WO2009006317A1 - Production and purification of esters of polyunsaturated fatty acids - Google Patents

Production and purification of esters of polyunsaturated fatty acids Download PDF

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Publication number
WO2009006317A1
WO2009006317A1 PCT/US2008/068613 US2008068613W WO2009006317A1 WO 2009006317 A1 WO2009006317 A1 WO 2009006317A1 US 2008068613 W US2008068613 W US 2008068613W WO 2009006317 A1 WO2009006317 A1 WO 2009006317A1
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WIPO (PCT)
Prior art keywords
composition
ester
acid
fatty acid
polyunsaturated fatty
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PCT/US2008/068613
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English (en)
French (fr)
Inventor
Krishna Raman
Jung Lee
Neil Leininger
Chris Luigart
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Martek Biosciences Corp
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Martek Biosciences Corp
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Publication date
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Priority to CA2692355A priority Critical patent/CA2692355C/en
Priority to CN200880104965A priority patent/CN101796014A/zh
Priority to MX2010000263A priority patent/MX2010000263A/es
Priority to MX2015006380A priority patent/MX374545B/es
Priority to EP08781103.0A priority patent/EP2173699A4/en
Priority to AU2008269989A priority patent/AU2008269989B2/en
Priority to JP2010515186A priority patent/JP2010532418A/ja
Publication of WO2009006317A1 publication Critical patent/WO2009006317A1/en
Anticipated expiration legal-status Critical
Priority to AU2014202880A priority patent/AU2014202880B2/en
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/52—Esters of acyclic unsaturated carboxylic acids having the esterified carboxyl group bound to an acyclic carbon atom
    • C07C69/587—Monocarboxylic acid esters having at least two carbon-to-carbon double bonds
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
    • A21D2/00—Treatment of flour or dough by adding materials thereto before or during baking
    • A21D2/08—Treatment of flour or dough by adding materials thereto before or during baking by adding organic substances
    • A21D2/14—Organic oxygen compounds
    • A21D2/16—Fatty acid esters
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/158—Fatty acids; Fats; Products containing oils or fats
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A23L33/12—Fatty acids or derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/03—Preparation of carboxylic acid esters by reacting an ester group with a hydroxy group
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/08—Preparation of carboxylic acid esters by reacting carboxylic acids or symmetrical anhydrides with the hydroxy or O-metal group of organic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/48—Separation; Purification; Stabilisation; Use of additives
    • C07C67/60—Separation; Purification; Stabilisation; Use of additives by treatment giving rise to chemical modification
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C11C3/003—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fatty acids with alcohols

Definitions

  • the invention relates to methods for producing and purifying esters of polyunsaturated fatty acids from triglyceride containing compositions.
  • the invention also relates to compositions comprising polyunsaturated fatty acids.
  • Omega-3 and omega-6 long chain polyunsaturated fatty acids are recognized as important dietary compounds for preventing arteriosclerosis and coronary heart disease, for alleviating inflammatory conditions and for retarding the growth of tumor cells.
  • Omega-6 PUFAs serve not only as structural lipids in the human body, but also as precursors for a number of factors in inflammation, such as prostaglandins and leukotrienes.
  • Long chain omega-3 and the omega-6 PUFAs represent important classes of PUFAs.
  • DHA docosahexaenoic acid
  • omega-3 LC-PUFAs include eicosapentaenoic acid (“EPA”), which is designated “20:5 n-3,” and omega-3 docosapentaenoic acid (“DPA n-3”), which is designated “22:5 n-3.”
  • Important omega-6 LC-PUFAs include arachidonic acid (“ARA”), which is designated “20:4 n-6,” and omega-6 docosapentaenoic acid (“DPA n- 6”), which is designated “22:5 n-6.”
  • PUFAs include vegetable oils, marine animal oils, fish oils and oilseeds.
  • oils produced by certain microorganisms have been found to be rich in LC- PUFAs.
  • the oils derived from each of these sources also contain substantial levels of saturated fatty acids and other undesirable impurities.
  • Numerous methods have been used to isolate or purify PUFAs and derivatives thereof from crude oils. Among these processes are fractional crystallization at low temperatures, urea adduct crystallization, extraction with metal salt solutions, super critical fluid fractionation on countercurrent columns and high performance liquid chromatography.
  • the present invention provides a method for purifying a composition comprising triglycerides having polyunsaturated fatty acid residues comprising reacting the composition in the presence of an alcohol and a base to produce an ester of a polyunsaturated fatty acid from the triglycerides and distilling the composition to recover a fraction comprising the ester of the polyunsaturated fatty acid.
  • the step of reacting the composition in the presence of an alcohol and a base is performed at a temperature from about 60° C to about 120° C.
  • the step of reacting the composition in the presence of an alcohol and a base is performed for a time from about 2 hours to about 12 hours.
  • the composition comprising triglycerides having polyunsaturated fatty acid residues has not been subjected to one or more treatments selected from the group consisting of refining, desolventization, deodorization, winterization, chill filtration, and bleaching.
  • the composition comprising triglycerides having polyunsaturated fatty acid residues has not been subjected to refining, desolventization, deodorization, winterization, chill filtration, and bleaching.
  • the composition comprising triglycerides having polyunsaturated fatty acid residues is from a source selected from the group consisting of a plant, a microorganism, an animal, and mixtures of the foregoing.
  • the source is a microorganism selected from the group consisting of algae, bacteria, fungi and protists.
  • the source is selected from the group consisting of plants selected from the group consisting of soybean, corn, rice, safflower, sunflower, canola, flax, peanut, mustard, rapeseed, chickpea, cotton, lentil, white clover, olive, palm, borage, evening primrose, linseed and tobacco and mixtures thereof.
  • the source is selected from the group consisting of a genetically modified plant and a genetically modified microorganism, wherein the genetic modification comprises the introduction of polyketide synthase genes.
  • the source is a microorganism selected from the group consisting of Thraustochytriales, dinoflagellates, and Mortierella.
  • the microorganism is Thraustochytriales, Schizochytrium or Thraustochytrium.
  • the microorganism is a dinoflagellate of the genus Crypthecodinium.
  • the source is an animal selected from aquatic animals.
  • the polyunsaturated fatty acid is a polyunsaturated fatty acid having a chain length of at least 18 carbons.
  • the polyunsaturated fatty acid is a polyunsaturated fatty acid selected from the group consisting of docosahexaenoic acid, docosapentaenoic acid, arachidonic acid, eicosapentaenoic acid, stearidonic acid, linolenic acid, alpha linolenic acid, gamma linolenic acid, conjugated linolenic acid and mixtures thereof.
  • the polyunsaturated fatty acid is docosahexaenoic acid.
  • the polyunsaturated fatty acid is arachadonic acid.
  • the base is a base of the formula RO-M, wherein M is a monovalent cation and RO is an alkoxide of a C 1 ⁇ alkyl alcohol.
  • the base is sodium ethoxide.
  • the alcohol is a C 1 ⁇ alkyl alcohol.
  • the alcohol is ethanol and the ester is an ethyl ester of the polyunsaturated fatty acid.
  • the step of distilling the composition to recover a fraction comprising the ester of the polyunsaturated fatty acid is performed under vacuum.
  • the step of distilling the composition to recover a fraction comprising the ester of the polyunsaturated fatty acid is performed at a temperature of less than about 170° C.
  • the fraction recovered comprises at least about 50 wt. %, 75 wt. %, 90 wt. %, or 95 wt. % ester of the polyunsaturated fatty acid.
  • the step of reacting the composition in the presence of an alcohol and a base produces an ester of a polyunsaturated fatty acid from the triglycerides by direct transesterification.
  • the method further comprises a) combining the fraction comprising the ester of the polyunsaturated fatty acid with urea in a medium; b) cooling or concentrating the medium to form a urea-containing precipitate and a liquid fraction; and c) separating the precipitate from the liquid fraction.
  • the medium further comprises an organic solvent that can solubilize the ester of the polyunsaturated fatty acid.
  • the organic solvent comprises an alkyl alcohol comprising from 1 to 4 carbon atoms.
  • the organic solvent comprises ethanol.
  • the medium is cooled to a temperature of from about O 0 C to about 25°C to form the urea-containing precipitate.
  • At least a portion of the urea-containing precipitate is formed under a non-oxidizing atmosphere.
  • the present invention also provides a method for producing an ester of a polyunsaturated fatty acid from a composition comprising triglycerides having polyunsaturated fatty acid residues comprising transesterifying the composition in the presence of an alcohol and a base to produce an ester of the polyunsaturated fatty acid from the triglycerides and distilling the composition to recover a fraction comprising the ester of the polyunsaturated fatty acid.
  • the present invention further provides a method for purifying a composition comprising triglycerides having polyunsaturated fatty acid residues comprising reacting the composition in the presence of an alcohol and a base to produce an ester of the polyunsaturated fatty acid from the triglycerides and separating a fraction comprising at least about 75% ester of the polyunsaturated fatty acid.
  • the step of separating comprises distilling.
  • the present invention also provides a method for preparing a composition comprising an ester of a polyunsaturated fatty acid comprising reacting a composition comprising triglycerides having polyunsaturated fatty acid residues in the presence of an alcohol and a base to produce an ester of a polyunsaturated fatty acid from the triglycerides, wherein the composition comprising triglycerides having polyunsaturated fatty acid residues has not been subjected to one or more treatments selected from the group consisting of refining, desolventization, deodorization, winterization, chill filtration, and bleaching.
  • the step of reacting the composition in the presence of an alcohol and a base is performed at a temperature from about 60° C to about 120° C.
  • the step of reacting the composition in the presence of an alcohol and a base is performed for a time from about 2 hours to about 12 hours.
  • the method further comprises a step of distilling the composition to recover a fraction comprising the ester of the polyunsaturated fatty acid.
  • the composition comprising triglycerides having polyunsaturated fatty acid residues is from a source selected from the group consisting of a plant, a microorganism, an animal, and mixtures of the foregoing.
  • the source is a microorganism selected from the group consisting of algae, bacteria, fungi and protists.
  • the source is selected from the group consisting of plants selected from the group consisting of soybean, corn, rice, safflower, sunflower, canola, flax, peanut, mustard, rapeseed, chickpea, cotton, lentil, white clover, olive, palm, borage, evening primrose, linseed and tobacco and mixtures thereof.
  • the source is selected from the group consisting of a genetically modified plant and a genetically modified microorganism, wherein the genetic modification comprises the introduction of polyketide synthase genes.
  • the source is a microorganism selected from the group consisting of Thraustochytriales, dinoflagellates, and Mortierella. In some embodiments, the source is a microorganism selected from the group consisting of Thraustochytriales, dinoflagellates, and Mortierella.
  • the microorganism is a dinoflagellate of the genus Crypthecodinium .
  • the source is an animal selected from aquatic animals.
  • the polyunsaturated fatty acid is a polyunsaturated fatty acid having a chain length of at least 18 carbons.
  • the polyunsaturated fatty acid is a polyunsaturated fatty acid selected from the group consisting of docosahexaenoic acid, docosapentaenoic acid, arachidonic acid, eicosapentaenoic acid, stearidonic acid, linolenic acid, alpha linolenic acid, gamma linolenic acid, conjugated linolenic acid and mixtures thereof.
  • the polyunsaturated fatty acid is docosahexaenoic acid.
  • the polyunsaturated fatty acid is arachadonic acid.
  • the base is a base of the formula RO-M, wherein M is a monovalent cation and RO is an alkoxide of a Ci .6 alkyl alcohol.
  • the base is sodium ethoxide.
  • the alcohol is a C 1 ⁇ alkyl alcohol.
  • the alcohol is ethanol and the ester is an ethyl ester of the polyunsaturated fatty acid.
  • the step of distilling the composition to recover a fraction comprising the ester of the polyunsaturated fatty acid is performed under vacuum.
  • the step of distilling the composition to recover a fraction comprising the ester of the polyunsaturated fatty acid is performed at a temperature of less than about 17O 0 C.
  • the fraction recovered comprises at least about 50 wt. %, 75 wt. %, 90 wt. %, or 95 wt. % ester of the polyunsaturated fatty acid.
  • the step of reacting the composition in the presence of an alcohol and a base produces an ester of a polyunsaturated fatty acid from the triglycerides by direct transesterification.
  • the present invention also provides a composition comprising at least about 90 wt. % ethyl ester of docosahexaenoic acid, wherein the composition further comprises at least about 0.1 wt. % of 4,7,10,13,16,19,22,25 octacosaoctaenoic acid (C28:8) or an ester thereof.
  • the composition comprises at least about 0.5 wt. %, 1.0 wt.
  • the composition further comprises at least about 0.1 wt. %, 0.3 wt. %, 0.4 wt. %, or 0.5 wt. % of docosapentaenoic acid (n-3) or an ester thereof.
  • the composition comprises at least about 92 wt. % or 95 wt. % ethyl ester of docosahexaenoic acid.
  • the composition further comprises less than about 1 wt. %, 0.5 wt. % or 0.25 wt. % eicosapentaenoic acid or an ester thereof.
  • the present invention further provides a composition comprising at least about 90 wt. % ethyl ester of docosahexaenoic acid, wherein the composition further comprises at least about 0.1 wt. % of docosapentaenoic acid (n-3) or an ester thereof.
  • the composition comprises at least about 0.3 wt. %, 0.4 wt. %, or 0.5 wt. % of docosapentaenoic acid (n-3) or an ester thereof.
  • the composition further comprises at least about 0.5 wt. %, 0.75 wt. %, 1.0 wt. %, or 1.2 wt. % of 4,7,10,13,16,19,22,25 octacosaoctaenoic acid (C28:8) or an ester thereof.
  • the composition comprises at least about 92 wt. % or 95 wt. % ethyl ester of docosahexaenoic acid.
  • the composition further comprises less than about 1 wt. %, 0.5 wt. % or 0.25 wt. % eicosapentaenoic acid or an ester thereof.
  • the present invention also provides a composition comprising at least about 90 wt. % ethyl ester of docosahexaenoic acid, wherein the composition further comprises at least one additional fatty acid or an ester thereof with a boiling point of about 150-170 0 C at a pressure of 0.8 mm Hg.
  • the present invention further provides a composition comprising at least about 70 wt. % ethyl ester of docosahexaenoic acid and at least about 25 wt. % ethyl ester of docosapentaenoic acid (n-6). In some embodiments, the composition further comprises less than about 4% of a saturated fatty acid or an ester thereof.
  • the saturated fatty acid or an ester thereof contains less than 20 carbons.
  • the saturated fatty acid or an ester thereof contains 14 or 16 carbons.
  • the present invention also provides a composition comprising at least about 90 wt. % of a combination of ethyl ester of docosahexaenoic acid and ethyl ester of docosapentaenoic acid (n-6).
  • the composition comprises at least about 10 wt. % ethyl ester of docosahexaenoic acid and at least about 10 wt. % ethyl ester of docosapentaenoic acid (n-6).
  • the composition further comprises less than about 4% of a saturated fatty acid or an ester thereof.
  • the saturated fatty acid or an ester thereof contains less than 20 carbons.
  • the saturated fatty acid or an ester thereof contains 14 or 16 carbons.
  • the present invention further provides a comprising at least about 90 wt. % of a combination of ethyl ester of docosahexaenoic acid and ethyl ester of docosapentaenoic acid (n-6), wherein the composition further comprises at least one additional fatty acid or an ester thereof with a boiling point of about 150-175 0 C at a pressure of 0.5 mm Hg.
  • the present invention also provides a method for preparing a composition comprising an ester of a polyunsaturated fatty acid comprising reacting a composition comprising triglycerides having polyunsaturated fatty acid residues in the presence of an alcohol and a base to produce an ester of a polyunsaturated fatty acid from the triglycerides, wherein the composition comprising triglycerides having polyunsaturated fatty acid residues comprises at least one characteristic selected from the group consisting of: at least about 300 ppm phosphorus, at least about 0.4 % free fatty acids, and a peroxide value of at least about 0.2 meq/kg.
  • the present invention also provides a method for purifying a composition comprising triglycerides having polyunsaturated fatty acid residues, wherein the composition comprises at least one characteristic selected from the group consisting of: at least about 300 ppm phosphorus, at least about 0.4 % free fatty acids, and a peroxide value of at least about 0.2 meq/kg, comprising a) reacting the composition in the presence of an alcohol and a base to produce an ester of a polyunsaturated fatty acid from the triglycerides; and b) distilling the composition to recover a fraction comprising the ester of the polyunsaturated fatty acid.
  • the present invention further provides a composition comprising at least about 60 wt. % esters of arachidonic acid.
  • the composition further comprises less than about 10 wt. % ⁇ . 77 ' ⁇ L J i * ⁇ t K 1 eicosapentaenoic acid.
  • esters of arachidonic acid are ethyl esters of arachidonic acid.
  • the present invention also provides a method of treating a subject with high levels of triglycerides comprising administering a composition according to claim 68, 81, 94, 95, 99 or 104 to the subject.
  • the present invention further provides a method of treating a subject with a neurological disorder, dementia or a pre-dementia related condition comprising administering a composition according to claim 68, 81, 94, 95, 99 or 104 to the subject. DESCRIPTION OF THE INVENTION
  • the present invention provides novel methods for the purification of compositions containing triglycerides having PUFA residues.
  • the invention includes reacting the composition in the presence of an alcohol and a base to produce an ester of a polyunsaturated fatty acid from the triglycerides, hi one embodiment, the invention advantageously and efficiently is conducted on relatively crude oils that have not been subjected to conventional processing methods that can include refining, bleaching, deodorizing and winterization.
  • the invention includes producing esters from triglycerides and then distilling the resulting composition to recover a fraction comprising the ester of the polyunsaturated fatty acid.
  • the fraction comprising the ester of the polyunsaturated fatty acid is further purified by urea crystallization.
  • the present invention allows the efficient and cost effective production of esters of PUFAs directly from crude or processed oils.
  • the starting material for the methods of the present invention is a composition comprising triglycerides having PUFA residues.
  • the terms “oils” and “compositions comprising triglycerides having PUFA residues” are used interchangeably throughout this application.
  • a "triglyceride” is an ester of three fatty acid residues and glycerol having a general chemical formula of CH ⁇ OOCR ⁇ CHlOOCR ⁇ CH ⁇ OOCR 3 ), wherein each of OOCR 1 , OOCR 2 , and OOCR 3 represents a fatty acid residue.
  • Suitable triglycerides contain at least one PUFA. hi some embodiments, the PUFA has a chain length of at least 18 carbons.
  • the PUFA can be docosahexaenoic acid C22:6 n-3 (DHA), omega-3 docosapentaenoic acid C22:5 n-3 (DPA), omega-6 docosapentaenoic acid C22:5 n-6 (DPA), arachidonic acid C20:4 n-6 (ARA), eicosapentaenoic acid C20:5 n- 3 (EPA), stearidonic acid (SDA), linolenic acid (LLA), alpha linolenic acid (ALA), gamma linolenic acid (GLA), conjugated linolenic acid (CLA) or mixtures thereof.
  • DHA docosahexaenoic acid C22:6 n-3
  • DPA omega-3 docosapentaenoic acid C22:5 n-3
  • DPA omega-6 docosapentaenoic acid C22:5 n-6
  • ARA
  • the PUFAs can also be present in any of the common forms found in natural lipids including but not limited to triacylglycerols, diacylglycerols, monoacylglycerols, phospholipids, free fatty acids, or in natural or synthetic derivative forms of these fatty acids (e.g. calcium salts of fatty acids, and the like).
  • Reference to an oil or other composition comprising triglycerides having PUFA residues can refer to either a composition comprising triglycerides having only a single type of LC PUFA residue such as DHA or a composition comprising triglycerides having a mixture of more than one type of LC PUFA residues such as more than one of DHA, EPA and ARA.
  • compositions comprising triglycerides having PUFA residues can be obtained from or derived from any suitable source, such as a plant (including oilseeds), a microorganism, an animal, or mixtures of the foregoing.
  • the microorganisms can be algae, bacteria, fungi or protists.
  • Microbial sources and methods for growing microorganisms comprising nutrients and/or PUFAs are known in the art (Industrial
  • microorganisms can be cultured in a fermentation medium in a fermentor. Oils produced by microorganisms can be used in the methods and compositions of the present invention.
  • organisms include those selected from the group consisting of golden algae (such as microorganisms of the kingdom Stramenopiles), green algae, diatoms, dinoflagellates (such as microorganisms of the order Dinophyceae including members of the genus Crypthecodinium such as, for example, Crypthecodinium cohnii), yeast, and fungi of the genera Mucor and Mortierella, including but not limited to Mortierella alpina and Mortierella sect, schmuckeri.
  • golden algae such as microorganisms of the kingdom Stramenopiles
  • green algae diatoms
  • dinoflagellates such as microorganisms of the order Dinophyceae including members of the genus Crypthecodinium such as, for example, Crypthecodinium cohnii
  • yeast and fungi of the genera Mucor and Mortierella, including but not limited to Mortierella alpina and Mortierella sect, schmuckeri.
  • Stramenopiles include microalgae and algae-like microorganisms, including the following groups of microorganisms: Hamatores, Proteromonads, Opalines, Develpayella, Diplophrys, Labrinthulids, Thraustochytrids, Biosecids, Oomycetes, Hypochytridiomycetes, Commation, Reticulosphaera, Pelagomonas, Pelagococcus, Ollicola, Aureococcus, Parmales, Diatoms, Xanthophytes, Phaeophytes (brown algae), Eustigmatophytes, Raphidophytes, Synurids, Axodines (including Rhizochromulinaales, Pedinellales, Dictyochales), Chrysomeridales, Sarcinochrysidales, Hydrurales, Hibberdiales, and Chromulinales.
  • Axodines including Rhizochromul
  • the Thraustochytrids include the genera Schizochytrium (species include aggregatum, limnaceum, mangrovei, minutum, octosporum), Thraustochytrium (species include arudimentale, aureum, benthicola, globosum, kinnei, motivum, multirudimentale, pachydermum, proliferum, roseum, striatum), Ulkenia (species include amoeboidea, kerguelensis, minuta, profunda, radiate, sailens, sarkariana, schizochytrops, visurgensis, yorkensis), Aplanochytrium (species include haliotidis, kerguelensis, profunda, stocchinoi), Japonochytrium (species include marinum), Althornia (species include crouchi ⁇ ), and Elina (species include marisalba, sinoriflca).
  • Schizochytrium
  • the Labrinthulids include the genera Labyrinthula (species include algeriensis, coenocystis, chattonii, macrocystis, macrocystis atlantica, macrocystis macrocystis, marina, minuta, roscoffensis, valkanovii, vitellina, vitellina paci ⁇ ca, vitellina vitellina, zopfl), Labyrinthomyxa (species include marina), Labyrinthuloides (species include haliotidis, yorkensis), Diplophrys (species include archeri), Pyrrhosorus* (species include marinus), Sorodiplophrys* (species include stercorea), Chlamydomyxa* (species include labyrinthuloides, montana).
  • Labyrinthula genera include algeriensis, coenocystis, chattonii, macrocy
  • Suitable microorganisms include those capable of producing lipids comprising omega-3 and/or omega-6 polyunsaturated fatty acids, and in particular microorganisms that are capable of producing oils containing DHA, DPA, EPA or ARA will be described. More particularly, preferred microorganisms are algae, such as Thraustochytrids of the order Thraustochytriales, including Thraustochytrium (including Ulkenia) and Schizochytrium and including Thraustochytriales which are disclosed in commonly assigned U.S. Patent Nos.
  • the microorganisms are selected from the group consisting of microorganisms having the identifying characteristics of ATCC number 20888, ATCC number 20889, ATCC number 20890, ATCC number 20891 and ATCC number 20892. Since there is some disagreement among experts as to whether Ulkenia is a separate genus from the genus Thraustochytrium, for the purposes of this application, the genus Thraustochytrium will include Ulkenia. Also preferred are strains of Mortierella schmuckeri (e.g., including ATCC 74371) and Mortierella alpina.
  • strains of Crypthecodinium cohnii including microorganisms having the identifying characteristics of ATCC Nos. 30021, 30334-30348, 30541-30543, 30555-30557, 30571, 30572, 30772-30775, 30812, 40750, 50050-50060, and 50297-50300.
  • Oleaginous microorganisms are also preferred.
  • "oleaginous microorganisms” are defined as microorganisms capable of accumulating greater than 20% of the dry weight of their cells in the form of lipids.
  • Genetically modified microorganisms that produce PUFA-containing oils are also suitable for the present invention. These can include naturally PUFA-producing microorganisms that have been genetically modified as well as microorganisms that do not naturally produce PUFAs but that have been genetically modified to do so.
  • Suitable organisms can be obtained from a number of available sources, including by collection from the natural environment.
  • the American Type Culture Collection currently lists many publicly available strains of microorganisms identified above.
  • any organism, or any specific type of organism includes wild strains, mutants, or recombinant types. Growth conditions in which to culture or grow these organisms are known in the art, and appropriate growth conditions for at least some of these organisms are disclosed in, for example, U.S. Patent No. 5,130,242, U.S. Patent No. 5,407,957, U.S. Patent No. 5,397,591, U.S. Patent No. 5,492,938, U.S. Patent No. 5,711,983 and U.S. Patent No.
  • the microorganisms are cultured in an effective medium, herein defined as any medium capable of promoting oil production.
  • the effective medium also promotes rapid microbial growth.
  • the microorganisms can be cultured in conventional fermentation modes, which include, but are not limited to, batch, fed-batch, and continuous.
  • Another source of oils suitable for the compositions and methods of the present invention includes a plant source, such as oilseed plants.
  • PUFA-producing plants in alternate embodiments, can include those genetically engineered to express genes that produce PUFAs and those that produce PUFAs naturally.
  • genes can include genes encoding proteins involved in the classical fatty acid synthase pathways, or genes encoding proteins involved in the PUFA polyketide synthase (PKS) pathway.
  • PKS PUFA polyketide synthase
  • the genes and proteins involved in the classical fatty acid synthase pathways, and genetically modified organisms, such as plants, transformed with such genes, are described, for example, in Napier and Sayanova, Proceedings of the Nutrition Society (2005), 64:387-393; Robert et al., Functional Plant Biology (2005) 32:473-479; or U.S. Patent Application Publication 2004/0172682.
  • Oilseed crops suitable for use in the present invention include soybeans, corn, rice, safflower, sunflower, canola, flax, peanut, mustard, rapeseed, chickpea, cotton, lentil, white clover, olive, palm oil, borage, evening primrose, linseed, and tobacco that have been genetically modified to produce a PUFA as described above.
  • Transformation techniques for microorganisms and plants are well-known in the art. Transformation techniques for microorganisms are well known in the art and are discussed, for example, in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press. A general technique for transformation of dinoflagellates, which can be adapted for use with Crypthecodinium cohnii, is described in detail in Lohuis and Miller, The Plant Journal (1998) 13(3): 427-435. A general technique for genetic transformation of Thraustochytrids is described in detail in U.S.
  • Patent Application Publication No. 20030166207 published September 4, 2003.
  • Methods for the genetic engineering of plants are also well known in the art. For instance, numerous methods for plant transformation have been developed, including biological and physical transformation protocols. See, for example, Mild et al., "Procedures for Introducing Foreign DNA into Plants” in Methods in Plant Molecular Biology and
  • oilseed plants When oilseed plants are the source of PUFA-containing oils, the seeds can be harvested and processed to remove any impurities, debris or indigestible portions from the harvested seeds. Processing steps vary depending on the type of oilseed and are known in the art. Processing steps can include threshing (such as, for example, when soybean seeds are separated from the pods), dehulling (removing the dry outer covering, or husk, of a fruit, seed, or nut), drying, cleaning, grinding, milling and flaking. After the seeds have been processed to remove any impurities, debris or indigestible materials, they can be added to an aqueous solution and then mixed to produce a slurry. In some embodiments, milling, crushing or flaking is performed prior to mixing with water. A slurry produced in this manner can be treated and processed the same way as described for a microbial fermentation broth.
  • Another biomass source of PUFA-containing oils suitable for the compositions and methods of the present invention includes an animal source.
  • animal sources include aquatic animals (e.g., fish, marine mammals, and crustaceans such as krill and other euphausids) and animal tissues (e.g., brain, liver, eyes, etc.) and animal products such as eggs or milk.
  • Techniques for recovery of PUFA-containing oils from such sources are known in the art. While in one embodiment of the invention the composition comprising triglycerides having PUFA residues can be a crude oil (discussed in more detail below), other such compositions useful in the present invention can be recovered from their sources by any suitable means known to those in the art.
  • oils can be recovered by extraction with solvents such as chloroform, hexane, methylene chloride, methanol and the like, or by supercritical fluid extraction.
  • solvents such as chloroform, hexane, methylene chloride, methanol and the like
  • the oils can be extracted using extraction techniques, such as are described in U.S. Patent No. 6,750,048 and PCT Patent Application Serial No. USO 1/01806, both filed January 19, 2001, and entitled "Solventless Extraction Process," both of which are incorporated herein by reference in their entirety. Additional extraction and/or purification techniques are taught in PCT Patent Application Serial No. PCT/IBOl/00841 entitled "Method for the
  • an oil obtained from a source described above can serve as the starting material for the methods of the present invention even when it has not been subjected to conventional processing.
  • refining e.g., physical refining, silica refining or caustic refining
  • desolventization e.g., desolventization, deodorization, winterization, chill filtration, and/or bleaching.
  • the composition containing triglycerides having PUFA residues has not been subjected to one or more treatments selected from refining, desolventization, deodorization, winterization, chill filtration, and bleaching and in further embodiments, the composition has not been subjected to any one of refining, desolventization, deodorization, winterization, chill filtration, and bleaching.
  • the composition comprising triglycerides having polyunsaturated fatty acid residues may be an oil having characteristics of oils that have not been subjected to conventional processing, such as refining, desolventization, deodorization, winterization, chill filtration, and bleaching.
  • a suitable oil can have a chemical or physical characteristic of an unprocessed oil.
  • the oil may contain an undesirable component (e.g., an impurity) at a level that is typically not present in a conventionally processed oil.
  • the oil may contain from about 300 ppm phosphorous to about 1000 ppm phosphorous.
  • the oil comprises at least about 300 ppm phosphorous; at least about 400 ppm phosphorous; at least about 500 ppm phosphorous; at least about 600 ppm phosphorous; at least about 650 ppm phosphorous; at least about 700 ppm phosphorous; at least about 750 ppm phosphorous; at least about 800 ppm phosphorous; at least about 850 ppm phosphorous; at least about 900 ppm phosphorous; at least about 950 ppm phosphorous; or at least about 1000 ppm phosphorous.
  • the oil may contain free fatty acids in a range of from about 0.4 wt. % to about 1.4 wt. %.
  • the oil comprises at least about 0.4 wt. % free fatty acids; at least about 0.6 wt. % free fatty acids; at least about 0.8 wt. % free fatty acids; at least about 0.9 wt. % free fatty acids; at least about 1.0 wt. % free fatty acids; at least about 1.1 wt. % free fatty acids; at least about 1.2 wt. % free fatty acids; at least about 1.3 wt. % free fatty acids; or at least about 1.4 wt. % free fatty acids.
  • the oil may contain a peroxide value ranging from about 0.2 meq/kg to about 2.5 meq/kg.
  • the oil comprises a peroxide value of at least about 0.2 meq/kg; a peroxide value of at least about 0.4 meq/kg; a peroxide value of at least about 0.6 meq/kg; a peroxide value of at least about 0.8 meq/kg; a peroxide value of at least about 1.0 meq/kg; a peroxide value of at least about 1.2 meq/kg; a peroxide value of at least about 1.4 meq/kg; a peroxide value of at least about 1.5 meq/kg; a peroxide value of at least about 1.6 meq/kg; a peroxide value of at least about 1.7 meq/kg; a peroxide value of at least about 1.8 meq/kg; a peroxide value of at least about 1.9 meq/kg; a peroxide value of at least about 2.0 meq/kg; a peroxide value of at least about 2.1 meq/kg; a peroxide value of at least about 1.9 meq/
  • the crude oil may be isolated from a microorganism using standard techniques, without being subjected to further refinement or purification.
  • the oil is a microbial oil that has only been subjected to solvent extraction, such as hexane extraction, isopropanol extraction, or the like.
  • compositions comprising triglycerides having polyunsaturated fatty acid residues may be subjected to further processing steps, such as refining, desolventization, deodorization, winterization, chill filtration, and/or bleaching.
  • processing steps such as refining, desolventization, deodorization, winterization, chill filtration, and/or bleaching.
  • oils include microbial oils that have been subjected to solvent extraction and one or more of these additional processing steps.
  • oils are minimally processed.
  • “Minimally processed” oils include microbial oils that have been subjected to solvent extraction and filtration. In certain embodiments, minimally processed oils are further subjected to winterization.
  • Methods of the present invention involve reacting compositions containing triglycerides having PUFA residues in the presence of an alcohol and a base to produce esters of the PUFAs from the triglycerides.
  • Alcohols suitable for use in the present invention include any lower alkyl alcohol containing from 1 to 6 carbon atoms (i.e., a Ci .6 alkyl alcohol). Without being bound by theory, it is believed that the use of lower alkyl alcohols in the methods of the present invention produces lower alkyl esters of the PUFAs. For example, the use of ethanol produces ethyl esters. In certain embodiments, the alcohol is methanol or ethanol. In these embodiments, the PUFA esters produced are a methyl ester and an ethyl ester of the PUFA, respectively. In processes of the present invention, the alcohol typically comprises between about 25 wt. % and about 50 wt. %; between about 30 wt.
  • the alcohol comprises about 38 wt. % of the mixture of the composition, the alcohol and the base.
  • the composition and the base can be added to either pure ethanol or pure methanol.
  • the amount of alcohol used may vary with the solubility of the oil or composition containing triglycerides having PUFA residues in the alcohol. Any base known in the art to be suitable for use as a reactant may be used in the present invention.
  • Bases of the formula RO-M, wherein M is a monovalent cation and RO is an alkoxide of a C 1 ⁇ alkyl alcohol are particularly suited for the present invention.
  • suitable bases include elemental sodium, sodium methoxide, sodium ethoxide, potassium methoxide, and potassium ethoxide.
  • the base is sodium ethoxide.
  • the base is typically added in an amount of between about 0.5 and about 1.5 molar equivalents of triglycerides, between about 0.7 and about 1.4 molar equivalents of triglycerides, between about 0.9 and about 1.3 molar equivalents of triglycerides, or between about 1.0 and about 1.2 molar equivalents of triglycerides to the reaction step with the composition and the alcohol.
  • the base is typically added in an amount of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 1.0, 1.01, 1.02, 1.03, 1.04, 1.05, 1.10, 1.15, 1.2, 1.3, 1.4, or 1.5 molar equivalents of triglycerides to the reaction step with the composition and the alcohol.
  • the base is added in an amount of 1.04 molar equivalents of triglycerides to the reaction step with the composition and the alcohol.
  • composition comprising triglycerides having polyunsaturated fatty acid residues, the alcohol and the base are reacted together at a temperature and for an amount of time that allows the production of an ester between the fatty acid residues and the alcohol. Suitable reaction times and temperatures may be determined by one of skill in the art to produce an ester. Without intending to be bound by theory, the PUFA residues are believed to be cleaved from the glycerol backbone of the triglyceride and esters of each PUFA residue are formed during the step of reacting.
  • the step of reacting the composition in the presence of an alcohol and a base is performed at a temperature from about 60° C to about 120° C, from about 70° C to about 110° C, from about 75° C to about 100° C, or from about 80° C to about 90° C. In further embodiments, the step of reacting the composition in the presence of an alcohol and a base is performed at a temperature of about 75° C, 80° C, 85° C, 90° C, or 95° C.
  • the step of reacting the composition in the presence of an alcohol and a base is performed for a time from about 2 hours to about 12 hours, from about 3 hours to about 11 hours, from about 4 hours to about 10 hours, from about 5 hours to about 9 hours, or from about 6 hours to about 8 hours.
  • the step of reacting the composition in the presence of an alcohol and a base is performed for about 5.5, 6, 6.5, 7, 7.5, 8, or 8.5 hours
  • the step of reacting the oil composition, alcohol and base may be conducted by refluxing the components to produce the PUFA esters.
  • the step of reacting the oil composition may be carried out at a temperature that does not result in the refluxing of the reaction components.
  • carrying out the step of reacting the oil composition under pressures greater than atmospheric pressure can increase the boiling point of the solvents present in the reaction mixture. Under such conditions, the reaction can occur at a temperature at which the solvents would boil at atmospheric pressure, but would not result in the refluxing of the reaction components. In some embodiments, the reaction is conducted at a pressure from about 5 to about 20 pounds per square inch (psi); from about 7 to about 15 psi; or from about 9 to about 12 psi. In certain embodiments, the reaction is conducted at a pressure of about 7, 8, 9, 10, 11, or 12 psi. Reactions conducted under pressure may be carried out at the reaction temperatures listed above.
  • reactions conducted under pressure may be carried out at about 70° C, 75° C, 80° C, 85° C, or 90° C.
  • the reaction mixture comprising PUFA esters can be further processed to obtain the PUFA esters from the mixture.
  • the mixture may be cooled, diluted with water, and the aqueous solution extracted with a solvent such as hexane to produce a composition comprising PUFA esters.
  • Techniques for washing and/or extracting crude reaction mixtures are known in the art.
  • PUFA esters are separated from the reaction mixture by distilling the composition to recover a fraction comprising the ester of the polyunsaturated fatty acid.
  • distillation is performed under vacuum.
  • distillation under vacuum allows the distillation to be accomplished at a lower temperature than in the absence of a vacuum and thus may prevent the degradation of the esters.
  • Typical distillation temperatures range from about 120° C to about 170° C.
  • the step of distilling is performed at a temperature of less than about 180° C, less than about 175° C, less than about 170° C, less than about 165° C, less than about 160° C, less than about 155° C, less than about 150° C, less than about 145° C, less than about 140° C, less than about 135° C, or less than about 130° C.
  • Typical pressures for vacuum distillation range from about 0.1 mm Hg to about 10 mm Hg. In some embodiments, the pressure for vacuum distillation is about 0.1, 0.5, 1, 1.5, 2, 2,5, 3, 3.5, or 4 mm Hg.
  • compositions that contain a high percentage of PUFA esters may contain between about 50 wt. % and about 100 wt. % of an ester of a PUFA, and in other embodiments, the composition can comprise at least about 50 wt. %, at least about 55 wt. %, at least about 60 wt. %, at least about 65 wt. %, at least about 70 wt. %, at least about 75 wt. %, at least about 80 wt. %, at least about 85 wt. %, at least about 90 wt. %, at least about 95 wt. %, at least about 99 wt. % of esters of a PUFA.
  • the PUFA esters are subjected to a urea crystallization step.
  • PUFA esters e.g., esters of DHA
  • saturated fatty acid esters formed by the transesterification of a glyceride source using the techniques discussed above, a precipitate forms that comprises the urea and at least a portion of the saturated fatty acid esters.
  • This precipitate comprises a substantially lesser fraction of the PUFA esters than the initial reaction mixture.
  • the bulk of the PUFA esters instead remain in solution and can therefore be easily separated from the precipitated saturated fatty acid esters.
  • the urea crystallization separation process comprises first forming a solution comprising fatty acid esters and urea.
  • the amount of urea preferably is 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 urea is typically about 1:2.
  • the solution also preferably comprises an organic solvent that can solubilize urea and the desired PUFA ester, and more preferably can solubilize urea and all the fatty acid esters in the mixture. Examples of suitable solvents include alkyl alcohols having from 1 to 4 carbons, with methanol and ethanol being more preferred, and ethanol being the most preferred.
  • the volumetric ratio of the mixture of fatty acid esters to solvent is preferably about 1 : 10.
  • urea preferably is dissolved in the solution. This may generally be achieved by heating the solution.
  • the solution preferably is not heated to a temperature above the boiling point of the organic solvent. Typically, the solution is heated to a temperature of about 60 0 C, 65°C, 70 0 C, 75 0 C or 8O 0 C.
  • the PUFA esters are added to the solution.
  • the mixture may be heated until solids dissolve.
  • the solution may be cooled to form a precipitate comprising urea adducts of fatty acid esters.
  • the solution is cooled to a temperature that is from about 0 0 C to about 25 0 C, such as from about 15°C to about 25 0 C.
  • the solution is cooled to a temperature of about 0 0 C about 5°C about 1O 0 C about 15°C, about 2O 0 C, about 25°C, or from about 2O 0 C to about 25°C.
  • a precipitate comprising urea is formed by concentrating the solution.
  • the solution may be concentrated, for example, by evaporating a portion of the solvent in the solution.
  • the amount of solvent removed preferably is sufficient to cause the urea concentration in the solution to exceed the saturation concentration.
  • the solution may be kept in a non-oxidizing atmosphere, such as an atmosphere consisting essentially of a noble gas, N 2 , or a combination thereof, with an atmosphere consisting essentially of N 2 being most preferred.
  • a non-oxidizing atmosphere such as an atmosphere consisting essentially of a noble gas, N 2 , or a combination thereof, with an atmosphere consisting essentially of N 2 being most preferred.
  • Use of such an atmosphere may aid in minimizing oxidation of carbon-carbon double bonds of the PUFA esters.
  • the precipitate may be separated from the liquid fraction enriched in PUFA esters. This may be achieved, for example, by filtration or centrifugation. In one embodiment, the precipitate may be subsequently washed with a small quantity of the organic solvent (preferably saturated with urea) to recover any residual unprecipitated desired PUFA ester that remains with the precipitate. This solvent, in turn, may be combined with the liquid fraction. The liquid fraction may be concentrated, combined with water, and then the esters therein may be extracted with a non-polar solvent from the resulting mixture.
  • the organic solvent preferably saturated with urea
  • the liquid fraction may be concentrated, for example, by evaporating a portion of the solvent from the liquid fraction (the amount of solvent evaporated, however, preferably is not so great as to cause further urea to precipitate).
  • the amount of water subsequently combined with the resulting concentrated liquid fraction may vary widely.
  • the volume ratio of water to concentrated liquid fraction is about 2:1 (in a particularly preferred embodiment, sufficient acid (preferably H 2 SO 4 ) is also introduced to neutralize the urea).
  • the non-polar solvent that may be used to extract the fatty acid esters from the resulting concentrated- mother-liquor/water mixture may be, for example, petroleum ether, pentane, hexane, cyclohexane, ethyl acetate, or heptane, with hexane being the most preferred.
  • the volumetric ratio of the non-polar solvent to the concentrated-mother-liquor/water mixture preferably is about 2:3.
  • the liquid fraction may also be extracted with a slightly polar organic solvent to maximize recovery of the fatty acid esters (which are slightly polar).
  • suitable slightly polar solvents include diethyl ether and ethyl acetate, with diethyl ether being most preferred.
  • the volumetric ratio of slightly polar solvent to the mother-liquor/water mixture is about 2:3. Following the extraction with this slightly polar solvent, the solvent preferably may be combined with the non-polar solvent used in the initial extraction.
  • any residual water may be removed from the extraction solvent by, for example, washing the solvent with brine and/or passing the solvent over an anhydrous salt (e.g., sodium sulfate).
  • the solution then preferably is concentrated by, for example, evaporating a portion of the solvent.
  • the methods of the present invention may be used to purify ethyl arachidonate (arachidonic acid ethyl ester) from a crude Mortierella alpina oil.
  • a crude oil obtained from Mortierella alpina by hexane extraction (typically with an ARA content of about 0.5 g/g oil) can be used directly without any further processing, such as winterization and/or RBD processing.
  • 150 mL of absolute ethanol can be added to 175 g (approximately 0.2 moles) of the crude oil in a one-liter flask under N 2 at room temperature. The mixture can be allowed to stir for 15 minutes to obtain a homogeneous solution.
  • 67 g of a 21% solution of NaOEt/EtOH (approximately 1.04 molar equivalents) can be then added to the solution, and the mixture can be allowed to reflux under N 2 for about 10 hours.
  • the progress of the reaction may be monitored by gas chromatography (GC) and/or thin-layer chromatography (TLC).
  • ethanol When the reaction is completed, approximately 75 mL of ethanol can be removed by distillation, and the mixture can be allowed to cool to room temperature under N 2 .
  • 300 mL of hexane can be added to the cooled mixture, and the mixture can be allowed to stir for 15 minutes at room temperature.
  • 300 mL of deionized water can be then added to the mixture, and the mixture can be allowed to stir for an additional 15 minutes.
  • the aqueous layer After removing and saving the organic layer, the aqueous layer can be washed twice with 300 mL portions of hexane.
  • the combined organic layer can be washed with 200 mL of a saturated NaCl solution.
  • a GC analysis of the organic layer may be used to determine the amount of ARA ethyl ester present in the crude product.
  • approximately 50% of the crude product is ARA ethyl ester, with the remaining materials being predominantly lower molecular weight ethyl esters.
  • the crude product may then be subjected to vacuum fractional distillation or other purification procedures.
  • a purity of greater than about 60% ARA ethyl ester may be achieved following the fractional distillation of the crude product.
  • the methods of the present invention result in the direct transesterification of triglycerides having PUFA residues to produce esters of the PUFAs.
  • Previous methods utilized long reaction times, large amounts of reagents, and subjected the oils to harsh conditions such as high temperatures and highly acidic conditions.
  • the methods disclosed herein thus provide a more efficient and economical purification process that yields a pure product.
  • the methods disclosed herein may be applied to crude oils as well as purified oils, resulting in an additional increase in efficiency and cost savings.
  • compositions produced by the methods described herein include compositions produced by the methods described herein.
  • such compositions can contain greater than about 50 wt. %, greater than about 55 wt. %, etc. of esters of a PUFA.
  • the compositions can contain at least about 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 wt. % of PUFA esters.
  • the compositions may further comprise less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.25 or 0.1 wt. % eicosapentaenoic acid.
  • compositions of the invention may include any PUFA esters as described above, namely, DHA, omega-3 DPA, omega-6 DPA, ARA, SDA, LLA, ALA, GLA, or CLA or combinations thereof.
  • the compositions may comprise ethyl esters.
  • the composition comprises at least about 89 wt. % DHA esters.
  • the composition comprises at least about 89 wt. % of a combination of DHA and DPA esters.
  • compositions of the present invention also include compositions that contain at least about 60, 65, 70, 75, 80, 85, 90, or 95 wt. % ARA esters.
  • the ARA esters may be ethyl esters of ARA.
  • the compositions may further comprise less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.25 or 0.1 wt. % eicosapentaenoic acid.
  • compositions comprising at least about 90 wt. % ethyl ester of docosahexaenoic acid (DHA) and at least about 0.1 wt. % of DHA.
  • DHA docosahexaenoic acid
  • compositions may be produced by the methods disclosed herein.
  • the amount of ethyl ester of DHA in the compositions may be at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99 wt. %.
  • the amount of C28:8 in the compositions may be at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5 wt. %.
  • the C28:8 may be present in triglyceride or ester form.
  • the C28:8 may be present in ethyl ester form.
  • the present invention also provides compositions comprising at least about 90 wt.
  • DHA docosahexaenoic acid
  • n-3 % ethyl ester of docosahexaenoic acid
  • the amount of ethyl ester of DHA in the compositions may be at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99 wt. %.
  • the amount of DPA (n-3) in the compositions may be at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 wt. % of DPA (n-3).
  • the DPA (n-3) may be present in triglyceride or ester form.
  • the DPA (n-3) may be present in ethyl ester form.
  • compositions comprise all three of the ethyl ester of DHA, C28:8 and DPA (n-3) in the concentration ranges specified above. In further embodiments, the compositions may comprise less than about 1.0, 0.9,
  • compositions may comprise less than about 0.25 wt. % EPA.
  • the EPA may be present in triglyceride or ester form.
  • the EPA may be present in ethyl ester form.
  • the compositions may comprise 0 wt. % EPA.
  • the present invention also provides compositions comprising at least about 90 wt. % ethyl ester of docosahexaenoic acid and at least one additional fatty acid or an ester thereof.
  • the amount of ethyl ester of DHA in the compositions may be at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99 wt. %.
  • the additional fatty acid may have a boiling point of about 150-170 0 C at a pressure of 0.8 mm Hg.
  • compositions comprising at least about 70 wt. % ethyl ester of docosahexaenoic acid (DHA) and at least about 25 wt. % ethyl ester of docosapentaenoic acid (n-6).
  • compositions of the present invention also include compositions that comprises at least about 90 wt. % of a combination of ethyl ester of docosahexaenoic acid and ethyl ester of docosapentaenoic acid (n-6).
  • the compositions may comprise at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99 wt.
  • compositions may comprise at least about 10 wt. % ethyl ester of docosahexaenoic acid and at least about 10 wt. % ethyl ester of docosapentaenoic acid (n- 6). Pn other embodiments, the compositions may comprise at least about 15 or 20 wt. % ethyl ester of docosahexaenoic acid and at least about 15 or 20 wt. % ethyl ester of docosapentaenoic acid (n-6).
  • compositions comprising at least about 90 wt. % of a combination of ethyl ester of docosahexaenoic acid and ethyl ester of docosapentaenoic acid (n-6), and at least one additional fatty acid or an ester thereof.
  • the compositions may comprise at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99 wt. % of a combination of ethyl ester of docosahexaenoic acid and ethyl ester of docosapentaenoic acid (n-6).
  • the additional fatty acid may have a boiling point of about 150-170 0 C at a pressure of 0.8 mm Hg.
  • the DHA/DPA (n-6) compositions described above may further comprise less than about 4% of a saturated fatty acid or an ester thereof. In certain embodiments, the compositions may comprise less than about 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.0% or 0.5% of a saturated fatty acid or an ester thereof.
  • the saturated fatty acid or an ester thereof may contain less than 20 carbons, such as, for example, a saturated fatty acid or an ester thereof that contains 19, 18, 17. 16, 15, 14, 13, 12, 11,10, 9 or 8 carbons. In certain embodiments, the saturated fatty acid or ester thereof may contain 14 or 16 carbons.
  • PUFA esters and compositions of the present invention may be used in pharmaceutical products.
  • the pharmaceutical products may contain PUFA esters without an additional pharmaceutically active agent.
  • the pharmaceutical product may comprise a pharmaceutically active agent.
  • pharmaceutically active agents include statins, anti-hypertensive agents, anti-diabetic agents, anti-dementia agents, antidepressants, anti-obesity agents, appetite suppressants and agents to enhance memory and/or cognitive function.
  • the pharmaceutical products may further comprises any pharmaceutically acceptable excipient, carriers, binders or other formulation components known in the art.
  • PUFA esters produced by the methods of the present invention and compositions of the present invention are suitable for use as therapeutic and experimental agents.
  • An embodiment of the present invention comprises the production of PUFA esters for treatment of PUFA-def ⁇ cient infants.
  • the PUFA esters can be included in a parenteral formulation that can be administered to an infant through parenteral routes to fortify the infant's supply of a PUFA.
  • Preferred parenteral routes include, but are not limited to, subcutaneous, intradermal, intravenous, intramuscular and intraperitoneal routes.
  • a parenteral formulation can include PUFA esters of the present invention and a carrier suitable for parenteral delivery.
  • a “carrier” refers to any substance suitable as a vehicle for delivering a molecule or composition to a suitable in vivo site of action.
  • carriers include, but are not limited to water, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solutions, Hank's solution and other aqueous physiologically balanced solutions.
  • Suitable carriers also include oil-based carriers, non-aqueous solutions, suspensions, and emulsions. Examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable organic esters such as ethyl oleate, polyethoxylated castor oil (cremaphor), and others known in the art.
  • Acceptable protocols to administer PUFA esters in an effective manner include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art depending upon a variety of variables, including the weight of the infant and the extent of PUFA deficiency.
  • Another embodiment of the present invention comprises the production of PUFA esters for treatment of adults, in particular pregnant mothers. The product may be used for augmenting long chain PUFA levels in milk of warm-blooded animals.
  • Acceptable protocols for administration of PUFA esters to adults includes parenteral feeding techniques or encapsulating PUFA esters of the present invention in a capsule, such as gelatin (i.e., digestible) capsule, for oral administration and/or in a liquid diet formulation.
  • a liquid diet formulation can comprise a liquid composition containing nutrients suitable for supplementing a diet or nutrients sufficient as a complete diet.
  • PUFA esters produced by the methods of the present invention and compositions of the present invention may also be used to treat subjects (e.g., humans or animals) with high levels of triglycerides, including subjects with triglyceridemia.
  • subjects with fasting triglycerides of 150mg/dL or above may benefit from treatment with the PUFA esters of the present invention, and, additionally, the elevation of post-parandial triglyercides may be reduced by treatment with the PUFA esters of the present invention.
  • individual PUFA esters may be administered to a subject to treat high levels of triglycerides.
  • the PUFA ester may be DHA or ARA.
  • combinations of PUFA esters may be administered to a subject to treat high levels of triglycerides.
  • the combination of PUFA esters may comprise omega-3 and omega-6 PUFAS such as DHA and DPA n-6.
  • the PUFA esters may comprise about 90% of a composition administered to the subject.
  • the PUFA esters may be administered with other components and excipients, such as the carriers described above.
  • the PUFA esters may also be used to treat subjects with diseases that can be associated with high levels of triglycerides, such as cardiovascular disease or hypertension.
  • PUFA esters and compositions of the present invention may be used to treat subjects with neurological disorders, dementia and pre-dementia related conditions.
  • Therapeutic compounds appropriate to use with the PUFA esters and compositions of the present invention include any therapeutic which can be used to protect an individual against any of the conditions or diseases discussed herein, and may include a protein, an amino acid, a drug, other natural products and a carbohydrate. Such therapeutic compounds will be well known to those of skill in the art for the particular disease or condition being treated.
  • Some preferred therapeutic compounds to combine with a composition or formulation of the invention include, but are not limited to: Tacrine (COGNEX); Donepezil (ARICEPT); Rivastigmine (EXELON); Galantamine (REMINYL); Memantine (AKATINOL); Neotropin; Nootropics; Alpha-tocopherol (vitamin E); Selegeline (ELDEPRYL); non-steroidal anti-inflammatory agents (NSAIDS); Gingko biloba; estrogen; ⁇ -secretase inhibitors; vaccines, including lipid or liposome- based vaccines, that dissolve plaques in the brain; B complex vitamins; calcium channel blockers; HMG CoA reductase inhibitors; statins and other anti-cholesterol drugs (e.g., ZOCOR (simvastatin), LIPITOR (atorvastatin calcium), LESCOL (fluvastatin), LOPED (gemfibrozil), or PRAVACHOL (pravastatin sodium)); policosanols
  • PUFA esters and compositions of the present invention can be administered topically or as an injectable, the most preferred route of administration is oral administration.
  • the PUFAs may be administered to individuals in the form of nutritional supplements and/or foods and/or pharmaceutical formulations and/or beverages.
  • a preferred type of food is a medical food (e.g., a food which is in a formulation to be consumed or administered externally under the supervision of a physician and which is intended for the specific dietary management of a disease or condition for which distinctive nutritional requirements, based on recognized scientific principles, are established by medical evaluation.)
  • a medical food e.g., a food which is in a formulation to be consumed or administered externally under the supervision of a physician and which is intended for the specific dietary management of a disease or condition for which distinctive nutritional requirements, based on recognized scientific principles, are established by medical evaluation.
  • the fatty acids are administered to infants as infant formula, weaning foods, jarred baby foods, human milk fortifier and/or infant cereals.
  • any biologically acceptable dosage forms, and combinations thereof, are contemplated by the inventive subject matter.
  • dosage forms include, without limitation, chewable tablets, quick dissolve tablets, effervescent tablets, reconstitutable powders, elixirs, liquids, solutions, suspensions, emulsions, tablets, multilayer tablets, bi-layer tablets, capsules, soft gelatin capsules, hard gelatin capsules, caplets, lozenges, chewable lozenges, beads, powders, granules, particles, microparticles, dispersible granules, cachets, douches, suppositories, creams, topicals, inhalants, aerosol inhalants, patches, particle inhalants, implants, depot implants, ingestibles, injectables, infusions, health bars, confections, cereals, cereal coatings, foods, nutritive foods, functional foods and combinations thereof.
  • a food that is enriched with the desired PUFA is selected from the group including, but not limited to: baked goods and mixes; chewing gum; breakfast cereals; cheese products; nuts and nut-based products; gelatins, pudding, and fillings; frozen dairy products; milk products; dairy product analogs; soft candy; soups and soup mixes; snack foods; processed fruit juice; processed vegetable juice; fats and oils; fish products; plant protein products; poultry products; and meat products.
  • the present invention also includes a method of making any of the above-described compositions of the invention, such as by combining the components of the composition into any suitable delivery form using any suitable method known in the art.
  • the methods of the present invention are suitable for use in an individual that is a member of the Vertebrate class, Mammalia, including, without limitation, primates, livestock and domestic pets (e.g., a companion animal). Most typically, an individual will be a human individual.
  • the term "individual” can be interchanged with the term “subject” or “patient” and refers to the subject of a protocol or method according to the invention. Accordingly, an individual can include a healthy, normal (non-diseased) individual, as well as an individual who has or is at risk of developing pre-dementia or dementia or a symptom or indicator thereof as described herein.
  • the PUFA esters produced by the methods of the present invention may be used to produce PUFA salts.
  • PUFA salts can be produced by reacting the PUFA esters of the present invention in the presence of an alkaline metal base such as an alkaline metal hydroxide (e.g., potassium hydroxide).
  • an alkaline metal base such as an alkaline metal hydroxide (e.g., potassium hydroxide).
  • the PUFA salts formed from the PUFA esters of the present invention can be used in a variety of applications, such as in foods, beverages, and pharmaceuticals.
  • the PUFA salts produced using the PUFA esters of the present invention are water-soluble and can be used directly in foods, beverages, and pharmaceuticals.
  • PUFA esters produced by the methods of the present invention can be used in any animal food material, particularly food materials for humans, to create a food product having enhanced concentrations of PUFAs.
  • the amount of fatty acids naturally in food products varies from one food product to another.
  • a food product of the present invention can have a normal amount of a PUFA or a modified amount of a PUFA. In the former instance, a portion of the naturally occurring lipids may be substituted by PUFA esters of the present invention. In the latter instance, naturally occurring lipids may be supplemented by PUFA esters of the present invention.
  • PUFA esters may be added to foods for infants, such as infant formula and baby food.
  • infant refers to infants and children less than about two years old, including, in particular, premature infants.
  • Certain PUFAs are particularly important component of infant formula and baby food because of the rapid growth of infants (i.e., doubling or tripling in weight during the first year of life).
  • An effective amount of PUFA ester to supplement infant formula is an amount that approximates the concentration of the PUFAs in human breast milk.
  • Preferred amounts of PUFA esters to add to infant formula or baby food range from between about 0.1 to about 1.0% of total fatty acids, more preferably from between about 0.1 to about 0.6% of total fatty acids, and even more preferably about 0.4% of total fatty acids.
  • Another aspect of the present invention includes a food product comprising a food material combined with PUFA esters of the present invention. PUFA esters may be added to a food material to create a food product having enhanced concentrations of PUFAs.
  • the term "food material” refers to any food type fed to humans or non-human animals. Also within the scope of the present invention is a method to make a food product comprising adding PUFA esters produced by methods of the present invention to a food material.
  • a suitable food material useful for the formation of a food product of the present invention includes animal food.
  • animal means any organism belonging to the kingdom Animalia and includes, without limitation, primates (e.g., humans and monkeys), livestock and domestic pets.
  • food product includes any product to be fed to such animals.
  • Preferred food materials to be consumed by humans include infant formula and baby food.
  • Preferred food materials to be consumed by domestic pets include dog foods.
  • PUFA esters produced by methods of the present invention can be added to a wide range of products such as baked goods, vitamin supplements, diet supplements, powdered drinks, etc. at various stages of production. Numerous finished or semi-finished powdered food products can be produced using the compositions of the present invention.
  • a partial list of food products comprising the products of the present invention includes doughs, batters, baked food items including, for example, such items as cakes, cheesecakes, pies, cupcakes, cookies, bars, breads, rolls, biscuits, muffins, pastries, scones, and croutons; liquid food products, for example, beverages, energy drinks, infant formula, liquid meals, fruit juices, multivitamin syrups, meal replacers, medicinal foods, and syrups; semi-solid food products such as baby food, yogurt, cheese, cereal, pancake mixes; food bars including energy bars; processed meats; ice creams; frozen desserts; frozen yogurts; waffle mixes; salad dressings; and replacement egg mixes.
  • baked goods such as cookies, crackers, sweet goods, snack cakes, pies, granola/snack bars, and toaster pastries
  • salted snacks such as potato chips, corn chips, tortilla chips, extruded snacks, popcorn, pretzels, potato crisps, and nuts
  • specialty snacks such as dips, dried fruit snacks, meat snacks, pork rinds, health food bars and rice/corn cakes
  • confectionary snacks such as candy.
  • This example illustrates a method of the present invention for purifying ethyl docosahexaneoate (DHA ethyl ester) from docosahexaneoic acid-containing single cell oil.
  • DHASCO ® -T oil (Martek Biosciences Corporation, Columbia, MD, having a DHA content of 0.4 g/g oil) in a one-liter flask under nitrogen (N 2 ) at room temperature.
  • DHASCO ® -T oil is prepared from the microalgae Crypthecodinium cohnii.
  • the combined organic layer was concentrated under reduced pressure.
  • the crude concentrate was then subjected to vacuum fractional distillation.
  • the lower molecular weight ethyl esters were collected at temperatures between 100 - 150° C and at a pressure of 0.8 mm Hg.
  • the major components of this fraction were oleic, saturated C-14, and C- 12 esters.
  • the DHA ethyl ester was collected at temperatures between 155 - 165° C and at a pressure of 0.8 mm Hg.
  • a GC analysis of the DHA ethyl ester fraction showed a purity of about 91.3% DHA (see Table 1). From the fractional distillation, 68 g (86% yield) of the DHA ethyl ester was obtained as a light yellow oil.
  • Table 1 GC Analyses of DHASCO ® -T Oil Transesterification and Distillation Products
  • This example illustrates a method of the present invention for purifying ethyl docosahexaneoate (DHA ethyl ester) from a crude Crypthecodinium cohnii oil.
  • DHA ethyl ester ethyl docosahexaneoate
  • a crude oil obtained from Crypthecodinium cohnii by hexane extraction (DHA content of 0.5 g/g oil) was used directly without any further processing, such as winterization and/or RBD processing.
  • 150 mL of absolute ethanol was added to 175 g (approximately 0.2 moles of triglycerides) of the crude oil in a one-liter flask under N 2 at room temperature. The mixture was allowed to stir for 15 minutes to obtain a homogeneous solution.
  • 67 g of a 21% solution of NaOEt/EtOH (approximately 1.04 molar equivalents of triglycerides) was then added to the solution, and the mixture was allowed to reflux under N 2 for about 10 hours.
  • the combined organic layer was concentrated under reduced pressure.
  • the crude concentrate was then subjected to vacuum fractional distillation.
  • the lower molecular weight ethyl esters were collected at temperatures between 100 - 150° C and at a pressure of 0.8 mm Hg.
  • the major components of this fraction were oleic, saturated C-14, and C- 12 esters.
  • the DHA ethyl ester was collected at temperatures between 155 -165° C and at a pressure of 0.8 mm Hg.
  • a GC analysis of the DHA ethyl ester fraction showed a purity of about 92% DHA (see Table 2). From the fractional distillation, 69 g (66% yield) of the DHA ethyl ester was obtained as a light yellow oil.
  • This example illustrates a method of the present invention for purifying ethyl docosahexaenoate (as a DHA ethyl ester/DPA ethyl ester mixture) from a crude Schizochytrium sp. oil.
  • a crude oil obtained from Schizochytrium sp. by hexane extraction was used directly without any further processing, such as winterization and/or RBD processing.
  • the combined organic layer was concentrated under reduced pressure.
  • the crude concentrate was then subjected to vacuum fractional distillation.
  • the lower molecular weight ethyl esters were collected at temperatures between 100 - 150° C and at a pressure of 0.8 mm Hg.
  • the major components of this fraction were saturated C-14, and C-16 ethyl esters.
  • the DHA ethyl ester/DPA ethyl ester mixture was collected at temperatures between 155 -170° C and at a pressure of about 0.5 mm Hg.
  • a GC analysis of the DHA/DPA ethyl ester fraction showed a combined purity of about 93% (see Table 3). From the fractional distillation, 85 g (85% yield) of the DHA/DPA ethyl ester was obtained as a very light yellow oil.
  • This example illustrates GC analyses of crude and purified PUFA ethyl esters from Crypthecodinium cohnii oil and Schizochytrium sp. oil.
  • a crude oil obtained from Schizochytrium sp. or Crypthecodinium cohnii by hexane extraction was used directly without any further processing, such as winterization and/or RBD processing.
  • the crude oils were then subjected to a transesterification reaction as described above in Examples 2 and 3.
  • the crude ethyl esters were then subjected to urea adduction as described above, or to distillation as described in Examples 2 and 3.
  • GC analyses were then performed on each sample along with a DPA ethyl ester product or DHA ethyl ester product (Nu-Chek Prep, Inc., Elysian, MN).
  • This example illustrates a method of the present invention for purifying ethyl docosahexaneoate (DHA ethyl ester) from a mixture of fatty acid ethyl esters of docosahexaneoic acid-containing single cell oil via urea crystallization.
  • DHA ethyl ester ethyl docosahexaneoate
  • the mixture was allowed to stand for additional 2 hours at 0 - 4°C.
  • the crystallized urea adduct was then filtered at 0 - 4 0 C.
  • the filtrate was diluted with 300 niL of water and the mixture was acidified with dilute sulfuric acid to a pH of 1 - 2.
  • the acidified solution was extracted with 300 mL x 3 of hexane.
  • the combined hexane extracts were washed with saturated NaCl solution.
  • the washed hexane solution was dried over anhydrous sodium sulfate and concentrated in vacuo to obtain 70 - 75% of theoretical yield.
  • GC analysis showed purity of above obtained DHA Ethyl ester around 90 - 96% (800 - 860 mg/g).

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AU2014202880A1 (en) 2014-06-19
CA2692355A1 (en) 2009-01-08
AU2008269989B2 (en) 2014-02-27
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CA2692355C (en) 2018-09-11

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