EP1841847A1

EP1841847A1 - Method for producing a dha-containing fatty acid composition

Info

Publication number: EP1841847A1
Application number: EP06701343A
Authority: EP
Inventors: Dirk Fabritius
Original assignee: Nutrinova Nutrition Specialties and Food Ingredients GmbH
Current assignee: Celanese Sales Germany GmbH
Priority date: 2005-01-26
Filing date: 2006-01-26
Publication date: 2007-10-10
Also published as: KR100990814B1; JP2013151689A; JP2008528743A; US20080175975A1; CA2595917C; DE102005003625A1; CA2595917A1; CN101107348A; KR20070094951A; WO2006079534A1; AU2006208643A1

Abstract

The invention relates to a method for producing a fatty acid composition which, based on the entire weight of the fatty acids and/or fatty acid derivatives contained in the fatty acid composition, contains at least 70.0 % by weight of docosahexaenoic acid and/or docosahexaenoic alkyl ester. The inventive method comprises the following steps: a) transesterifying a biomass obtained fromUlkenia sp. with an alcohol, thereby producing at least one docosahexaenoic alkyl ester and at least one saturated fatty acid ester, b) producing a solution which contains urea, at least a part of the biomass from step a) and at least one organic solvent, c) cooling or concentrating the solution from step b), thereby producing i) a precipitate which contains urea and at least a part of the saturated fatty acid esters, and ii) a liquid fraction, and d) separating the precipitate i) from the liquid fraction ii). The invention also relates to the fatty acid composition obtainable according to the inventive method and to the use thereof.

Description

Process for the preparation of a DHA-containing fatty acid composition

The present invention relates to a method for producing a fatty acid composition which contains, based on the total weight of the fatty acids contained in the fatty acid composition and / o ^"of the fatty acid derivatives at least 70.0 wt .-% docosahexaenoic acid and / or docosahexaenoic.

Highly unsaturated polyunsaturated fatty acids (PUFAs) are essential fatty acids in human metabolism. PUFAs can be divided into two large groups. In addition to the group of ω-6 PUFAs, which is formulated starting from linoleic acid, there is the group of ω-3 PUFAs, which is built up from the α-linolenic acid.

PUFAs are important building blocks of cell membranes, retina and meninges and precursors of important hormones such as prostaglandins, thromboxanes and leukotrienes.

In addition to the function as building blocks, more and more has been shown over the last few years that PUFAs directly have many positive effects on the human organism or diseases.

A variety of clinical studies have shown that PUFAs, e.g. In cancer, rheumatoid arthritis, hypertension and atopic dermatitis and many other diseases can make an important contribution to the cure or relief. The use of docosahexaenoic acid (DHA; all-cis

4,7,10,13,16,19 docosahexaenoic acid) star (as opposed to the free DHA), often particularly advantageous because such esters (especially the ethyl esters and triglycerides) tend to have a pleasant taste and are easily spoiled by the Digestive system to be absorbed. These findings were responsible for the fact that international institutions and authorities have issued recommendations that regulate the daily intake of PUFAs. PUFAs can not be de novo synthesized by humans because they lack the enzyme systems that can introduce a double bond into the carbon chain at positions> C9 (missing Δ12-desaturase). Only through the supply of so-called precursor fatty acids (precursors, eg α-linolenic acid) through the diet, the human is able to synthesize highly unsaturated fatty acids. However, whether this amount is sufficient to meet the need for high-unsaturated fatty acids is controversial.

Most of the essential fatty acids are absorbed through the diet. In particular, vegetable oils are enriched with ω-6 fatty acids, (for example, evening primrose oil contains γ-linolenic acid (GLA)), but only up to a chain length of Cl 8, and fish oils or oils from microorganisms with ω-3 fatty acids (eg, salmon oil contains eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA; all-cis 4,7,10,13,16,19 docosahexaenoic acid)). In general, fish oils and microorganism oils are the only commercial source of highly unsaturated fatty acids. In general, however, the content of the desired PUFA is low and these are present in a mixture, and antagonistic PUFAs may also be included. In order to take the recommended daily dose of PUFAs, therefore, a high amount of oil must be taken. In particular, this applies to those patients who must take high doses of PUFAs (for example, in cystic fibrosis). In order to achieve a targeted effect of the individual PUFAs, enriched or high-purity PUFAs must be used. There is therefore a high demand for high-purity PUFAs in the prior art.

Numerous methods have been used singly or in combination to isolate (or at least concentrate) and recover certain fatty acids and their derivatives from a variety of naturally occurring sources. These processes include fractional crystallization at low temperatures, molecular distillation, urea-hydrofusion, Extraction with metal salt solutions, supercritical fluid fractionation on countercurrent columns and HPLC methods.

In W.W. Christi, Lipid Analysis, p.147-149 (Pergamon Press, 1976) discloses a general method in which urea is used to separate methyl esters of saturated fatty acids from a mixture which also contains methyl esters of polyunsaturated fatty acids. According to Christ, when urea is allowed to crystallize in the presence of several different long-chain aliphatic compounds, hexagonal crystals are formed which include the aliphatic compounds (so-called "urea complexes"). The aliphatic compounds can then be easily separated from the solution by filtration.

Christie states that the methyl esters of saturated fatty acids are more likely to form urea complexes than the methyl esters of unsaturated fatty acids of the same chain length, and that the methyl esters of unsaturated fatty acids having trans double bonds form urea complexes more readily than the methyl esters of the corresponding cis double bond unsaturated fatty acids. Christie also describes the use of urea crystallization to concentrate methyl esters of polyunsaturated fatty acids from a mixture containing methyl esters of polyunsaturated fatty acids and methyl esters of saturated fatty acids. In this way, apparently fatty acid compositions can be obtained with a mass yield of 20%, but in the document missing specific information on the experimental procedure and the PUF A yield. Furthermore, no information on the quality of the products, for example, the peroxide content, it can be taken.

Another document describing the separation of fatty acid methyl esters using urea crystallization is T. Nakahara, T. Yokochi, T. Higashihara, S. Tanaka, T. Yaguchi, D. Honda "Production of Docosahexaenoic and Docosapentaenoic Acids by Schizochytrium sp. isolated from Yap Islands, JAOCS, Vol. 73, No. 11, pp. 1421-26 (1996) Nakahara et al. describe the preparation of a mixture of fatty acid methyl esters by adding Schizochytrium sp. Cells are washed and dried and then methyl esterified directly with methanol in the presence of 10% HCl. Nakahara et al. report that 34.9% of the resulting methyl ester contained DHA residues and 8.7% of the resulting methyl ester contained DPA residues. To concentrate these polyunsaturated fatty acid esters, methanol and urea are added to the mixture. The mixture is then heated to 60 ° C to dissolve the urea in order, and then cooled to 10 ⁰ C, to crystallize the urea. According to Nakahara et al. Thus, a mixture containing 73.3% DHA methyl ester and 17.7% DPA methyl ester was obtained. Further information on ^{'experimental} procedure, the yield and quality of the products obtained the document can not be removed.

WO 01/51598 A1 discloses a process for the preparation of an enriched mixture of polyunsaturated fatty acid esters, in which an oil of Schizochytrium sp. transesterified with an alcohol (methanol). The fatty acid esters are then dissolved together with urea in a medium and cooled or concentrated to at least partially separate the saturated fatty acid esters which precipitate together with the urea. In this way, an oil can be obtained which, according to gas chromatography, contains 23.4% by weight of ω-6 DPA methyl ester, 65.2% by weight of ω-3-DHA methyl ester, 2.9% by weight of myristic acid. Methyl ester and 1.5 wt .-% palmitic acid methyl ester.

A disadvantage of the procedures described above is in particular the use of toxic methanol to esterify the fatty acids. Therefore, the fatty acid compositions described in these references are not suitable for food applications. Furthermore, the disclosure of the methods, in particular the method of Christie and the method of Nakahara et al., Is so incomplete and incomplete that they can not be reworked. In addition, in particular for the in Nakahara et al. be assumed that due to the W 2

relatively high increase in the DHA content, a comparatively low overall yield is obtained.

The growing use of polyunsaturated fatty acids, especially of DHA, and their esters in medicine and nutrition causes the

A desire for a most cost-effective and reliable method for producing a fatty acid composition having a very high proportion of polyunsaturated fatty acids, in particular to Docosahexaensäure and / or Docosahexaensäurealkylester and at the same time the lowest possible proportion of saturated fatty acids.

In view of this prior art, it was therefore an object of the present invention to provide such a method. The process of the invention should allow the preparation of the fatty acid composition in the simplest possible way, on a large scale and inexpensively.

At the same time, the process should provide a fatty acid composition with the highest possible purity and quality, in particular with the lowest possible acid value and / or with the lowest possible heavy metal content.

In addition, the process should be as gentle as possible and in particular to fatty acid compositions with the lowest possible peroxide - lead.

Furthermore, the process according to the invention should be possible as far as possible using solvents which are as suitable as possible for food. In particular, the use of hazardous substances should be avoided as far as possible.

The fatty acid compositions obtainable by the process should have the lowest possible ethyl carbamate content, in particular the Application of fatty acid compositions in the food industry without hesitation.

These and other objects, which are not quoted literally, but which can be deduced as a matter of course from the contexts discussed herein or which result inevitably, are solved by a method having all the features of present claim 1. Advantageous modifications of the method according to the invention are disclosed in US Pat the dependent claims described on claim 1. The claims of the product category protect the fatty acid composition obtainable by the process according to the invention and the use claims indicate particularly advantageous fields of application of the fatty acid composition according to the invention.

By providing a process for the preparation of a fatty acid composition which, based on the total weight of the fatty acids and / or fatty acid derivatives present in the fatty acid composition, contains at least 70.0% by weight of all-cis 4,7,10, 13,16,19 docosahexaenoic acid and / or allcis 4,7,10,13,16,19 docosahexaenoic acid alkyl ester, wherein: a) one from Ulkenia sp. transesterifying available biomass with at least one alcohol to form at least one docosahexaenoic acid alkyl ester and at least one saturated fatty acid ester, b) preparing a solution containing urea, at least part of the transesterified biomass from step a) and at least one organic solvent, c ) cooling or concentrating the solution of step b) to i) a precipitate containing urea and at least a portion of the saturated fatty acid esters, and ii) forming a liquid fraction, d) the precipitate i) of the liquid fraction ii) disconnects, fails to provide a novel and useful process for preparing a fatty acid composition which, based on the total weight of the fatty acids and / or fatty acid derivatives present in the fatty acid composition, is at least 70.0 wt. % Docosahexaenoic acid and / or docosahexaenoic acid alkyl ester. This was particularly surprising because enrichment of, in particular, the DHA and / or the DHA ester is achieved according to the invention, although the compounds to be separated according to the invention are very complex molecules and there are only minimal structural differences between them.

At the same time, the procedure according to the invention has a number of further advantages:

The method according to the invention can be carried out in a simple manner, on an industrial scale and at low cost. > The inventive method is extremely efficient and makes the

Fatty acid compositions accessible with comparatively high yields.

The process according to the invention provides a fatty acid composition with comparatively high purity and quality, in particular with a comparatively low acid number and with a comparatively low heavy metal content. The acid number of the fatty acid composition, as measured by AOCS Official Method Ja 8-87, is preferably less than or equal to 1.5 mg KOH per g of fatty acid composition, desirably less than or equal to 0.8 mg KOH per g of fatty acid composition, preferably smaller or equal to 0.2 mg KOH per g fatty acid composition, in particular less than or equal to 0.06 mg KOH per g fatty acid composition.

The process according to the invention is comparatively gentle and leads to fatty acid compositions with a comparatively low peroxide content. The peroxide value of the fatty acid composition, measured according to AOCS Official Method Cd-3d 63, is preferably less than or equal to 0.5 meq. per kg of fatty acid composition, suitably less than or equal to 0.1 meq. per kg of fatty acid composition, preferably less than or equal to 0.05 meq. per kg of fatty acid composition, in particular less than or equal to 0.01 meq. per kg fatty acid composition. The heavy metal content of the fatty acid composition, measured in accordance with LMBG §35 L06.00-7, is preferably less than or equal to 0.7 mg per kg fatty acid composition, advantageously less than or equal to 0.4 mg per kg fatty acid composition, preferably smaller or equal to 0.3 mg per kg of fatty acid composition, in particular less than or equal to 0.2 mg per kg of fatty acid composition.

The fatty acid compositions obtainable by the process are distinguished by a comparatively low cadmium content. The cadmium content of the fatty acid compositions obtainable according to the invention, measured in accordance with LMBG §35 L06.00-7, is preferably less than or equal to 0.20 mg per kg of fatty acid

Composition, suitably less than or equal to 0.10 mg per kg of fatty acid composition, preferably less than or equal to 0.05 mg per kg of fatty acid composition, in particular less than or equal to 0.03 mg per kg of fatty acid composition. > The fatty acid compositions obtainable by the process are characterized by a comparatively low lead content. The lead content of the fatty acid compositions obtainable according to the invention, measured in accordance with LMBG §35 L06.00-7, is preferably less than or equal to 0.20 mg per kg fatty acid composition, advantageously less than or equal to 0.10 mg per kg fatty acid composition , preferably less than or equal to 0.05 mg per kg fatty acid composition, in particular less than or equal to 0.03 mg per kg fatty acid composition. > The fatty acid compositions obtainable by the process are characterized by a comparatively low mercury content. The mercury content of the fatty acid compositions obtainable according to the invention, measured according to LMBG §35 L06.00-7, is preferably less than or equal to 0.10 mg per kg fatty acid composition, suitably less than or equal to 0.05 mg per kg fatty acid composition, preferably less than or equal to 0.01 mg per kg fatty acid composition, in particular less than or equal to 0.005 mg per kg fatty acid composition.

> The fatty acid compositions obtainable by the process are distinguished by a comparatively low arsenic content. The arsenic content of the fatty acid compositions obtainable according to the invention, measured in accordance with LMBG §35 L06.00-7, is preferably less than or equal to 0.20 mg per kg of fatty acid

Composition, suitably less than or equal to 0.10 mg per kg of fatty acid composition, preferably less than or equal to 0.05 mg per kg of fatty acid composition, in particular less than or equal to 0.03 ^■ mg per kg of fatty acid composition. > The fatty acid compositions obtainable by the process are characterized by a comparatively low copper content. The copper content of the fatty acid compositions obtainable according to the invention, measured according to LMBG §35 L06.00-7, is preferably less than or equal to 0.25 mg per kg fatty acid composition, advantageously less than or equal to 0.20 mg per kg fatty acid composition , preferably less than or equal to 0.10 mg per kg of fatty acid composition, in particular less than or equal to 0.06 mg per kg of fatty acid composition. ) ^r> obtainable by the process of fatty acid compositions are characterized content of iron by a comparatively low.

The iron content of the fatty acid compositions obtainable according to the invention, measured in accordance with LMBG §35 L06.00-7, is preferably less than or equal to 0.25 mg per kg fatty acid composition, advantageously less than or equal to 0.20 mg per kg fatty acid composition , preferably less than or equal to 0.10 mg per kg of fatty acid composition, in particular less than or equal to 0.06 mg per kg of fatty acid composition. > The fatty acid compositions obtainable by the process are characterized by a comparatively low nickel content. The nickel content of the fatty acid compositions obtainable according to the invention, measured according to LMBG §35 L06.00-7, is preferably less than or equal to 0.25 mg per kg fatty acid

Composition, expediently less than or equal to 0.20 mg per kg of fatty acid composition, preferably less than or equal to 0.10 mg per kg of fatty acid composition, in particular less than or equal to 0.06 mg per kg of fatty acid composition. > In the method according to the invention comparatively food-safe solvents are used.

The fatty acid compositions obtainable by the process according to the invention furthermore have a comparatively low ethylcarbamate content and are therefore particularly suitable for applications in the foodstuffs sector.

The present invention relates to a process for preparing a fatty acid composition which, based on the total weight of the fatty acids and / or fatty acid derivatives present in the fatty acid composition, preferably based on the total weight of the fatty acids and / or fatty acid esters contained in the fatty acid composition, in particular based on the total weight of the fatty acids and / or fatty acid triglycerides contained in the fatty acid composition, at least 70.0% by weight of all-cis 4,7,10,13,16,19 docosahexaenoic acid and / or all-cis 4,7, 10,13,16,19 docosahexaenoic acid alkyl ester.

The term "fatty acid composition" in this context includes both compositions containing free fatty acids and compositions containing fatty acid derivatives, preferably fatty acid esters, especially fatty acid triglycerides, wherein the fatty acid residues may in principle be the same or different. According to the invention, fatty acids denote aliphatic carboxylic acids which may be saturated or mono- or polyunsaturated and preferably have from 6 to 30 carbon atoms.

In the method according to the invention is a from Ulkenia sp. available

Biomass used as starting material. From Ulkenia sp. Available biomasses are known per se. According to the invention, both biomasses of Ulkenia sp. Wild-type strains as well as biomass of mutant or recombinant Ulkenia sp. Strains that efficiently produce DHA (all-cis 4,7,10,13,16,19 docosahexaenoic acid) and / or DPA (all-cis 4,7,10,13,16 docosapentaenoic acid). Such mutant or recombinant strains include microorganisms which, compared to the percentage of the original Ulkenia sp. Wild-type strain, using the same substrate, a higher percentage of DHA and / or DPA in fats and / or compared to that obtained by the original Ulkenia sp. Wild-type strain, using the same substrate, containing a higher total amount of lipids.

According to a particularly preferred embodiment of the present invention, a Ulkenia sp. Dry mass used as starting material. According to another particularly preferred embodiment of the present invention, an oil from Ulkenia sp. used as starting material.

Oils from Ulkenia sp. are conveniently obtained by cultivating the microorganism rich in DHA, harvesting the biomass from the culture, digesting and isolating the oil. A very particularly favorable method in this context is described in WO 03/033631 A1, the disclosure of which is hereby incorporated by reference.

To isolate the oil, extraction processes with organic solvents, in particular hexane, or with supercritical fluids are preferably used. Conveniently, the oil is extracted from the biomass by percolation of the dried biomass with hexane. such Extractions with organic solvents are described inter alia in WO 9737032, in WO 9743362 and EP 515460. A particularly detailed presentation can also be found in the Journal of Dispersion Science and Technology, 10, 561-579, 1989 "Biotechnological. Processes for the Production of PUFAs".

Alternatively, the extraction can also be carried out without solvent. A particularly favorable process in this context is described in EP-A-1178118. In this process, a solvent is avoided by preparing an aqueous suspension of the biomass and separating the oil phase from the aqueous phase by centrifugation.

The composition of Ulkenia sp. Biomass available can vary widely. Preferably, it contains at least one glyceride, in particular a triglyceride, which comprises at least one polyunsaturated fatty acid radical. According to a particularly preferred embodiment, at least 10%, more preferably at least 25% and especially at least 30% of the fatty acid residues in the biomass are DHA residues.

A "glyceride", as used herein, is an ester of glycerin and at least one fatty acid wherein one to three hydroxyl groups of the glycerol have been esterified with one or more fatty acid residues Radicals may be the same or different.

For many suitable starting materials, the majority of the glycerides are

Triglycerides, ie esters of three fatty acid residues and glycerine. Each fatty acid residue may be either saturated (ie all bonds between the carbon atoms are single bonds) or unsaturated (ie there is at least one carbon-carbon double or triple bond). The nature of the unsaturated fatty acid residues are sometimes characterized herein with a ω. This number indicates the position of the first double bond, when counting from the terminal methyl group of the fatty acid or fatty acid residue.

According to the invention from Ulkenia sp. available biomass initially transesterified with at least one alcohol. The goal of the transesterification step is to cleave the fatty acid residues from the glycerol backbone of the glycerides in the starting material and form separate esters from each of the residues (at least one docosahexaenoic acid alkyl ester and at least one saturated fatty acid ester) such that the esters are separated can be.

According to the invention, the transesterification is preferably carried out using at least one alcohol of the formula R ¹ -OH, where R ^{1 is} a linear or branched alkyl radical having 1 to 20, preferably 1 to 6, in particular 1 to 4, carbon atoms. Particularly preferred are the methyl esters and ethyl esters, especially the ethyl esters.

According to a first preferred embodiment of the invention, the transesterification is catalyzed by at least one base. Preferred bases include sodium methoxide, potassium methoxide, elemental sodium, sodium hydroxide and potassium hydroxide. Preferably, the volume ratio of the biomass to the base / alcohol mixture is 1: 1 to 1: 5. The concentration of the base in the alcohol is preferably 0.1 to 2 M. According to a preferred variant, the transesterification reaction is carried out at room temperature (ie at a temperature in the range of about 20-25 ^° C.) for 6-20 hours. According to a further preferred variant, the transesterification reaction at a temperature above room temperature, preferably at a temperature of at least 40 ⁰ C, more preferably at a temperature of 70 to 150 ⁰ C, in particular at a temperature above the boiling point of one or more components in the Mixture (under reflux), carried out.

According to a second preferred embodiment of the invention, the transesterification is catalyzed by at least one acid, preferably by the biomass at a temperature of about 0 to about 15O ⁰ C in a mixture which incubates the at least one alcohol and at least one acid, preferably HCl, preferably under an inert gas atmosphere and in the absence of water. According to a preferred variant, the triglyceride / acid / alcohol mixture is refluxed for at least 2 hours. According to a further preferred variant, the triglyceride / acid / alcohol mixture is maintained at a temperature of 0 to 5O ⁰ C for at least 12 hours.

Since the acid-catalyzed transesterification is usually reversible, the

Preferably, alcohol is presented in a large excess so that the reaction proceeds substantially until complete conversion. Preferably, the triglyceride concentration in the alcohol / acid mixture is 0.1 to 15% by weight. The concentration of the acid, preferably HCl, in the alcohol / acid mixture is preferably 4 to 15% by weight. Such a mixture can be prepared by many different methods known in the art, such as, for example, by introducing gaseous hydrogen chloride into dry alcohol or by adding acetyl chloride to alcohol. Although HCl is most preferred in the present invention, other acids may alternatively be used. Such an acid is H ₂ SO ₄ , which is preferably used at a concentration of 0.5 to 5% by weight in the alcohol. It should be kept in mind, however, that H ₂ SO _{4 is} a strong oxidizing agent, and therefore preferably only in combination with short reflux times (ie, less than 6 hours), low concentrations (ie, less than 5 wt%), and low temperatures (ie, less 15O ⁰ C) is used. Another one

An example of a suitable acid is boron fluoride, which is preferably used at a concentration of 1-20% by weight. However, HCl is preferred over boron fluoride because boron fluoride is more prone to form undesired by-products.

The transesterification reaction is preferably carried out under an inert gas atmosphere (eg noble gas and / or N ₂ ). Furthermore, an antioxidant (eg Ascorbyl palmitate or propyl galate) may be added to the reaction mixture to prevent autooxidation.

During the transesterification, at least one organic solvent is preferably added. In particular, preferred solvents include those compounds which are capable of dissolving the fatty acid esters to be transesterified. When the starting material contains a plurality of fatty acid esters to be transesterified, the organic solvent is preferably capable of dissolving all the fatty acid esters to be transesterified. Very particularly suitable solvents according to the invention include dichloromethane, acetonitrile, ethyl acetate and diethyl ether, in particular dichloromethane.

After transesterification, the esters are preferably separated from the reaction mixture by addition of water. Often, the esters (which are organic) float on top of the reaction mixture and can easily be separated from the remaining reaction mixture. This is especially true for large-scale, industrial applications.

Alternatively, a liquid-liquid solvent extraction can be used to separate the esters from the remaining reaction mixture. This extraction can vary within a wide range. According to a preferred variant, water is added to the mixture and the esters are extracted with a non-polar solvent. If the transesterification has been catalyzed by at least one base, the water preferably comprises a sufficient amount of acid, preferably HCl, citric acid or acetic acid, especially HCl, to neutralize the mixture or more preferably to give the mixture a weakly acidic pH. The ratio of the total volume of the non-polar solvent to the volume of the reaction mass (including the added water) can also be varied within a wide range, and is more preferably from 1: 3 to 4: 3. According to a particularly preferred embodiment, the mixture is extracted with several fractions of the non-polar organic solvent, which are finally combined. Particularly suitable non-polar solvents according to the present invention include petroleum ether, pentane, hexane, cyclohexane and heptane, with hexane and petroleum ether being most preferred.

The non-polar solvent may also contain a small amount of a weakly polar organic solvent, such as diethyl ether. The use of such a polar component tends to improve the extraction of fatty acid esters from the aqueous layer because such esters are also weakly polar. If a weakly polar organic component is used, the volumetric concentration of the weakly polar component to the nonpolar component is preferably not greater than about 20%, more preferably not greater than 10%, and most preferably from 5% to 10%.

The resulting organic extraction solvent layer may be washed to remove, for example, any acid residues and / or remaining water. The removal of acid residues is preferably accomplished by washing the layer with an aqueous solution containing a weak base, e.g. For example, potassium carbonate. Remaining water removal can be achieved, for example, by washing the layer with a brine (i.e., a saturated brine) and / or by drying with an anhydrous salt (e.g., sodium sulfate or magnesium sulfate).

After extraction, the fatty acid esters can be concentrated in the non-polar solvent layer. According to a preferred

Embodiment of this invention, the esters are concentrated by evaporating a portion of the non-polar solvent. , ^'

The transesterification of a from Ulkenia sp. Biomass available in addition to the DHA alkyl ester usually provides other fatty acid esters. Many of these fatty acid esters, especially the saturated fatty acid esters, have unknown and / or adverse medical properties and nutritional properties. It is therefore necessary, as completely as possible to remove the saturated fatty acid esters from the transesterification reaction mixture. The process according to the invention therefore comprises a urea crystallization in which b) first produces a solution containing urea, at least part of the transesterified biomass from step a) and at least one organic solvent, c) cooling the solution from step b) or concentrated to i) a precipitate containing urea and at least a portion of the saturated fatty acid esters, and ii) forming a liquid fraction, d) separating the precipitate i) from the liquid fraction ii).

When urea crystallizes in a solution containing polyunsaturated fatty acid esters (eg, esters of DHA) and saturated fatty acid esters obtained by transesterification using the method described above, a precipitate forms which comprises the urea and at least a portion thereof containing the saturated fatty acid ester. However, this precipitate comprises a much lower proportion of polyunsaturated fatty acid esters than the starting solution. The majority of polyunsaturated fatty acid esters therefore remain in solution and can be easily separated from the precipitated saturated fatty acid esters.

The urea crystallization separation process of the present invention comprises first forming a solution containing the fatty acid esters and urea. The amount of urea is preferably proportional to the

Total amount of saturated fatty acids to be separated from the solution. The mass ratio of the mixture of fatty acid esters to the

Urea is preferably 1: 1 to 1: 4. The solution also preferably comprises at least one organic solvent which dissolves urea and the desired DHA ester, more preferably urea and all fatty acid esters in the mixture. In this context, particularly suitable solvents include alcohols having 1 to 4 carbon atoms, with methanol and ethanol, especially ethanol, being particularly preferred. The volumetric ratio of the mixture of the fatty acid esters to the solvent is preferably 1: 5 to 1:20.

Preferably, substantially all of the urea is dissolved in the solution. This can generally be achieved by heating the solution, preferably to a temperature of greater than 50.degree. According to a very particularly preferred embodiment of the invention the solution is prepared by dissolving the urea and the fatty acid ester mixture from each other, preferably, dissolved separately under heating, particularly at temperatures greater than 5O ⁰ C in the solvent and the resulting solutions then mixed together ,

To separate the saturated fatty acid esters, the solution containing the fatty acid esters and the urea is preferably cooled to form a precipitate comprising urea. Preferably, the solution is cooled to a temperature of less than 40 ° C, preferably less than or equal to 30 ⁰ C, in particular less than or equal to 25 ° C, the temperature conveniently greater than 10 ⁰ C, preferably greater than or equal to 15 ⁰ C, suitably greater or equal to 20 ° C, is. The cooled solution is preferably allowed to stand for a certain period of time, typically not longer than about 20 hours, preferably for 5 to 20 hours, at the cooled temperature with occasional stirring.

According to another preferred embodiment of this invention, a urea-containing precipitate is formed by concentrating the solution containing the fatty acid esters and the urea. For example, the solution can be concentrated by adding a portion of the solvent evaporated in the solution. The amount of solvent removed is preferably sufficient to effect a urea concentration in the solution that exceeds the saturation concentration.

The urea crystallization is conveniently carried out under an inert gas atmosphere (eg noble gases and / or N ₂ )

After the urea-containing precipitate has formed, the precipitate is preferably separated from the liquid fraction enriched with polyunsaturated esters. This is preferably achieved by filtration or centrifugation. According to a particularly preferred embodiment, the precipitate is then (preferably saturated with urea) with a small amount of the organic solvent washed ^'by adhering to the precipitate, polyunsaturated recover fatty acid esters. This washing solution is in turn preferably combined with the liquid fraction.

The liquid fraction is preferably concentrated, combined with water, and the esters contained in the liquid fraction are preferably extracted with a non-polar solvent. For example, the liquid fraction may be concentrated by vaporizing a portion of the solvent from the liquid fraction, wherein the vaporized amount of the solvent is preferably not so large that further urea precipitates. The amount of water added to the concentrated liquid fraction may vary within a wide range. Preferably that is

Volume ratio of water to the concentrated liquid fraction 4: 1 to 1: 1.

In a most preferred embodiment, a sufficient amount of acid, preferably HCl, is also added to neutralize the urea. Particularly suitable nonpolar solvents for the purposes of the present invention include petroleum ether, pentane, hexane, cyclohexane, Ethyl acetate and heptane, with hexane being most preferred. The volumetric ratio of the nonpolar solvent to the concentrated liquid fraction / water mixture is preferably 1: 5 to 5: 1.

According to a particularly preferred embodiment of the present invention, the liquid fraction is also extracted with a weakly polar organic solvent to maximize the recovery of the fatty acid esters (which, as noted previously, are weakly polar). Low polar solvents particularly useful in the present invention include diethyl ether and ethyl acetate, with diethyl ether being most preferred. Preferably, the volumetric ratio of the weakly polar solvent to the concentrated liquid fraction / water mixture is 1: 5 to 5: 1. After extraction with the weakly polar solvent, the extracts are preferably combined.

After extraction, the extracts may be dried by, for example, washing with a brine and / or using an anhydrous salt (e.g., sodium sulfate). The solution is then preferably concentrated, for example by partial or complete evaporation of the solvent.

The inventive method is characterized in particular by an extremely efficient separation of the saturated fatty acid esters. Therefore, in the context of the present invention, the transesterified biomass is preferably directly, i. without further intermediate steps, subjected to urea crystallization.

Although not usually necessary, it is also possible to increase the proportion of polyunsaturated fatty acids in the transesterified biomass by partial separation of the other ingredients prior to urea crystallization, in order to increase the efficiency of the inventive method even further. This can be done in a manner known per se, with the use of extraction methods, in particular the extraction with W

nonpolar solvents (as described above), as well as winterization, have proven particularly useful.

Winterization involves cooling a solution containing the transesterified biomass to a temperature which causes at least a portion of the saturated fatty acid ester to precipitate, while a much lower proportion of the polyunsaturated fatty acid ester precipitates. Preferably the solution is at a temperature less, more preferably to a temperature in the range -30 to -10 ⁰ C, in particular to a temperature in the range of -25 to -15 ° C, cooled 0 ° C. The solution is preferably maintained at these temperatures for up to 20 hours and under an inert gas atmosphere.

The winterization is preferably carried out in an organic solvent which dissolves the DHA ester and at least one saturated fatty acid ester in the fatty acid ester mixture. Particularly suitable solvents include methanol and ethanol, with methanol being most preferred. Preferably, the volumetric ratio of the fatty acid ester mixture to the organic solvent is 1: 5 to 1:20.

After formation of the precipitate, the solution is preferably separated from the precipitate to form a liquid fraction enriched in the desired polyunsaturated fatty acid esters. This is preferably achieved by filtration or centrifugation. After the liquid fraction is separated, it is conveniently concentrated by evaporation of the solvent in a rotary evaporator.

Possible fields of application of the fatty acid compositions obtainable according to the invention are immediately obvious to the person skilled in the art. They are particularly suitable for all applications that are pre-defined for PUFAs and PUFA esters. In this case, the fatty acid compositions according to the invention can usually be used directly. For some applications, however, it is necessary to previously add the fatty acid ester (s) in the liquid phase saponify. This can be achieved, for example, by reaction with KOH in ethanol.

The fatty acid compositions obtainable according to the invention are used in particular as an active ingredient or component in pharmaceutical fatty acid compositions, as a constituent in cosmetic preparations, as a food additive or food ingredient, as a component of functional foods and for the production of higher-concentration PUFA derivatives, such as esters and acids.

The invention will be explained in more detail by examples, without this being intended to limit the inventive concept.

example 1

1. transesterification

To a mixture of 560.5 g Ulkenia sp. Crude oil and 292 g of absolute ethanol, while stirring, a solution of 13.12 g of Na-ethylate in 228 g absolute

Ethanol added dropwise. The resulting mixture is stirred for 2.5 hours. Thereafter, 4466 g of water are added and the batch is allowed to stand for one hour. After 1 hour, an additional 171 g of water are added. The batch is allowed to stand for an additional 12 hours, whereupon 2 phases are formed. The oil phase is separated and the ethanol and water residues are removed on a rotary evaporator. 405.7 g of interesterified ethyl ester oil are obtained.

2. urea precipitation

680 g of urea are dissolved in 4430 ml of ethanol at 77 ⁰ C (6 liter four-necked round bottom flask with stirrer, thermometer and condenser). Parallel 404 g interesterified Ethylesteröl in 443 ml of ethanol are vormkubiert at 7O ⁰ C and added to the urea solution. The batch is allowed to stand for 12 hours. The precipitate formed is separated and the remaining liquid phase is concentrated on a rotary evaporator to 1.5 liters. Thereafter, 1.5 liters of 2 molar hydrochloric acid and 2.5 liters of water are added to the liquid phase. The organic phase is separated off and dried at 45 ^° C. on the vacuum pump. There are obtained 230 g of purified oil. The fatty acid profile of the oil is shown in Table 1 and the characteristics of the quality of the oil (acid value, peroxide value, heavy metal content) are given in Table 2.

Example 2 1. Transesterification

1 kg Ulkenia sp. Dry biomass are stirred with 2.5 liters of 10% ethanolic sulfuric acid at 75 ⁰ C under nitrogen for 48 hours. The batch is cooled to 50 ^° C. and extracted with 3.5 liters of hexane. The hexane phase is separated off and the solvent (hexane) is removed on a rotary evaporator. 390.1 g of interesterified ethyl ester oil are obtained.

2. urea precipitation

599 g of urea are dissolved in 3.9 liters of ethanol at 77 ° C (6 liter four-necked round bottom flask with stirrer, thermometer and condenser). Parallel 390 g interesterified Ethylesteröl in 390 ml of ethanol are preincubated at 7O ⁰ C and added to the urea solution. The batch is allowed to stand for 12 hours. The precipitate formed is separated and the remaining liquid phase is concentrated on a rotary evaporator to 1.5 liters. Thereafter, 1.5 liters of 2 molar hydrochloric acid and 2.5 liters of water are added to the liquid phase. The organic phase is separated off and dried at 45 ^° C. on the vacuum pump. There are obtained 216.2 g of purified oil. The fatty acid profile of the oil is shown in Table 1 and the characteristics of the quality of the oil (acid value, peroxide value, heavy metal content) are given in Table 2. Table 1: Fatty acid profile of the purified oil (in% by weight)

Table 2: Quality of the purified oil

: measured according to AOCS Official Method Yes 8-87

² : measured according to AOCS Official Method Cd-3d 63 (American OiI Chemists

Society)

³ : measured according to LMBG §35 L06.00-7 (Lebensmittelbedarfsständegesetz)

Claims

W 2Patent claims:

1. A process for the preparation of a fatty acid composition containing, based on the total weight of the fatty acids and / or fatty acid derivatives contained in the fatty acid composition, at least 70.0% by weight of docosahexaenoic acid and / or docosahexaenoic acid alkyl ester in which: a) one from Ulkenia sp. Transesterification of available biomass with at least one alcohol to form at least one Docosahexaensäurealkylester and at least one saturated fatty acid ester, b) preparing a solution containing urea, at least part of the transesterified biomass from step a) and at least one organic solvent, c) the solution from step b), cooled or concentrated to iii) a precipitate containing urea and at least a portion of the saturated fatty acid esters, and iv) to form a liquid fraction, d) the precipitate i) from the liquid fraction ii ) separates.

2. The method according to claim 1, characterized in that as biomass. an oil from Ulkenia sp. starts.

3. The method according to claim 1, characterized in that as biomass Ulkenia sp. Bio-solids.

4. The method according to at least one of the preceding claims, characterized in that one uses for the transesterification of an alcohol of the formula R ¹ -OH, wherein R ^{1 is} a linear or branched alkyl radical having 1 to 20

Carbon atoms.

5. The method according to at least one of the preceding claims, characterized in that durchfuhrt the transesterification in the presence of at least one base.

6. The method according to at least one of claims 1 to 4, characterized in that the transesterification in the presence of at least one

Acid carried out.

7. The method according to at least one of the preceding claims, characterized in that the organic solvent from step b) at least. an alkyl alcohol having 1 to 4 carbon atoms.

8. The method according to at least one of the preceding claims, characterized in that in step c) the solution is cooled to a temperature not greater than equal to 15 ⁰ C.

9. The method according to claim 8, characterized in that the solution is cooled to a temperature in the range of 15 ⁰ C to 25 ° C.

10. The method according to at least one of the preceding claims, characterized in that in step b) the transesterified biomass from step a) used directly.

11. The method according to at least one of the preceding claims, characterized in that one uses in step b) an oil which is obtained from the transesterified biomass from step a) by the proportion of polyunsaturated fatty acids in the interesterified biomass by partial separation of the other ingredients.

12. The method according to claim 11, characterized in that one increases the proportion of polyunsaturated fatty acids in the transesterified biomass by extraction process.

13. The method according to claim 11 or 12, characterized in that one increases the proportion of polyunsaturated fatty acids in the transesterified biomass by winterization process.

14. The method according to at least one of the preceding claims, characterized in that one saponifies the fatty acid ester or esters in the liquid phase.

15. fatty acid composition obtainable according to at least one of the preceding claims.

16. Fatty acid composition according to claim 15, characterized in that it has an acid number, measured according to AOCS Offϊcial Method Ja 8-87, of less than or equal to 1.5 mg KOH per g of Fettsäure.-composition.

17. Fatty acid composition according to claim 15 or 16, characterized in that it has a peroxide value, measured according to AOCS Official Method Cd-3d 63, of less than or equal to 0.5 meq. per kg of fatty acid composition.

18. fatty acid composition according to claim 15, 16 or 17, characterized in that it has a heavy metal content, measured in accordance with LMBG §35 L06.00-7, of less than or equal to 0.7 mg per kg of fatty acid composition.

19. Use of a fatty acid composition according to any one of claims 15 to 18 as an active ingredient or ingredient in pharmaceutical compositions.

20. Use of a fatty acid composition according to any one of claims 15 to 18 as a component in cosmetic preparations.

21. Use of a fatty acid composition according to any one of claims 15 to 18 as a food additive and / or as a food ingredient.

22. Use of a fatty acid composition according to any one of claims 15 to 18 as a component of animal feed.