fatty acids and most mono-unsaturated fatty acids from the rest of the unsaturated fatty acids.

In the five main fractional crystallization process, petroleum ether, cyclohexane, or methylene chloride is used instead of acetone.
The method of Example 1 was repeated. When using petroleum ether and cyclohexane.

The same results were obtained using acetone. Precipitation of EPA or DNA occurred at the same temperature as with acetone. When methylene chloride was used as the solvent, substantially 100% pure NA was precipitated at -38 to -40°C, as in Example 1. However, assimilation of methylene chloride begins before EPA precipitates; therefore, methylene chloride is not a suitable solvent.

11. Kill the salmon at 0 to 4 degrees Celsius. Remove its internal organs and cut the meat into slices. Immediately spray all over the slices with a 1:1 mixture of γ-tocopherol and ascorbyl palmitate.

The antioxidant sprayed slices were then mixed in a blender for 1-2 minutes, and the blended mixture was transferred to a centrifuge for 7,000-10 minutes. 1 at OOOrpm
Process for 0-15 minutes. After centrifugation, the oil phase is separated and mixed with 1% aqueous hydrochloric acid in a 1:1 ratio.

After thorough mixing, allow the phases to separate and separate the oil phase using a separatory funnel. The mixing step with dilute hydrochloric acid is repeated several times to remove all methylamine, and the oil is then washed several times with distilled water and then separated to remove any remaining acid. . Then 0.02% gamma-tocopherol and 0 ascorbyl palmitate based on the weight of the oil.
．． Antioxidants are further added in an amount of 0.02%.

The oil thus obtained is then treated exactly as in the case of cod liver oil in Example 1.

As in Example 1, 100% all cis D1
The same <100% all-cis EPA as 1A is obtained.

110 cc of fish oil, 80 cc of 95% ethanol and 2 ml of water
Mix with 0cc. Then, 23 g of potassium hydroxide is added. The mixture is heated to reflux in the flask with continuous stirring. Nitrogen is bubbled through the solution. The addition of trace amounts of ascorbic acid, ascorbyl palmitate, and gamma-tocopherol to both the organic and aqueous phases of the mixture prevents the oil from oxidizing.

Hydrolysis is complete after 60-90 minutes of reflux. The mixture is cooled and poured onto crushed ice, followed by the addition of 200 cc of water. This mixture is shaken with a water-immiscible organic solvent to wash away unsaponifiable substances such as cholesterol, vitamin A, vitamin D, and hydrocarbons. After removing the organic phase in a separatory funnel, add 4 moles of sulfuric acid or its equivalent to 120
Acidify the aqueous phase using ~140 wR. The free acid mixture is extracted with 1 portion of petroleum ether, separated and dried over magnesium sulfate.

Completely evaporate the petroleum ether in vacuum.
- ad over vacuum), dissolve the free fatty acids in 100 cc of ethanol and add 75 g of urea in absolute ethanol. Stir these mixtures well and cool. Inclusion crystal of urea and saturated fatty acid (i
nclusion crystal) will appear.

These are filtered and the supernatant is evaporated until new inclusion crystals appear. The supernatant is further cooled until more clathrate crystals form and these crystals are filtered off. The remaining supernatant, which contains most of the polyunsaturated free fatty acids, is evaporated. The residue from the supernatant is dissolved in petroleum ether and 5% HCI and separated.

The organic phase is then washed with water.

Free fatty acids have a fatty acid to petroleum ether ratio of 1:
10 in petroleum ether and cooled overnight to -20°C. On the 6th day, the solution is filtered and discarded. The filtrate is reduced to one third of its original volume and cooled again to -20°C. New crystals of mono-unsaturated fatty acids appear and are filtered out. The filtrate is then reduced to 1/9 of its original volume and - requires cooling. Either new crystals or a new phase consisting of di-unsaturated fatty acids appear; either the separated fatty acid phase is separated in a separatory funnel or the crystals are filtered out and discarded. The filtrate is cooled again to -30°C.
No separate phases or crystals appear. The solvent is then evaporated and the remaining liquid consists of EPA and DHA.

100 g of isolated linseed oil is treated as in Example 5 to form a mixed solution of precipitated urea complex and polyunsaturated free fatty acids. A cooling step and a volume reduction step are then carried out in the same manner as described in Example 5.

The first volume reduction step (volume reduce,
on 5tep), palmitic acid (palmiti
c acid) crystals appear.

In the second volume reduction step, liquid oleic acid (oleic acid)
A separated phase of oleic acid (acid 1iquid) or crystals of oleic acid appear.

In the third volume reduction step, liquid cis-linoleic acid (ci
A separated phase of s-linoleic acid 1quid or crystals of cis-linoleic acid appears.

The only fatty acids found in the filtrate after the third step are α-
Alpha-1 inolenic acid
).

It will be apparent to those skilled in the art that various modifications can be made without departing from the scope of the present invention, and that the present invention should not be considered limited to only what is described in this specification. That's probably the case.

Agent Patent Attorney 1) Parent Male

Claims (1)

[Claims] 1. From marine animal oil, (all-z)-5,8,1
1,14,17-eicosapentaenoic acid (EPA) and (
A method for separating a substantially pure mixture of all-z)-4,7,10,13,16,19-docosahexaenoic acid (DHA), comprising: extracting polyunsaturated free fatty acids, or mixtures of their methyl or ethyl esters, from the oil; (2) mixing the fatty acids or esters with urea and a polar organic solvent, the solvent comprising urea and the fatty acids or esters; and the urea is present in a coordination complex with substantially all of the fatty acids or esters that may be complexed therewith.
(3) removing the precipitate formed after the mixing step and collecting the filtrate; (4) removing residual solvent and urea from the filtrate to obtain a pure (5) Dissolve the pure fatty acid or ester in an organic solvent to form a solution; (6) Slowly cool the solution until a first precipitate forms; (7) removing from the filtrate remaining after removing the precipitate an amount of solvent sufficient to substantially reduce the volume of the solution and increase the concentration of the solution; (8) a second precipitate; (9) adding a sufficient amount of solvent to substantially reduce the volume of the solution and increase its concentration; From the filtrate remaining after removal of the second precipitate, remove; (10) slowly cool the filtrate until a separate liquid or solid phase is formed, and remove the separate phase; and (11) A residual liquid phase containing a mixture of essentially pure EPA and DHA is retained. 2. In claim 1, the temperature at which the solution is cooled in steps 8 and 10 to obtain the precipitate is
The method of the preceding paragraph, wherein sufficient solvent is removed in steps 7 and 9 such that the temperature is substantially the same as that at which the solution is cooled in step 6. 3. The method of claim 2, wherein the temperature is about -10 to 30°C. 4. In claim 1, steps 7 and 9
The method of claim 1, wherein the solvent is removed from the solution in each step, reducing the volume of the solution by a factor of about 2 to 9. 5. The method of claim 4, wherein in steps 7 and 9, the amount of solution is reduced by a factor of about 3. 6. Substantially pure (all-z) from marine animal oils
-5,8,11,14,17-eicosapentaenoic acid (
EPA) comprising: (1) from an oil a polyunsaturated free fatty acid;
(2) mixing the fatty acid or ester with urea and a polar organic solvent, the solvent being present in an amount sufficient to dissolve the urea and the fatty acid or ester; and the urea is complexed with it.
(exed) in an amount sufficient to form a coordination complex with substantially all of the possible fatty acids or esters;
(3) removing the precipitate formed after the mixing step and collecting the filtrate; (4) removing residual solvent and urea from the filtrate to obtain the pure fatty acid or ester; (5) removing the pure fatty acid or ester; Dissolve the fatty acid or ester in an organic solvent to form a solution; (6) slowly cool it and remove any new solid or liquid phase that separates from the solution during this cooling process; and (7) substantially Retain a phase containing essentially pure EPA. 7. The method of separating substantially pure EPA from marine animal oil in claim 6 further includes substantially pure (all of Z) -4,7,10,13,1
Adding a method for separating 6,19-docosahexaenoic acid (DHA) and further providing substantially pure DHA after the slow cooling and subsequent removal step in the method.
The method according to the same item, comprising the step of maintaining a phase containing. 8. Substantially pure from marine animal oils (all Z)
- A method for separating 4,7,10,13,16,19-docosahexaenoic acid (DHA), comprising: (1) polyunsaturated free fatty acids, or their methyl or extracting a mixture of ethyl esters; (2) mixing the fatty acid or ester with urea and a polar organic solvent, the solvent being present in an amount sufficient to dissolve the urea and the fatty acid or ester; and (3) removing the precipitate that forms after the mixing step; , and collecting the filtrate; (4) removing residual solvent and urea from the filtrate to obtain a pure fatty acid or ester; (5) dissolving the pure fatty acid or ester in an organic solvent to form a solution; (6) ) cooling slowly and removing any new solid or liquid phase that separates from the solution during this cooling step; and (7) retaining a phase containing substantially pure DHA.
(retaining). 9. In any one of claims 1, 6, or 8, the organic solvent used in the dissolution step consists of ethanol, methanol, acetone, cyclohexane, petroleum ether, or ethylene chloride. The method described in the same paragraph, which is selected from the group. 10. In any one of claims 1, 6, or 8, the step (1) consists of the following step.
The method described in the same paragraph: (a) peroxidation and cis-trans conversion (cis-tra
(b) removing unsaponifiables from the hydrolysis product by washing with an organic solvent and removing the aqueous phase; and (c) acidifying the collected aqueous phase to release the organic phase (
release) and recover the organic phase containing pure fatty acids or esters. 11. Claim 10, wherein prior to the hydrolysis step, oxidation is prevented by adding one selected from the group consisting of ascorbic acid, ascorbyl palmitate, gamma tocopherol, and mixtures thereof. The method described in section. 12. The method of claim 10, wherein nitrogen is bubbled through the oil throughout the hydrolysis step. 13. In any one of claims 1, 6, or 8, the polar organic solvent used in the mixing step with urea is methanol, ethanol, isopropanol, petroleum ether, benzene, trichloro The method according to the same item, wherein the compound is selected from the group consisting of ethylene and methyl isobutyl ketone. 14. The method according to claim 13, wherein the solvent used in the mixing step with urea is ethanol. 15. In any one of claims 1, 6, or 8, the urea mixing step is carried out at a temperature sufficient to dissolve the urea and the fatty acid or ester in the organic solvent used. , and further comprising a step of cooling the mixture after the step of mixing with urea to produce a precipitate. 16. In any one of claims 1, 6, or 8, the step of extracting the polyunsaturated fatty acid mixture from the oil comprises removing triglycerides in the oil using lipase or a strong base. The method described in the same section, which includes a step of hydrolyzing. 17. In claim 16, the hydrolyzing agent detaches only the first and third branches of the triglyceride to be hydrolyzed, so that the middle branch is substantially on the glyceride skeleton. The method according to the same item, wherein the hydrolysis step consists of hydrolysis only for a period of time long enough to remain intact. 18. In any one of claims 1, 6, or 8, the step (1) includes a step of hydrolyzing triglycerides in the oil by transesterification using ethanol or methanol. The method described in the same paragraph. 19. Claim 18, wherein the transesterification step consists of refluxing the oil with absolute ethanol or methanol in the presence of trace amounts of sodium metal, sodium ethoxide or sodium methoxide. Yes, the method described in the same section.