CA1303416C - Refined fish oil concentrate and the production process for same - Google Patents
Refined fish oil concentrate and the production process for sameInfo
- Publication number
- CA1303416C CA1303416C CA000523914A CA523914A CA1303416C CA 1303416 C CA1303416 C CA 1303416C CA 000523914 A CA000523914 A CA 000523914A CA 523914 A CA523914 A CA 523914A CA 1303416 C CA1303416 C CA 1303416C
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- Prior art keywords
- fatty acid
- concentrate
- process according
- urea
- compounds
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B3/00—Refining fats or fatty oils
- C11B3/001—Refining fats or fatty oils by a combination of two or more of the means hereafter
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- Life Sciences & Earth Sciences (AREA)
- Microbiology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Fats And Perfumes (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Abstract Refining of fish waste product so that a concentrate of .omega.3-fatty acid alkyl ester is formed with 20-30% eicosa-pentaenoic acid alkyl ester and 35-50% docosahexaenoic acid alkyl ester (both by weight) free from cholesterol. The .omega.3-concentrate is produced through urea precipitation of the non- .omega.3-fatty acid esters so that the filtrate from the pre-cipitation may be extracted by means of hexane for the trans-mission of the .omega.3-fatty acid esters and the cholesterol to the hexane extract, Hexane is thereafter removed. The remaining concentrate of the fatty acid esters with the chol-esterol is cooled to a temperature of not lower than -50°C, whereby the cholesterol is crystallized. The remainder is a .omega.3-concentrate with the composition mentioned above.
Description
~IL303~
i , The present invention concerns a refined fish oil concentrate as well as the production process for same. In the refining process, cholesterol and useful biproducts such as urea adducts of fatty acid compounds are produced, in addition to higher unsaturated fatty acids.
It is known that waste products from the fish refining indu-stry contain usable products, among others fatty acids, chol-esterol, proteins and enzymes. These are either fat-soluble or water-soluble. Such waste products are normally referred to as fish entrails.
Through the processing of fish entrails, the water-soluble portion containing proteins and en~ymes may be separated rom the fat-soluble portion. The present invention conce~ns only the fat-soluble portion of the waste product~, but it can also be used for other refined fish oils such as occur for instance in the fish product industry~ In the following this wlll be called fish~oil~product.
It is known that certain essential fatty acids in ~ish oil have a medicinal effect and are used in the prevention and cure of thrombotic illnesses, for instance ischemic heart disease. In addition, these compounds lower the cholestsrol level in the blood.
Among the above-mentioned fatty acids, the following may be specified as suitable for the medicinal purpose referred to:
elcosapentaenoic acid (EPA) and docosahexaenoic acid ~DHA~.
~3 C334:~
Both fatty acids are ~ 3-fatty acids of the C-20 and C-~2 acids. Their nomenclature according to the IUPAC system is:
for the eicosapentaensoic acid (EDA) cis - 5, 8, 11, 14, 17 - eicosapentaenoic acid and for the docosahexaenoic acid (D~A) cis - 4, 7, 10, 13, 16, 19 - docosahexaenoic acid which is abbreviated to:
eicosapentaenoic acid 20:5 ~3 and docosahexaenoic acid 22:6 ~3 where 20 and 22 indicate the number of C-atoms in the mole-cule of fatty acid, 5 and 6 the number of unsaturated bondings, and ~ 3 that the last unsaturated bonding i9 posi-tioned in a distance of 3 carbon atoms from the ~-position.
In the following we will be using the description EPA and 20:5 ~3 for the eicosapentaenoic acid and DHA and 22:6 ~3 for the docosahexaenoic acid.
The fat soluble portion of cod entrails usually contains 10 ~ 25~ of the essential fatty acid compounds EPA and DE~, as well as 2 - 4% cholesterol. The remainder is mainly fatty acid compounds with lower unsaturation, such as pure fatty acids or their glycerides.
The purpose of the invention is to separate the essential fatty acid compounds from cholesterol and the remaining fatty acid compounds.
Another purpose of the invention is to produce from a non-uniform raw material containing marine fatty acids and/or esters of these fatty acids, as well as cholesterol as main .
136)34~L6 components after trans-esterification with a lower alcohol, and under alkaline conditions produce a concentrate con-taining the essential ~ 3-fatty acid compounds, EPA and DHA, as well as isolate the cholesterol and the urea adduct of the saturated and lower unsaturated fatty acid compounds, which then appear in their own product fractions.
It has long been known that the easiest way to separate fatty acids is by means of extraction or distillation when they appear in the form of esters, for instance as methyl or ethyl esters. Before treatment, fatty waste product~ from the fish industry must have been subjected to trans-esterification and es~erification, for instance with methanol for production of fatty acid methyl esters.
This basic material is well suited for further separation of essential fatty acid compounds and cholesterol from the remaining less important fatty acid compounds.
In US Patent Publication No. 4.145.446 a method is described for precipitation of fatty acid compounds in raw material by means of urea. The purpose is to obtain a product containing proteins and fats, suitable for fodder. The production of the urea adduct is brought about by having a solution of melted urea at 60 - 140 C to which is added melted fatty acid or a mixture of fats heated to a temperature of 35-105 so that the ratio fat/fatty acids to urea will be from 40/60 to 60/40 units by weight.
Another patent, SU 950.393 describes a method for production of cholesterol from for instance fish waste products by hydrolysing the fatty acid compounds and converting them to soaps. These are then subjected to an extraction of trichlor-ethylene at room temperature, whereby the cholesterol is com-bined with the trichlorethylene and this compound is then subject to further separation~
13~)3~16 ~;~
GB 1.240.513 also concerns a separation technique by means of urea where the raw material consists of pure methyl and ethyl esters of the C16-C18fatty acids. Urea precipitation occurs in a neutral environment with a surplus of the rele-vant alcohol. The purpose is to be able to obtain a stronger concentration of the r -linolenic acid. The above-mentioned fatty acid esters do not contain any higher fatty acids other than C-18 in the form of stearic acid, oleic acid, linoleic acid and linolenic acid, which after the urea precipitation and separation of the urea adduct from the rest of the material has obtained a higher content of Y linolenic acid by means chromotography.
The higher unsaturated fatty acid compounds 20:5 ~3 and 22:6 ~3 may be concentrated according to a method described in J 59-071396 where the fatty acid compounds men-tioned are extracted by means of polar solvents, suc~ as ace-tone, methyl ethyl ketone, methanol, ethanol, and similar solvents, whereby a soluble and an insoluble extract are formed. Thereafter the extract is further processed to obtain essential fatty acid compounds.
According to the invention it now appears possible - in a remarkably simple manner - to optimize the procedure to increase the concentration of ~3-fatty acid compounds and cholesterol by means of a simplified process. This is based on a fractionated precipitation of the less interesting fatty acid compounds with urea, since urea tends to form an adduct with fatty acids which do not belong to the ~3 type. Nor doe~ cholesterol form an adduct with urea. The procedure used previously was to isolate the fatty acid compounds before these were esterified separately, and then they were sub-jected to a fractionated precipitation with urea. This proce-dure is unnecessary with our invention.
~L3a33~ 6 4a ~6625-53 Accordiny to the present invention there is provided a process for the production of a refined fish oil concentrate containing at least 20% eicosapentaenoic acid ~EPA) and at least 35~ docosehexaensoic acid (DHA) by weigh~, the remainder o~ the concentrate includlng other unsaturated fatty acid compounds, and the fatty acid compounds of the concentrate being mainly present as alkyl esters of lower alcohols, which process comprlses the steps of: (a) esterifying and/or trans-esterifying the fat/fatty acid fraction of fish oil product at room temperature with a lower alcohol in an alkaline environment containing amounts of a base sufficient only to catalyze the esterification and~or trans-esterification reaction; (b) heating the resu~ting alkyl ester product with an excess of urea in an alkanol to fro~ 55-~0C; (c) cooling the product of step (b) to 0C to precipitate urea fatty acid alkyl ester adduct and thereaf~er separating off ~aid adduct to leave a solution mainly containing 3-fatty acid esters and an un~aponifiable portion; (d) separating from the solution remaining from step (c) the 3-fatty acid alkyl esters and the unsaponifiable portion, (e) removing any solvent from the mixture obtained in step (d); and (f) cooling the concentrate obtained in step ~e) to crystallize out cholesterol and to precipitate out other undefined unsaponifiable compounds, thereby to leave a refined fish oil concentrate.
~L3q:~3~6 A special feature of this process i~ that fatty acid com-pounds are not produced prior to precipitation of urea, but precipitate from the same non-uniorm mixture of components like they are found in the raw material.
Another special feature i9 that the precipitation of ~rea takes place in an alkaline environment, and in such a way that the alkaline environment is created through applying the base only in catalytic quantities such as a catalytic agent for the trans-esterification of glycerides to methyl e~ters and not as a means of saponification of the fatty acids.
A third special feature is that the trans-esterification takes place at room temperature.
A result of trans-esterification at low temperature and in an environment with low alkalinity is that isomerization of the double compounds is avoided, which results in a more uniform product with no toxic effect. At the same time transcon-figurations are avoided. The remaining solution is thereafter extracted by means of non-polar solvents, ~mong others hex-ane, whereby the ~3-atty acids as well as cholesterol will be found in the hexane phase.
Thereafter the non-polar phase is subjected to evaporation of the hexane under moderate conditions, for instance by means of vacuum distillation. The remaining ~3-concentrate now contains all the cholesterol, and it becomes apparent that the cholesterol does not dissolve easily in this concentrate and may crystallize in cooling. The ~3-concentrate which is left will - according to the simplified process invented -contain 20 - 30 per cent EPA by weight and 35-50% DHA by weight~ The rest consists mainly of non-essential fatty acid compounds and are not important for our purposer ~3~39L~
For a better understanding of the invention, we refer to the block diagram in Fig. 1, where each block represents a step in the process and is marked with a reference number. The flow of the material to and from each block and between the blocks is marked by solid and dotted lines. In addition, each material is characterized by a letter~
The basic material is fat and/or fatty acids from fish and especially fat and/or fatty acids obtained from the fish processing industry in connection with ensilage and or auto-catalysis process~s, but the process may also be used for other forms of marine fat. This fatty raw material is called a fish oil product in the claims.
Such fat/fatty acids have a high content of saturated, unsaturated and polyunsaturated fatty acids with a chain length C 18, C 20 and C 22 as well as a certain amount of cholesterol, vitamins and other fat soluble products which are undefinable, usually characterized as unsaponifiable, as well as fatty acid compounds with shorter chain lengths.
Box 1.
Fat/atty acids (A) from fish with a content of i.a. chol-esterol, also called the fish oil product, was placed in a container for trans-esterification with an alcohol with a low boiling point (B), for in tance methanol or ethanol, prefer-ably methanol, and a catalytic agent as well as auxiliary compounds (C) to obtain a faster esterification and trans-esterification in order to prevent oxydization and dis-coloration. Potassium hydroxide may be used as a catalytic agent and in order to prevent oxidization, especially when heavy metals are pr~sent, such as chromium, iron, cobalt, nickel and copper, small amounts of the sodium salt of ethyl--enediaminetetra-acetic acid (EDTA-Na3) may be added. The esterification and trans-esterification taXe place under moderate conditions and stirring at about 20 C or some ~3~3~
hours. The formation of alkyl esters is nearly complete when the ester product has changed its appearance from opalescent -to clear. The slear solution (Dl) therefore contains alkyl esters of the fatty acids, glycerol, alkanol, as well as some water from the esterification of the free fatty acids.
Box 2.
The clear solution is then heated to a temperature of 65-68C, whereafter a fixed amount of urea (E) and alkanol (B) is added and stirred in until everything i completely dissolved. The amount of urea depends on the composition o~
the fatty acids so that if the raw material (A) contains 6-8%
EPA by weight, urea is added in the ratio 3 parts urea by weight to 1 part alkyl ester. In order to ensure that the components are completely soluble, 9 parts alkanol by weïght is added.
When everything is dissolved, the solution is slowly cool~d to approx. 20 C. An adduct of urea fatty ester (F) is crystalllized and then removed by means of for in~tance decanting and filtration, whereafter ~.he filter mass is cooled to 0 C in order to crystallize a larger portion of the adduct (F). The adduct is then separated by a known method so that the remaining filter mass (G) contains the essential fatty acid fractions and the unsaponifiable frac-tions.
Box 3.
The slightly alkaline filter mass (G) is saturated with hex-ane and is extracted by means of this solvent through a known technique as for instance by a continuing fluid-to-fluid counter current process, whereafter a further quantity of adduct of urea fatty ester may be crystallized. By means of this extraction two fluids are formed, consisting of hexane ~I) and a residu~ ~K).
~L3~3~
Box 4.
The hexane extract (I), which contains the alkyl-fat-ty esters of the polyunsaturated fatty acids 18:4 ~3, 20:5 ~3~and 22:6 ~3 as well as cholesterol as the most important com-ponents, is washed in a diluted hydrochloxic acid (L) in order to neutraliæe possible potassium soaps of the essential polyunsaturated fatty acids in the hexane extract. The washing water is decanted.
Hexane (H) is thereafter removed by evaporation of the extract (I) so that a concentrate is produced which is free from solvents (N) and which contains the compounds that are essential for the invention, the fatty acid compounds EPA and DHA as well as cholesterol.
~he dehydrated extract normally contains 20-30 % EPA, 35-45 DHA, 10-20% other polyunsaturated fatty acids, as well as 5-15% cholesterol and undefined compounds, all by weight, but the composition referred to will depend on the type of fish used, the time of year the fish is caught, and the type and condition of the raw material.
Box 5.
The concentrate of alkyl fatty acid ester (~) is thereafter cooled to approx. minus 25 C, whereafter cholesterol (0) is crystallized. This is centrifuged/filtered.
Further impurities which are present in the concentrate (N) may be removed by cooling the mixture to a temperature of lower than minus 25 C, whereafter certain undefined com-pounds are precipitated and filtered by means of a known method. The remainin~ ~3-concentrate (P) thus contains 20-35% EPA, 35-50% DHA and 15-25~ of other polyunsaturated ~3-fatty acid compounds (all by weight) as well as unsatur-ated fatty acid compounds which are not essential for the invention.
~3~34~1~6 Product (P) which contains the alkyl esters of the corre-sponding ~3-fatty acids may be utilized as it is or the con-centration of EPA and DHA may be increased.
Since the product contain~ only small amounts of other fatty acids with the same chain length as EPA and DHA, it is well suited for separation of the essential fatty acids, EPA and DHA by means of supercritical fluid extraction.
Another method for increasing the concentration is by means of preparative liquid chromatography by which method a more than 90% purity of the essential fatty acids is obtained.
Box 6.
The alkaline residue (K) is acidified by means of con-centrated hydrochloric acid (L) to a ph =2, whereafter a hex~
ane fraction (R) is precipitated in an upper layer which is separated~ One may also subject the acid solution (S) to furt~er extraction by means of hexane if this should be necessary, whereafter the hexane extracts are gathered. The hexane fractions (R) contain free fat1y acids as well as some of their alkyl esters and fairly high percentages of EPA and DHA, but also a fairly substantial portion of C 18-, C
20- and C 22-fatty acids with lower unsaturation. This acid solution contains water, alcohol, alkanol, glycerol, urea and other products which may be retrieved by a separate process which is not described here.
Box 7.
The fatty acid components of the hexane fraction are increased by evaporating hexane (H) in a separate piece of equipment.
Box 8.
The remaining solution is esterified using lower alkanols, for instance methanol or ethanol by means of an appropriate ~L303~1L~
catalytic agent ~V) which for instance may be dehydrated hydrochloric acid, acetic acid chloride or boron-trichloride.
The resultant alkyl ester (D2) from the fatty acid components (T) from box 7 may be processed in various ways, for instance returned to box 2 for urea precipitation of the less unsatur-ated fatty acids.
Example To 50 kg fish oil product (A) from cod entrails (containing 8~ EPA, 11~ DHA, and 2.3 % cholesterol all by weight) 400 1 methanol (B) and 10 g EDTA Na3 (C) were added in a reactor~
Potassium hydroxide (C) dissolved in methanol was added for neutralisation of free fatty acids in a quantity corre-sponding to a colour reaction of pH 12 on a moist pH-paper.
Thereafter 50 1 methanol (B) were added.
The whole mixture was subjected to stirring for 15 hours at 20C in order to bring about a trans-esterification of the glycerides to methyl esters and esteriication of the free fatty acids to methyl esters.
When the tr~ns-esterification was complete, the temperature was increased to 65 - 68 C and 140 kg urea (E), as well as a fixed amount of methanol (B) were stirred in while being heated until every hing seemed to be dissolved.
Then the solution was slowly cooled to room temperature (approx. 20C), whereafter a urea adduct of fatty acid was precipitated (F). It contained the major portion o the saturated and lower unsaturated fatty acid methyl esters.
The urea adduct was separated from the solution by decanting and filtering according to an ordinary, known technique.
Result: 100.1 kg urea adduct (F).
~3~34~6 Therea~ter the solution was cooled to 0 - 4 C, whereby an additional 5.1 kg urea adduct (F) could be filtrated from the solution.
This filtrate (G) contained ~3-polyunsaturated fatty acid methyl esters, cholesterol and a residue of unwanted fatty acid fractions with lower unsaturated C 18, C 20 and C 22 fatty acid methyl esters. To this filtrate we added hexane for saturation, whereby a further amount of urea add~ct (22 kg) could be separated. This hexane-saturated solution was extracted in a counter-current with hexane so that the hexane extract (I) finally made up approx. 300 1. The remaining unextracted solution is called (K). The hexane extract was thereafter evaporated. The yield of ~3-~atty acid methyl ester concentrate : 10.2 kilos.
The concentrate (~) which contained 23% EPA , 41% DHA and 8%
cholesterol, all by weight, was thereafter cooled to minus 25C, whereby pure cholesterol (0) crystallized and was removed by means of centrifuging during which time the resi-due in the centrifuge was washed with hexane with a lower temperature in order to remove the fatty acid methyl esters from the cholesterol crystals. Yield: 760 g pure cholesterol.
The concentrated filtrate (P3 contained 25% EPA-methylester, 43% DHA-me~hylester by weight based on the fatty acid portion and traces of cholesterol.
The above-mentioned extracted residue (K) was cleansed with a solution of concentrated hydrochloric acid, whereby one hex ane phase could be filtered off. Additional hexane was added to the,batch, stirred and then precipitated. The hexane frac-tions were put together and the hexane evaporated. To 7.7 kilos of the remaining fatty acid and the methyl fatty acid fraction , 15 liter 2~ methanolic solution of boron trichloride was added.
~L3~34~;
Yield: 6 kilos methyl fatty acid esters, containing 13%
EP~-metylester, 17% DHA-metylester and approximately 2% chol-esterol, all by weight.
The methyl fatty acid concentrate, containing methanol, was returned to the process for treatment with urea.
With this invention it has been possible to produce a very pure ~3-concentrate of fatty acid alkyle esters, where the essential anti-thrombotic fatty acid components eicosa-pentaenoic acid (EPA) and docosahexaenoic acid (DHA) is present in a strong concentration Further, by means of the procedure invented, it has been possible in a simple manner to separate very pure and crystalline cholesterol. An additional product is a urea adduct of fatty acid components, but this is of no interest to the invention.
Another advantage with the invention is that it is possible to produce a urea adduct based on the same procedure where a trans-esterification has been done from glycerides to alkanol esters without following the cumbersome procedure by first producing the fatty acids, esterify these with alkanol and then separate them by means of the fractionated urea preci-pitation.
By following the procedure invented, it is also possible to avoid the formation of emulsions in the phases, and the phase separation is thereby facilitated during later extraction stages.
i , The present invention concerns a refined fish oil concentrate as well as the production process for same. In the refining process, cholesterol and useful biproducts such as urea adducts of fatty acid compounds are produced, in addition to higher unsaturated fatty acids.
It is known that waste products from the fish refining indu-stry contain usable products, among others fatty acids, chol-esterol, proteins and enzymes. These are either fat-soluble or water-soluble. Such waste products are normally referred to as fish entrails.
Through the processing of fish entrails, the water-soluble portion containing proteins and en~ymes may be separated rom the fat-soluble portion. The present invention conce~ns only the fat-soluble portion of the waste product~, but it can also be used for other refined fish oils such as occur for instance in the fish product industry~ In the following this wlll be called fish~oil~product.
It is known that certain essential fatty acids in ~ish oil have a medicinal effect and are used in the prevention and cure of thrombotic illnesses, for instance ischemic heart disease. In addition, these compounds lower the cholestsrol level in the blood.
Among the above-mentioned fatty acids, the following may be specified as suitable for the medicinal purpose referred to:
elcosapentaenoic acid (EPA) and docosahexaenoic acid ~DHA~.
~3 C334:~
Both fatty acids are ~ 3-fatty acids of the C-20 and C-~2 acids. Their nomenclature according to the IUPAC system is:
for the eicosapentaensoic acid (EDA) cis - 5, 8, 11, 14, 17 - eicosapentaenoic acid and for the docosahexaenoic acid (D~A) cis - 4, 7, 10, 13, 16, 19 - docosahexaenoic acid which is abbreviated to:
eicosapentaenoic acid 20:5 ~3 and docosahexaenoic acid 22:6 ~3 where 20 and 22 indicate the number of C-atoms in the mole-cule of fatty acid, 5 and 6 the number of unsaturated bondings, and ~ 3 that the last unsaturated bonding i9 posi-tioned in a distance of 3 carbon atoms from the ~-position.
In the following we will be using the description EPA and 20:5 ~3 for the eicosapentaenoic acid and DHA and 22:6 ~3 for the docosahexaenoic acid.
The fat soluble portion of cod entrails usually contains 10 ~ 25~ of the essential fatty acid compounds EPA and DE~, as well as 2 - 4% cholesterol. The remainder is mainly fatty acid compounds with lower unsaturation, such as pure fatty acids or their glycerides.
The purpose of the invention is to separate the essential fatty acid compounds from cholesterol and the remaining fatty acid compounds.
Another purpose of the invention is to produce from a non-uniform raw material containing marine fatty acids and/or esters of these fatty acids, as well as cholesterol as main .
136)34~L6 components after trans-esterification with a lower alcohol, and under alkaline conditions produce a concentrate con-taining the essential ~ 3-fatty acid compounds, EPA and DHA, as well as isolate the cholesterol and the urea adduct of the saturated and lower unsaturated fatty acid compounds, which then appear in their own product fractions.
It has long been known that the easiest way to separate fatty acids is by means of extraction or distillation when they appear in the form of esters, for instance as methyl or ethyl esters. Before treatment, fatty waste product~ from the fish industry must have been subjected to trans-esterification and es~erification, for instance with methanol for production of fatty acid methyl esters.
This basic material is well suited for further separation of essential fatty acid compounds and cholesterol from the remaining less important fatty acid compounds.
In US Patent Publication No. 4.145.446 a method is described for precipitation of fatty acid compounds in raw material by means of urea. The purpose is to obtain a product containing proteins and fats, suitable for fodder. The production of the urea adduct is brought about by having a solution of melted urea at 60 - 140 C to which is added melted fatty acid or a mixture of fats heated to a temperature of 35-105 so that the ratio fat/fatty acids to urea will be from 40/60 to 60/40 units by weight.
Another patent, SU 950.393 describes a method for production of cholesterol from for instance fish waste products by hydrolysing the fatty acid compounds and converting them to soaps. These are then subjected to an extraction of trichlor-ethylene at room temperature, whereby the cholesterol is com-bined with the trichlorethylene and this compound is then subject to further separation~
13~)3~16 ~;~
GB 1.240.513 also concerns a separation technique by means of urea where the raw material consists of pure methyl and ethyl esters of the C16-C18fatty acids. Urea precipitation occurs in a neutral environment with a surplus of the rele-vant alcohol. The purpose is to be able to obtain a stronger concentration of the r -linolenic acid. The above-mentioned fatty acid esters do not contain any higher fatty acids other than C-18 in the form of stearic acid, oleic acid, linoleic acid and linolenic acid, which after the urea precipitation and separation of the urea adduct from the rest of the material has obtained a higher content of Y linolenic acid by means chromotography.
The higher unsaturated fatty acid compounds 20:5 ~3 and 22:6 ~3 may be concentrated according to a method described in J 59-071396 where the fatty acid compounds men-tioned are extracted by means of polar solvents, suc~ as ace-tone, methyl ethyl ketone, methanol, ethanol, and similar solvents, whereby a soluble and an insoluble extract are formed. Thereafter the extract is further processed to obtain essential fatty acid compounds.
According to the invention it now appears possible - in a remarkably simple manner - to optimize the procedure to increase the concentration of ~3-fatty acid compounds and cholesterol by means of a simplified process. This is based on a fractionated precipitation of the less interesting fatty acid compounds with urea, since urea tends to form an adduct with fatty acids which do not belong to the ~3 type. Nor doe~ cholesterol form an adduct with urea. The procedure used previously was to isolate the fatty acid compounds before these were esterified separately, and then they were sub-jected to a fractionated precipitation with urea. This proce-dure is unnecessary with our invention.
~L3a33~ 6 4a ~6625-53 Accordiny to the present invention there is provided a process for the production of a refined fish oil concentrate containing at least 20% eicosapentaenoic acid ~EPA) and at least 35~ docosehexaensoic acid (DHA) by weigh~, the remainder o~ the concentrate includlng other unsaturated fatty acid compounds, and the fatty acid compounds of the concentrate being mainly present as alkyl esters of lower alcohols, which process comprlses the steps of: (a) esterifying and/or trans-esterifying the fat/fatty acid fraction of fish oil product at room temperature with a lower alcohol in an alkaline environment containing amounts of a base sufficient only to catalyze the esterification and~or trans-esterification reaction; (b) heating the resu~ting alkyl ester product with an excess of urea in an alkanol to fro~ 55-~0C; (c) cooling the product of step (b) to 0C to precipitate urea fatty acid alkyl ester adduct and thereaf~er separating off ~aid adduct to leave a solution mainly containing 3-fatty acid esters and an un~aponifiable portion; (d) separating from the solution remaining from step (c) the 3-fatty acid alkyl esters and the unsaponifiable portion, (e) removing any solvent from the mixture obtained in step (d); and (f) cooling the concentrate obtained in step ~e) to crystallize out cholesterol and to precipitate out other undefined unsaponifiable compounds, thereby to leave a refined fish oil concentrate.
~L3q:~3~6 A special feature of this process i~ that fatty acid com-pounds are not produced prior to precipitation of urea, but precipitate from the same non-uniorm mixture of components like they are found in the raw material.
Another special feature i9 that the precipitation of ~rea takes place in an alkaline environment, and in such a way that the alkaline environment is created through applying the base only in catalytic quantities such as a catalytic agent for the trans-esterification of glycerides to methyl e~ters and not as a means of saponification of the fatty acids.
A third special feature is that the trans-esterification takes place at room temperature.
A result of trans-esterification at low temperature and in an environment with low alkalinity is that isomerization of the double compounds is avoided, which results in a more uniform product with no toxic effect. At the same time transcon-figurations are avoided. The remaining solution is thereafter extracted by means of non-polar solvents, ~mong others hex-ane, whereby the ~3-atty acids as well as cholesterol will be found in the hexane phase.
Thereafter the non-polar phase is subjected to evaporation of the hexane under moderate conditions, for instance by means of vacuum distillation. The remaining ~3-concentrate now contains all the cholesterol, and it becomes apparent that the cholesterol does not dissolve easily in this concentrate and may crystallize in cooling. The ~3-concentrate which is left will - according to the simplified process invented -contain 20 - 30 per cent EPA by weight and 35-50% DHA by weight~ The rest consists mainly of non-essential fatty acid compounds and are not important for our purposer ~3~39L~
For a better understanding of the invention, we refer to the block diagram in Fig. 1, where each block represents a step in the process and is marked with a reference number. The flow of the material to and from each block and between the blocks is marked by solid and dotted lines. In addition, each material is characterized by a letter~
The basic material is fat and/or fatty acids from fish and especially fat and/or fatty acids obtained from the fish processing industry in connection with ensilage and or auto-catalysis process~s, but the process may also be used for other forms of marine fat. This fatty raw material is called a fish oil product in the claims.
Such fat/fatty acids have a high content of saturated, unsaturated and polyunsaturated fatty acids with a chain length C 18, C 20 and C 22 as well as a certain amount of cholesterol, vitamins and other fat soluble products which are undefinable, usually characterized as unsaponifiable, as well as fatty acid compounds with shorter chain lengths.
Box 1.
Fat/atty acids (A) from fish with a content of i.a. chol-esterol, also called the fish oil product, was placed in a container for trans-esterification with an alcohol with a low boiling point (B), for in tance methanol or ethanol, prefer-ably methanol, and a catalytic agent as well as auxiliary compounds (C) to obtain a faster esterification and trans-esterification in order to prevent oxydization and dis-coloration. Potassium hydroxide may be used as a catalytic agent and in order to prevent oxidization, especially when heavy metals are pr~sent, such as chromium, iron, cobalt, nickel and copper, small amounts of the sodium salt of ethyl--enediaminetetra-acetic acid (EDTA-Na3) may be added. The esterification and trans-esterification taXe place under moderate conditions and stirring at about 20 C or some ~3~3~
hours. The formation of alkyl esters is nearly complete when the ester product has changed its appearance from opalescent -to clear. The slear solution (Dl) therefore contains alkyl esters of the fatty acids, glycerol, alkanol, as well as some water from the esterification of the free fatty acids.
Box 2.
The clear solution is then heated to a temperature of 65-68C, whereafter a fixed amount of urea (E) and alkanol (B) is added and stirred in until everything i completely dissolved. The amount of urea depends on the composition o~
the fatty acids so that if the raw material (A) contains 6-8%
EPA by weight, urea is added in the ratio 3 parts urea by weight to 1 part alkyl ester. In order to ensure that the components are completely soluble, 9 parts alkanol by weïght is added.
When everything is dissolved, the solution is slowly cool~d to approx. 20 C. An adduct of urea fatty ester (F) is crystalllized and then removed by means of for in~tance decanting and filtration, whereafter ~.he filter mass is cooled to 0 C in order to crystallize a larger portion of the adduct (F). The adduct is then separated by a known method so that the remaining filter mass (G) contains the essential fatty acid fractions and the unsaponifiable frac-tions.
Box 3.
The slightly alkaline filter mass (G) is saturated with hex-ane and is extracted by means of this solvent through a known technique as for instance by a continuing fluid-to-fluid counter current process, whereafter a further quantity of adduct of urea fatty ester may be crystallized. By means of this extraction two fluids are formed, consisting of hexane ~I) and a residu~ ~K).
~L3~3~
Box 4.
The hexane extract (I), which contains the alkyl-fat-ty esters of the polyunsaturated fatty acids 18:4 ~3, 20:5 ~3~and 22:6 ~3 as well as cholesterol as the most important com-ponents, is washed in a diluted hydrochloxic acid (L) in order to neutraliæe possible potassium soaps of the essential polyunsaturated fatty acids in the hexane extract. The washing water is decanted.
Hexane (H) is thereafter removed by evaporation of the extract (I) so that a concentrate is produced which is free from solvents (N) and which contains the compounds that are essential for the invention, the fatty acid compounds EPA and DHA as well as cholesterol.
~he dehydrated extract normally contains 20-30 % EPA, 35-45 DHA, 10-20% other polyunsaturated fatty acids, as well as 5-15% cholesterol and undefined compounds, all by weight, but the composition referred to will depend on the type of fish used, the time of year the fish is caught, and the type and condition of the raw material.
Box 5.
The concentrate of alkyl fatty acid ester (~) is thereafter cooled to approx. minus 25 C, whereafter cholesterol (0) is crystallized. This is centrifuged/filtered.
Further impurities which are present in the concentrate (N) may be removed by cooling the mixture to a temperature of lower than minus 25 C, whereafter certain undefined com-pounds are precipitated and filtered by means of a known method. The remainin~ ~3-concentrate (P) thus contains 20-35% EPA, 35-50% DHA and 15-25~ of other polyunsaturated ~3-fatty acid compounds (all by weight) as well as unsatur-ated fatty acid compounds which are not essential for the invention.
~3~34~1~6 Product (P) which contains the alkyl esters of the corre-sponding ~3-fatty acids may be utilized as it is or the con-centration of EPA and DHA may be increased.
Since the product contain~ only small amounts of other fatty acids with the same chain length as EPA and DHA, it is well suited for separation of the essential fatty acids, EPA and DHA by means of supercritical fluid extraction.
Another method for increasing the concentration is by means of preparative liquid chromatography by which method a more than 90% purity of the essential fatty acids is obtained.
Box 6.
The alkaline residue (K) is acidified by means of con-centrated hydrochloric acid (L) to a ph =2, whereafter a hex~
ane fraction (R) is precipitated in an upper layer which is separated~ One may also subject the acid solution (S) to furt~er extraction by means of hexane if this should be necessary, whereafter the hexane extracts are gathered. The hexane fractions (R) contain free fat1y acids as well as some of their alkyl esters and fairly high percentages of EPA and DHA, but also a fairly substantial portion of C 18-, C
20- and C 22-fatty acids with lower unsaturation. This acid solution contains water, alcohol, alkanol, glycerol, urea and other products which may be retrieved by a separate process which is not described here.
Box 7.
The fatty acid components of the hexane fraction are increased by evaporating hexane (H) in a separate piece of equipment.
Box 8.
The remaining solution is esterified using lower alkanols, for instance methanol or ethanol by means of an appropriate ~L303~1L~
catalytic agent ~V) which for instance may be dehydrated hydrochloric acid, acetic acid chloride or boron-trichloride.
The resultant alkyl ester (D2) from the fatty acid components (T) from box 7 may be processed in various ways, for instance returned to box 2 for urea precipitation of the less unsatur-ated fatty acids.
Example To 50 kg fish oil product (A) from cod entrails (containing 8~ EPA, 11~ DHA, and 2.3 % cholesterol all by weight) 400 1 methanol (B) and 10 g EDTA Na3 (C) were added in a reactor~
Potassium hydroxide (C) dissolved in methanol was added for neutralisation of free fatty acids in a quantity corre-sponding to a colour reaction of pH 12 on a moist pH-paper.
Thereafter 50 1 methanol (B) were added.
The whole mixture was subjected to stirring for 15 hours at 20C in order to bring about a trans-esterification of the glycerides to methyl esters and esteriication of the free fatty acids to methyl esters.
When the tr~ns-esterification was complete, the temperature was increased to 65 - 68 C and 140 kg urea (E), as well as a fixed amount of methanol (B) were stirred in while being heated until every hing seemed to be dissolved.
Then the solution was slowly cooled to room temperature (approx. 20C), whereafter a urea adduct of fatty acid was precipitated (F). It contained the major portion o the saturated and lower unsaturated fatty acid methyl esters.
The urea adduct was separated from the solution by decanting and filtering according to an ordinary, known technique.
Result: 100.1 kg urea adduct (F).
~3~34~6 Therea~ter the solution was cooled to 0 - 4 C, whereby an additional 5.1 kg urea adduct (F) could be filtrated from the solution.
This filtrate (G) contained ~3-polyunsaturated fatty acid methyl esters, cholesterol and a residue of unwanted fatty acid fractions with lower unsaturated C 18, C 20 and C 22 fatty acid methyl esters. To this filtrate we added hexane for saturation, whereby a further amount of urea add~ct (22 kg) could be separated. This hexane-saturated solution was extracted in a counter-current with hexane so that the hexane extract (I) finally made up approx. 300 1. The remaining unextracted solution is called (K). The hexane extract was thereafter evaporated. The yield of ~3-~atty acid methyl ester concentrate : 10.2 kilos.
The concentrate (~) which contained 23% EPA , 41% DHA and 8%
cholesterol, all by weight, was thereafter cooled to minus 25C, whereby pure cholesterol (0) crystallized and was removed by means of centrifuging during which time the resi-due in the centrifuge was washed with hexane with a lower temperature in order to remove the fatty acid methyl esters from the cholesterol crystals. Yield: 760 g pure cholesterol.
The concentrated filtrate (P3 contained 25% EPA-methylester, 43% DHA-me~hylester by weight based on the fatty acid portion and traces of cholesterol.
The above-mentioned extracted residue (K) was cleansed with a solution of concentrated hydrochloric acid, whereby one hex ane phase could be filtered off. Additional hexane was added to the,batch, stirred and then precipitated. The hexane frac-tions were put together and the hexane evaporated. To 7.7 kilos of the remaining fatty acid and the methyl fatty acid fraction , 15 liter 2~ methanolic solution of boron trichloride was added.
~L3~34~;
Yield: 6 kilos methyl fatty acid esters, containing 13%
EP~-metylester, 17% DHA-metylester and approximately 2% chol-esterol, all by weight.
The methyl fatty acid concentrate, containing methanol, was returned to the process for treatment with urea.
With this invention it has been possible to produce a very pure ~3-concentrate of fatty acid alkyle esters, where the essential anti-thrombotic fatty acid components eicosa-pentaenoic acid (EPA) and docosahexaenoic acid (DHA) is present in a strong concentration Further, by means of the procedure invented, it has been possible in a simple manner to separate very pure and crystalline cholesterol. An additional product is a urea adduct of fatty acid components, but this is of no interest to the invention.
Another advantage with the invention is that it is possible to produce a urea adduct based on the same procedure where a trans-esterification has been done from glycerides to alkanol esters without following the cumbersome procedure by first producing the fatty acids, esterify these with alkanol and then separate them by means of the fractionated urea preci-pitation.
By following the procedure invented, it is also possible to avoid the formation of emulsions in the phases, and the phase separation is thereby facilitated during later extraction stages.
Claims (10)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. Process for the production of a refined fish oil concentrate containing at least 20% eicosapentaenoic acid (EPA) and at least 35% docosehexaensoic acid (DHA) by weight, the remainder of the concentrate including other unsaturated fatty acid compounds, and the fatty acid compounds of the concentrate being mainly present as alkyl esters of lower alcohols, which process comprises the steps of:
(a) esterifying and/or trans-esterifying the fat/fatty acid fraction of fish oil product at room temperature with a lower alcohol in an alkaline environment containing amounts of a base sufficient only to catalyze the esterification and/or trans-esterification reaction;
(b) heating the resulting alkyl ester product with an excess of urea in an alkanol to from 55-90°C;
(c) cooling the product of step (b) to 0°C to precipitate urea fatty acid alkyl ester adduct and thereafter separating off said adduct to leave a solution mainly containing 3-fatty acid esters and an unsaponifiable portion;
(d) separating from the solution remaining from step (c) the 3-fatty acid alkyl esters and the unsaponifiable portion, (e) removing any solvent from the mixture obtained in step (d); and (f) cooling the concentrate obtained in stop (e) to crystallize out cholesterol and to precipitate out other undefined unsaponiflable compounds, thereby to leave a refined fish oil concentrate.
(a) esterifying and/or trans-esterifying the fat/fatty acid fraction of fish oil product at room temperature with a lower alcohol in an alkaline environment containing amounts of a base sufficient only to catalyze the esterification and/or trans-esterification reaction;
(b) heating the resulting alkyl ester product with an excess of urea in an alkanol to from 55-90°C;
(c) cooling the product of step (b) to 0°C to precipitate urea fatty acid alkyl ester adduct and thereafter separating off said adduct to leave a solution mainly containing 3-fatty acid esters and an unsaponifiable portion;
(d) separating from the solution remaining from step (c) the 3-fatty acid alkyl esters and the unsaponifiable portion, (e) removing any solvent from the mixture obtained in step (d); and (f) cooling the concentrate obtained in stop (e) to crystallize out cholesterol and to precipitate out other undefined unsaponiflable compounds, thereby to leave a refined fish oil concentrate.
2. Process according to Claim 1, wherein the concentrate obtained in step (f) is further treated in order to increase the concentration of EPA and DHA therein.
3. Process according to Claim 1 or 2, wherein, in step (b), the fatty acid alkyl esters 60-80°C, whereby the urea fatty acid alkyl ester adduct in the main does not contain 3-fatty acid compounds and unsaponifiable compounds.
4. Process according to Claim 1 or 2 wherein, in step (d), the separation is effected by extraction with hexane.
5. Process according to Claim 4, wherein, prior to step (e) the hexane extract is cleansed with a dilute acid.
6. Process according to Claim 1, 2 or 5, wherein step (f) is carried out by first cooling the 3-fatty acid alkyl ester concentrate to a temperature not lower than -25°C, whereby cholesterol is crystallized out, and thereafter to -50°C whereby the remaining portion of the unsaponifiable compounds precipitates.
7. Process according to Claim 1, 2 or 5, wherein methanol is used in step (a), whereby the fatty acid compound of the concentrate obtained are mainly present as methyl esters.
26625-53 3. Process according to Claim 1, 2 or 5, wherein, the refined fish oil concentrate obtained in step (f) contains 20-30%
by weight of EPA and 35-50% by weight DHA.
by weight of EPA and 35-50% by weight DHA.
9. A process according to Claim 1 step (d) wherein the separation is effected by solvent extraction.
10. A process according to Claim 5 wherein said dilute acid is hydrochloric acid.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| NO855147A NO157302C (en) | 1985-12-19 | 1985-12-19 | PROCEDURE FOR THE PREPARATION OF A FISH OIL CONCENTRATE. |
| NO855.147 | 1985-12-19 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1303416C true CA1303416C (en) | 1992-06-16 |
Family
ID=19888637
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000523914A Expired - Lifetime CA1303416C (en) | 1985-12-19 | 1986-11-27 | Refined fish oil concentrate and the production process for same |
Country Status (15)
| Country | Link |
|---|---|
| EP (1) | EP0255824B1 (en) |
| AR (1) | AR242111A1 (en) |
| AU (1) | AU6621186A (en) |
| CA (1) | CA1303416C (en) |
| DD (1) | DD261805A1 (en) |
| DE (1) | DE3668467D1 (en) |
| IE (1) | IE59171B1 (en) |
| IS (1) | IS1425B6 (en) |
| MA (1) | MA20840A1 (en) |
| MX (1) | MX168698B (en) |
| NO (1) | NO157302C (en) |
| NZ (1) | NZ218500A (en) |
| PT (1) | PT83991B (en) |
| WO (1) | WO1987003899A1 (en) |
| ZA (1) | ZA868927B (en) |
Families Citing this family (40)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB8819110D0 (en) * | 1988-08-11 | 1988-09-14 | Norsk Hydro As | Antihypertensive drug & method for production |
| JPH05506869A (en) * | 1990-03-12 | 1993-10-07 | ソラ，アイナル | Methods for enriching fats with respect to polyunsaturated fatty acids and phospholipids and applications of fats thus enriched |
| SE9303446D0 (en) * | 1993-10-20 | 1993-10-20 | Trikonex Ab | A novel urea fractionation process |
| GB9404483D0 (en) * | 1994-03-08 | 1994-04-20 | Norsk Hydro As | Refining marine oil compositions |
| TW425285B (en) | 1996-06-10 | 2001-03-11 | Viva America Marketing Inc | Fish oil and garlic nutritive supplement |
| US6313167B1 (en) | 1997-06-16 | 2001-11-06 | Nippon Suisan Kaisha Ltd. | Composition having capability of removing risk factor during exercise |
| NO309795B1 (en) * | 1998-07-01 | 2001-04-02 | Norsk Hydro As | Method for stabilizing oils and their use, method for stabilizing pigments, and method for preparing for |
| IT1308613B1 (en) | 1999-02-17 | 2002-01-09 | Pharmacia & Upjohn Spa | ESSENTIAL FATTY ACIDS IN THE PREVENTION OF CARDIOVASCULAR EVENTS. |
| NO311041B1 (en) * | 1999-12-22 | 2001-10-01 | Norsk Hydro As | Stabilization of pigments and polyunsaturated oils and oil concentrates |
| US6395778B1 (en) | 2000-01-11 | 2002-05-28 | Omegatech, Inc. | Process for making an enriched mixture of polyunsaturated fatty acid esters |
| AT414205B (en) | 2000-06-20 | 2006-10-15 | Vis Vitalis Lizenz & Handels | PROCESS FOR PRODUCING UNSATURATED FATTY ACID DRY CONCENTRATE |
| ITMI20010129A1 (en) | 2001-01-25 | 2002-07-25 | Pharmacia & Upjohn Spa | ESSENTIAL FATTY ACIDS IN THE THERAPY OF HEART INSUFFICIENCY AND HEART FAILURE |
| EP2295529B2 (en) | 2002-07-11 | 2022-05-18 | Basf As | Use of a volatile environmental pollutants-decreasing working fluid for decreasing the amount of pollutants in a fat for alimentary or cosmetic use |
| SE0202188D0 (en) | 2002-07-11 | 2002-07-11 | Pronova Biocare As | A process for decreasing environmental pollutants in an oil or a fat, a volatile fat or oil environmental pollutants decreasing working fluid, a health supplement, and an animal feed product |
| ITMI20032247A1 (en) * | 2003-11-19 | 2005-05-20 | Tiberio Bruzzese | INTERACTION OF POLAR DERIVATIVES OF COMPOUNDS INSATURATED WITH INORGANIC SUBSTRATES |
| EP1899453B1 (en) | 2005-06-07 | 2013-12-18 | Ocean Nutrition Canada Limited | Eukaryotic microorganisms for producing lipids and antioxidants |
| ITMI20051560A1 (en) * | 2005-08-10 | 2007-02-11 | Tiberio Bruzzese | COMPOSITION OF N-3 FATTY ACIDS WITH HIGH CONCENTRATION OF EPA AND E-O DHA AND CONTAINING N-6 FATTY ACIDS |
| US9023616B2 (en) | 2006-08-01 | 2015-05-05 | Dsm Nutritional Products Ag | Oil producing microbes and method of modification thereof |
| EP2519332B1 (en) | 2009-12-30 | 2014-03-05 | BASF Pharma (Callanish) Limited | Simulated moving bed chromatographic separation process for the purification of polyunsaturated fatty acids |
| ITMI20102297A1 (en) | 2010-12-15 | 2012-06-16 | Prime Europ Therapeuticals | PROCEDURE FOR STABILIZING FAT POLYINSATURED ACIDS BY MEANS OF METAL HYDRATES |
| ES2661234T3 (en) * | 2011-03-08 | 2018-03-28 | Cognis Ip Management Gmbh | A procedure for the distillation of fatty acid esters |
| GB201111591D0 (en) | 2011-07-06 | 2011-08-24 | Equateq Ltd | Further new process |
| GB201111589D0 (en) | 2011-07-06 | 2011-08-24 | Equateq Ltd | New modified process |
| GB201111594D0 (en) | 2011-07-06 | 2011-08-24 | Equateq Ltd | New improved process |
| GB201111595D0 (en) | 2011-07-06 | 2011-08-24 | Equateq Ltd | Improved process |
| GB201111601D0 (en) | 2011-07-06 | 2011-08-24 | Equateq Ltd | New process |
| GB201300354D0 (en) | 2013-01-09 | 2013-02-20 | Basf Pharma Callanish Ltd | Multi-step separation process |
| PT2970926T (en) | 2013-03-13 | 2018-03-22 | Dsm Nutritional Products Ag | Engineering microorganisms |
| US9428711B2 (en) | 2013-05-07 | 2016-08-30 | Groupe Novasep | Chromatographic process for the production of highly purified polyunsaturated fatty acids |
| EP2883860B1 (en) | 2013-12-11 | 2016-08-24 | Novasep Process | Chromatographic method for producing polyunsaturated fatty acids |
| WO2015104464A1 (en) | 2014-01-07 | 2015-07-16 | Novasep Process | Process for purifying aromatic amino acids |
| US9163198B2 (en) * | 2014-01-17 | 2015-10-20 | Orochem Technologies, Inc. | Process for purification of EPA (eicosapentanoic acid) ethyl ester from fish oil |
| WO2016028235A1 (en) * | 2014-08-18 | 2016-02-25 | Chiang Mai University | A system and method for extracting and/or concentrating vitamin e |
| JP6816012B2 (en) | 2015-03-26 | 2021-01-20 | ティベリオ ブリュジーズ | Purification method for polyunsaturated fatty acids |
| AR104042A1 (en) | 2015-03-26 | 2017-06-21 | Mara Renewables Corp | HIGH-DENSITY PRODUCTION OF BIOMASS AND OIL USING GLUCEROL IN GROSS |
| KR102678363B1 (en) | 2015-05-13 | 2024-06-26 | 이팍스 노르웨이 에이에스 | Very long chain polyunsaturated fatty acids from natural oils |
| CN107849514A (en) | 2015-07-13 | 2018-03-27 | 玛拉可再生能源公司 | Strengthen the microalgae metabolism of xylose |
| US10851395B2 (en) | 2016-06-10 | 2020-12-01 | MARA Renewables Corporation | Method of making lipids with improved cold flow properties |
| CN111315855B (en) | 2017-09-14 | 2023-07-25 | 发酵生物技术有限公司 | Improved method for extracting cholesterol from fish oil waste residue |
| US10196586B1 (en) * | 2018-02-14 | 2019-02-05 | Golden Omega S.A. | Feed ingredient |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IT1176916B (en) * | 1984-10-10 | 1987-08-18 | Elvira Pistolesi | PHARMACEUTICAL OR DIETETIC COMPOSITION WITH HIGH ANTI-THROMBOTIC AND ANTI-ARTERIOSCLEROTIC ACTIVITY |
-
1985
- 1985-12-19 NO NO855147A patent/NO157302C/en not_active IP Right Cessation
-
1986
- 1986-11-21 IS IS3159A patent/IS1425B6/en unknown
- 1986-11-21 AU AU66211/86A patent/AU6621186A/en not_active Withdrawn
- 1986-11-21 IE IE306486A patent/IE59171B1/en not_active IP Right Cessation
- 1986-11-21 WO PCT/NO1986/000077 patent/WO1987003899A1/en active IP Right Grant
- 1986-11-21 DE DE8686906964T patent/DE3668467D1/en not_active Expired - Lifetime
- 1986-11-21 EP EP86906964A patent/EP0255824B1/en not_active Expired - Lifetime
- 1986-11-25 ZA ZA868927A patent/ZA868927B/en unknown
- 1986-11-27 CA CA000523914A patent/CA1303416C/en not_active Expired - Lifetime
- 1986-12-03 NZ NZ218500A patent/NZ218500A/en unknown
- 1986-12-08 MX MX004578A patent/MX168698B/en unknown
- 1986-12-17 DD DD86297792A patent/DD261805A1/en not_active IP Right Cessation
- 1986-12-19 PT PT83991A patent/PT83991B/en not_active IP Right Cessation
- 1986-12-19 AR AR86306258A patent/AR242111A1/en active
- 1986-12-19 MA MA21072A patent/MA20840A1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| ZA868927B (en) | 1987-08-26 |
| EP0255824A1 (en) | 1988-02-17 |
| IS3159A7 (en) | 1987-06-20 |
| PT83991A (en) | 1987-01-01 |
| IS1425B6 (en) | 1990-03-28 |
| PT83991B (en) | 1989-01-17 |
| IE863064L (en) | 1987-06-19 |
| MA20840A1 (en) | 1987-07-01 |
| NO157302B (en) | 1987-11-16 |
| DD261805A1 (en) | 1988-11-09 |
| IE59171B1 (en) | 1994-01-12 |
| EP0255824B1 (en) | 1990-01-24 |
| NZ218500A (en) | 1989-03-29 |
| DE3668467D1 (en) | 1990-03-01 |
| WO1987003899A1 (en) | 1987-07-02 |
| AR242111A1 (en) | 1993-03-31 |
| NO855147L (en) | 1987-06-22 |
| NO157302C (en) | 1988-02-24 |
| MX168698B (en) | 1993-06-04 |
| AU6621186A (en) | 1987-07-15 |
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