(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY(PCT)
`
`(19) World Intellectual Property Organization { A
`
`International Bureau
`
`(43) International Publication Date
`29 November 2007 (29.11.2007)
`
` fe) TANIA MIURATT
`
`(10) International Publication Number
`WO 2007/136281 Al
`
`(51) International Patent Classification:
`
`Notclassified
`
`(21) International Application Number:
`PCT/NZ2007/000122
`
`(22) International Filing Date:
`
`24 May 2007 (24.05.2007)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`547429
`
`24 May 2006 (24.05.2006) NZ
`
`(71) Applicant (for all designated States except US): INDUS-
`TRIAL RESEARCH LIMITED [NZ/NZ]; Gracefield
`Research Centre, 69 Gracefield Road, Lower Hutt (NZ).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): CATCHPOLE,
`Owen John [NZ/NZ]; 14/189 The Terrace, Wellington
`(NZ). GREY, John Bertram [NZ/NZ]; 146 Barnard
`Street, Wadestown, Wellington (NZ). MACKENZIE,An-
`drew Douglas [NZ/NZ]; 27 MonowaiRoad, Johnsonville,
`Wellington (NZ). TALLON,Stephen John [NZ/NZ]; 60
`Glen Road, Stokes Valley, Lower Hutt (NZ).
`
`(74) Agent: BALDWINS; P O Box 852, Wellington (NZ).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH,
`CN, CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES,
`FI, GB, GD, GE, GH, GM, GT, HN, HR, HU,ID,IL, IN,
`IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR,
`LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, Mx,
`MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, PT, RO,
`RS, RU, SC, SD, SE, SG, SK, SL, SM, SV, SY, TJ, TM,
`TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE,ES, FT,
`FR, GB, GR, HU,IE,IS, IT, LT, LU, LV, MC, MT, NL, PL,
`PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM,
`GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`
`with international search report
`before the expiration of the time limit for amending the
`claims and to be republished in the event of receipt of
`amendments
`
`For two-letter codes and other abbreviations, refer to the "Guid-
`ance Notes on Codes and Abbreviations" appearing at the begin-
`ning of each regular issue of the PCT Gazette.
`
`(54) Title: EXTRACTION OF HIGHLY UNSATURATEDLIPIDS WITH LIQUID DIMETHYL ETHER
`
`(57) Abstract: A process for obtaining lipids containing highly unsaturated fatty acids from plant or animal material, including
`contacting the material with liquid dimethyl ether to give a dimethyl ether solution containing lipids and a residue of plant or animal
`material, separating the solution from the residue of plant or animal material, and recovering lipids from the solution.
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`1
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`EXTRACTION OF HIGHLY UNSATURATEDLIPIDS WITH LIQUID DIMETHYL ETHER=eEENARAIPIEIQUIDDIMETHYLETHER
`
`
`
`
`
`TECHNICAL FIELD
`
`the invention relates to the
`In particular,
`This invention relates to separation technology.
`extraction of materials, such as dried or partially dried plants or seeds (including marine or
`terrestrial species), or animal products (including marine or terrestrial species), with liquid
`dimethyl ether (DME), and optionally also with near-critical carbon dioxide, to obtain an extract
`rich in highly unsaturated lipids, especially highly unsaturated complex lipids, and optionally, a
`residue that is useful as a nutraceutical or for extracting watersoluble enzymes and/orproteins.
`
`BACKGROUND
`Highly unsaturatedlipids (lipids having 3 or more sites of unsaturation, and 18 or more carbons
`in the fatty acid chain) have a variety of metabolic roles within the human body. They are
`essential in the development of the brain and eyesight for infants, and may also be beneficial for
`cardiovascular health, mental health, and immune and inflammatory conditions. The biological
`properties of theselipids are usually dependent on the type of fatty acids that are present, and
`those containing highly unsaturated fatty acids are the most bioactive.
`In general, these highly
`unsaturated fatty acids are only found in significant quantities in complex lipids of terrestrial
`plants and animals, but may also appearin both neutral and complex lipids of marine animals.
`
`Phospholipids are a subset of complexlipids. They are essential componenisof all mammalian
`cell membranes,and play an importantrole in maintaining the fluidity of the cell membrane, and
`passage of molecules through the membrane. The highly unsaturated arachidonic acid (C20:4
`w-6) is absent from, or present in very low concentrationsin, secondary products derived from
`animals, such as phospholipids from non-human milk. Arachidonic acid is vital
`for the
`developmentof infants, and so infant formula made from non-human milk is supplemented with
`this fatty acid. There is a need, therefore, to obtain sourcesofthis fatty acid for this purpose.
`The complex lipids of many animal tissues, especially organs and glands,are rich in arachidonic
`acid, as are eggs.
`
`Mosses and ferns are also known to contain high levels of arachidonic acid in complex lipid
`form.
`It is therefore desirable to find an extraction technology which can recover this highly
`unsaturated fatty acid (HUFA) in a complexlipid form, especially since the complexlipid form of
`the fatty acid gives protection against oxidation.
`
`Marine organisms (micro and macro algae, fish flesh, eggs and livers, molluscs, invertebrates)
`are rich sources of the HUFAseicosapentaenoic (C20:5 w-3) and docosahexaenoic acid (C20:6
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`w-3) in neutral and/or complexlipid form. These fatty acids are also required for infant formula
`supplementation, and for use in controlling neurological disorders, cardiovascular disease,
`inflammation, and lipid content in the blood.
`It is also desirable to find an extraction technology
`which can recover these polyunsaturated fatty acids.
`
`Similarly, seeds from certain plants, especially those from pinus and podocarp trees, contain
`complexlipids rich in non-methylene interrupted polyunsaturated fatty acids (C20:3 and C20:4).
`Non-methylene interrupted fatty acids are used for controlling satiety and as possible anti-
`inflammatory agents. There is a need therefore to find an extraction technology which can
`recover these polyunsaturated fatty acids.
`
`The extraction of neutrallipids using supercritical CO2 is well known, especially in the extraction
`of seed oils. A disadvantage of these processes in generalis that large high pressure vessels
`(typically 300 bar or higher pressure is used) are required to contain the raw material, which
`makesthe production plant very expensive. High flow rates and long extraction times are also
`required, as the oils have very low solubility in supercritical CO2 (typically 1 g of oil per 100 g of
`solvent). There are fewer publications concerning the extraction of lipids from marine species.
`US 6,083,536 describes a process for the extraction of non-polarlipids from crude freeze-dried
`mussel powder to give a non-polar lipid fraction useful for the treatment of inflammatory
`conditions.
`Fresh mussel
`is stabilised with tartaric acid prior to freeze-drying and CO,
`extraction. No compositional data of the extract is given, and no complex lipids are extracted,
`as they are insoluble in COz.
`
`US 4,367,178 describes a process for purifying crude soy lecithin by using supercritical CO, to
`extract neutral
`lipids and leave behind insoluble phospholipids,
`thereby concentrating the
`phospholipids
`in the lecithin.
`The crude lecithin had been produced by conventional
`degummingof soy oil. The use of co-solvents such as ethanol to increase the solvent powerof
`supercritical CO2 has been proposed to overcomethelimitations of COp.
`
`EP 1,004,245 A2 describes a process in which dried eggis first extracted with supercritical CO,
`to remove neutrallipids, and is then either extracted with supercritical CO, and an organic co-
`solvent (ethanol) that is a liquid at room temperature or the organic solvent (without CO.) to
`extract the phospholipids. Both options have the disadvantage of incomplete phospholipid
`extraction.
`In addition, both leave solvent residues in the defatted egg powder, which results in
`denaturation of protein. The neutral egg lipids obtained by supercritical CO, extraction have
`negligible levels of highly unsaturated fatty acids, as shown in example 3.
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`Arntfield et al. (JAOCS, 69, 1992, 823 — 825) show that egg proteinis substantially denatured
`after extraction with CO. and methanol as a co-solvent. The use of ethanol with supercritical
`CO, results in incomplete extraction of phospholipids. Phosphatidyl choline is the most readily
`extracted phospholipid, but all other phospholipids have very low or no solubility and are not
`extracted (Teberliker et al, JAOCS, 78, 2002, 115 — 119). Schriener ef al. (Journal of Food
`Lipids, 13, 2006, 36 — 56) show that the majority of highly unsaturated fatty acids in egg yolk
`lipids are in phosphatidyl ethanolamine, which is not extracted in this process.
`
`PCT publication WO 02/092540 discloses medicinal uses ofpolarlipids containing HUFAs, and
`biends of polarlipids with other oils. The extraction method is disclosed as using alcohol and
`centrifugation, but no further details are given.
`It
`is also disclosed that the polarlipid-rich
`fraction could be obtained as a by-productof edible seed oil extraction by the industrial process
`of degumming.
`
`A processfor the extraction of phospholipids containing HUFA from wet phospholipid-containing
`maierial
`is described in PCT publication WO 2005/072477. An aliphatic alcohol, and in
`particular,
`isopropanol and/or n-propanol,
`is used. The material containing phospholipids is
`contacted with a waier soluble aliphatic alcohol at a temperature sufficiently high that the
`phospholipids dissolve in the solvent, while the proteins, which become denatured, precipitate
`from solution.
`
`DMEhaspreviously been usedin the extraction oflipids from raw egg yolk (US 4,157,404) and
`dried egg powder (US 4,234,619). The process causesthefractionation of the lipid and protein
`components into separate streams. US 4,157,404 describes the extraction of lipids from raw
`egg yolk (50-55 % moisture content), but the proteins are denatured in the process. The
`described process also requires that the lipid and water mixture recovered is then dehydrated to
`a water content of 20 % or less, which then results in phase separation of neutral-rich and
`complexlipid/water-rich phases. US 4,234,619 discloses that proteins are not denaturedif the
`egg is dry, but the phospholipids can then only be partially extracted.
`In the processes
`described, DME was used in a temperature range of -30°C to 40°C, spray dried whole egg
`powder was used and only a maximum 70 % yield of phospholipids was obtained. The desired
`product of the invention was an egg powder that contained at
`least 30 % ofits original
`phospholipids content, and no cholesterol. A process for the recovery and concentration of
`highly unsaturated fatty acids is not disclosed. Further, the separation of neutral lipids and
`complexlipids in the totallipid extract into separate fractions was not discovered because of the
`low extraction and separation temperatures used.
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`PCT publication WO 2004/066744 describes the extraction oflipids from an aqueous dairy
`stream using nearcritical extraction where DMEis the solvent. The publication also discloses
`that neither supercritical CO nor liquid DME can extractlipids in useful yields from dry whey
`protein concentrate (WPC) dairy powders. The process does not disclose a method for
`extracting HUFA polarlipids from dry animal or plant tissue. Whey proteins are not found in
`animal orplant tissues, andthelipids obtained do not contain highly unsaturated fatty acids.
`
`NZ 535894 describes the extraction oflipids from spray dried dairy products containing milk fat
`globular membraneproteins, which is a dairy lipoprotein/lipid/lactose mixture arising from the
`production of skim milk powder. The proteins are associated with the cream fraction of milk,
`and are not found in animal orplant tissue. Attempts to extract lipids from this dairy powder
`stream with high lactose contents (where high lactose content means at least 30% by mass of
`the total powder) by extraction using liquid DME were unsuccessful, and it was necessary to
`reduce the lactose content prior to production of the powder. There is no disclosure of a
`method for extracting HUFAlipids from dry animalor plant tissue, because the lipids contain no
`HUFAs. The residual powderafter extraction still contains around 6 — 8 % complexlipids.
`
`PCT publication WO 2006/058382 broadly describes a process for obtaining an extract from a
`range of materials using liquid DME. There is, however, no description of the extraction of
`HUFAs, nor the separation of complex lipids from neutral lipids. The process described is a
`simple conventional process which usesliquid DME.
`Indeed, the sole process described in any
`detail is a process that usesliquid DME for obtaining an extract from Jojoba seeds which do not
`contain HUFAs.
`
`is evident that the type of proteins and other complex carbohydrates present in products
`It
`derived from animal and plant materials (and the method by which the material
`is dried)
`determines whether or notlipids can be successfully extracted. The proteins and complex
`carbohydrates that are presentin plant or animal tissuesdiffer substantially from those found in
`secondary products derived from animals, such as milk.
`It is therefore generally not possible to
`predict with any certainty whether extraction oflipids, and especially complex lipids containing
`highly unsaturated fatty acids,
`is possible from plant or animal tissue containing proteins and
`carbohydrates associated with cells and tissue using dimethyl ether.
`
`liquid DME can be used as an efficient
`the applicant has discovered that
`Surprisingly,
`extractant for obtaining HUFAsfrom plant or animal material, andin particular that residual DME
`in a lipid extract consisting of neutral and complex lipids enables formation of a gum-like phase
`containing complexlipids whichis then easily separated from the neutrallipids.
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`is an object of the invention to provide a process for obtaining lipids containing highly
`It
`unsaturated fatty acids, or at least to provide a useful alternative to other processes.
`
`STATEMENTSOF INVENTION
`
`the invention provides a process for obtaining lipids containing highly
`In a first aspect
`unsaturated fatty acids from plant or animal material, including the steps:
`(i)
`contacting the material with liquid dimethyl ether to give a dimethyl ether solution
`containinglipids and a residue of plant or animal material:
`separating the solution from the residue of plant or animal material; and
`recoveringlipids from the solution.
`
`(ii)
`(iii)
`
`In certain preferred embodiments of the invention, the solution formed after contact with the
`material in step (i) contains neutral lipids and complexlipids.
`
`Preferably the neutral lipids are recovered from the solution together with the complex lipids.
`The neutrallipids are then preferably separated from the complexlipids.
`
`The complex lipids may form a gum phase with dissolved dimethyl ether during the recovery
`Step (iii). Preferably the gum phase containing complexlipids is separated from the solution
`containing neutrallipids.
`
`lipids are separated from the complex lipids by phase separation.
`Preferably the neutral
`Centrifugation may also be used to aid separation. Heating may be used priorto centrifugation.
`The complexlipids are then preferably dried by vacuum drying.
`,
`
`The process of the invention also preferably includes treating the lipids recovered from the
`solution in step(iii) with supercritical CO. according to the following steps:
`(iv)
`contacting the lipids recovered from the solution in step(iii) with supercritical CO. to
`give a COzsolution containing neutrallipids and a residue of complexlipids:
`separating the CO, solution containing neutral lipids from the residue of complex
`lipids; and
`
`(v)
`
`(vi)
`
`recovering the neutrallipids from the COzsolution.
`
`In certain embodiments of the invention, the plant or animal material to be contacted with liquid
`dimethyl etherin step(i) is first treated with near-critical CO, according to the following steps:
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`6
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`a. contacting the material with near-critical CO, to give a CO, solution containing
`neutrallipids and a residue of plant or animal material:
`separating the CO,solution from the residue of plant or animal material; and’
`recovering the neutral lipids from the CO. solution.
`
`b.
`c.
`
`In a preferred process of the invention, the plant or animal material is dried or partially dried
`before use. Preferably the plant or animal material is dried to less than 30 % by weight of water
`in the material, more preferably to not
`less than 5% by weight of water in the material.
`Preferably the plant or animal material is dried by freeze drying or by spray drying.
`
`In certain embodiments of the invention, the plant or animal material is wet biomass that has
`been frozen. Typically, the frozen wet biomassis ground prior to extraction.
`
`Preferably one or more of the complex lipids are phospholipids, gangliosides, glycolipids,
`cerebrosides, or sphingolipids, typically phospholipids. The phospholipids may include any one
`or more
`of
`phosphatidyl
`choline,
`phosphatidyl
`serine,
`phosphatidyl
`ethanolamine,
`
`
`sphingomyelin, cardiolipin,—plasmalogens, alkylacyiphospholipids,|phosphonolipids,
`lysophospholipids, ceramide aminoethylphosphonate and phosphatidic acid. The glycolipids
`may include galactolipids, gangliosides,
`sulphoquinovoysldiacylglycerides,
`tauroglycolipds,
`glycosphingophospholipids, and mannosy|lipids.
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`Preferably the highly unsaturated fatty acids contained in the complex lipids include, but are not
`limited to, any one or more of arachidonic acid (AA), alpha- and gamma-linolenic acid, pinolenic
`acid, sciadonic acid, columbinic acid, dihomolinolenic acid, eicosatetraenoic acid, juniperonic
`acid,
`stearidonic acid, eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and
`docosahexaenoic acid (DHA).
`
`is obtained from any one of the group
`is also preferred that the plant or animal material
`lt
`consisting of animal organs, animal glands, marine macro- and micro-algae,lipid-bearing micro-
`organisms cultured by fermentation, especially filamentous fungi, algae, yeast and bacteria;
`Shellfish, fish, marine invertebrates, eggs, plant seeds, plant leaves, plant needles, fern fronds,
`mossand lichen.
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`In preferred embodiments of the invention, the liquid dimethyl ether is near-critical dimethy!
`ether.
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`7
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`In another aspectthe invention provideslipids containing highly unsaturated fatty acids obtained
`by the processof the invention.
`
`In a further aspect the invention provides complex lipids obtained by the process of the
`invention.
`
`In another aspect the invention provides neutrallipids obtained by the processofthe invention.
`
`In yet another aspect the invention provides plant or animal material from which lipids containing
`highly unsaturated fatty acids have been extracted by the process ofthe invention.
`
`The invention also provides the use of the plant-or animal material, from which complexlipids
`containing highly unsaturated fatty acids have been extracted by the process of the invention,
`as a nutraceutical, a food supplement, or as a source of enzymes.
`
`DETAILED DESCRIPTION
`
`Definitions
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`
`Fatty acid means any saturated or unsaturated aliphatic carboxylic acid typically having a
`hydrocarbon chain of 6 or more carbon atoms. Fatty acids are classified according to the
`numberof carbon atoms (e.g. C20), numberof sites of unsaturation (e.g. C20:4), the position of
`the first site of unsaturation from the methyl end of the fatty acid (e.g. C20:4 w-3), and how
`many carbons separate the sites of unsaturation. Normally one carbon separates the sites of
`unsaturation, (known as “methyleneinterrupted’), and is signified in the shortened nomenclature
`only whenit is conjugated (no carbons separating thesites of unsaturation), orit is separated by
`more than one carbon (known as “non-methylene interrupted”) and the positions of the carbons
`from the methyl end of the fatty acid are noted (e.g. 5,11,14 C20:3).
`Fatty acids are
`constituents of both neutral and complexlipids.
`In neutral lipids, only fatty acids are bound to
`glycerol via an ester or ether bond. Fatty acids can also be present in an unbound state, and
`are then referred to as “free fatty acids”.
`In complex lipids, fatty acids and other (polar)
`constituents are attached to glycerol.
`
`Polyunsaturated fatty acid (PUFA) meansa fatty acid having 2 or moresites of unsaturation.
`
`Highly unsaturated fatty acid (HUFA) means a fatty acid having 3 or more sites of
`unsaturation, and 18 or more carbon atoms in the fatty acid chain. Examples include
`arachidonic acid (AA), alpha- (ALA) and gammaz-linolenic acid (GLA), pinolenic acid, sciadonic
`acid, columbinic acid, dihomolinolenic acid, dihomopinolenic acid, juniperonic acid, stearidonic
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`,
`
`acid, eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid
`(DHA).
`
`Complexlipidsarelipids consisting of at least three building blocks,including fatty acids (and
`closely related ether, amine and hydrocarbon derivatives); a polar phosphorous group (usually a
`phosphate ester or acid), and/or amino alcohols, and/or carbohydrates; and glycerol. Complex
`lipids include, but are notlimited to, phospholipids, gangliosides, glycolipids, cerebrosides, and
`sphingolipids.
`Examples of phospholipids include phosphatidyl choline (PC), phosphatidyl
`serine (PS), phosphatidyl ethanolamine (PE), phosphatidyl inositol (Pl), sphingomyelin (SM),
`cardiolipin (CL), plasmalogens, lysophospholipids, and phosphatidic acid.
`
`Neutrallipids are lipids consisting of one or two building blocks, neither of which contain polar
`phosphorous groupsor carbohydrates. The building blocks includefatty acids, glycerol, sterols,
`fatty alcohols, amines, carotenoids and naturally occurring hydrocarbons. Neutrallipids include,
`but are not
`limited to,
`fatty acids, mono-,
`di- and triacyiglycerides,
`ceramides, N-
`acylethanolamines, sterols and sterol esters, carotenoids and carotenoid esters.
`
`DME-hydrated complex lipid means a complexlipid that has formed a weak association with
`DME, analogousto a lipid hydrated with water molecules.
`
`Critical point means the point at which the liquid and vapour state of a substance become
`identical.
`
`Supercritical means the pressure-temperature region above the critical point of a substance.
`-Above, but close to, the critical point of a substance, the substanceis in a fluid state that has
`properties of both liquids and gases. The fluid has a density similar to a liquid, and viscosity
`and diffusivity similar to a gas.
`
`Subcritical means the pressure-temperature region equal to or above the vapourpressure for a
`substance, but below the critical temperature. The terms “liquefied gas” and “compressed
`liquefied gas” can be used to describe the same region, in which the vapour pressure of the gas
`is at least 3 bar at the extraction temperature.
`
`Near-critical means the pressure-temperature region close to the critical point of a substance,
`and thus includes both subcritical and supercritical.
`Near-critical
`includes the reduced
`temperature range 0.70 < T, < 1.25 (where T,
`is the temperature divided by the critical
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`temperature, T. of the DME); and the pressure ranges P > P, (where P,is the vapour pressure)
`for T < T, and P > P, (where P,is the critical pressure) for T = T,.
`
`Nutraceutical means a productisolated or purified from foods, and generally sold in medicinal
`forms not usually associated with food and demonstrated to have a physiological benefit or
`provide protection against chronic disease.
`
`The Invention
`
`The invention provides a process for obtaining lipids containing highly unsaturated fatty acids
`from plant or animal material, including the steps:
`(i)
`contacting the material with liquid dimethyl ether to give a dimethyl ether solution
`containing lipids and a residue of plant or animal material:
`separating the solution from the residue of plant or animal material; and
`recovering lipids from the solution.
`
`(ii)
`(iii)
`
`The plant or animal material may be any animal tissue or plant tissue that contains lipids having
`HUFAs.
`In particular, the process is directed to animal organs and glands, marine macro and
`microalgae,
`lipid-bearing micro-organisms cultured by fermentation, especially filamentous
`fungi, algae, yeast and bacteria; small marine animals (shellfish and invertebrates), eggs, and
`seeds of plants. The plant or animal tissue may include parts or the whole material of a plant or
`animal that includes cellular material, protein,
`lipid and carbohydrate, but does not
`include
`secondary products derived from plant or animals such as milk.
`
`DMEis a gas at normal room temperatures and pressures, but in liquid form is known to be an
`effective solvent for the extraction of substances from natural products. The liquid DME usedin
`the processofthe invention is typically near-critical DME. Preferably, the pressure of the liquid
`DMEis at least equal to the vapour pressure at the temperature of the extraction, and more
`preferably is at least 10 bar greater than the vapour pressure. The temperature is preferably in
`the range 273 — 373 K, and more preferably in the range 313 — 353 K. Higher extraction
`temperatures give higher yields of complexlipids that are enriched in highly unsaturated fatty
`acids. A typical extraction temperature is approximately 333 K. A typical extraction pressure at
`this temperature is 40 bar, which is sufficiently above the vapour pressure of DME to ensure
`maximum extraction of water if the biomass is wet.
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`The lipids obtained by the process are generally a mixture of complexlipids having a range of
`associated HUFAs. The composition of the mixture will be largely dependent on the source of
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`If the plant or animal material also contains neutrallipids,
`the plant or animal material used.
`then the neutrallipids will also be extracted in the process.
`
`The applicant has discovered that residual DME inalipid extract consisting of neutral and
`complexlipids gives rise to the formation of a gum-like phase containing complexlipids and a
`liquid phase containing neutral
`lipids, providing that the neutral
`lipids do not contain high
`concentrations (more than 5 % by mass) of free fatty acids and/or partial glycerides. The gum
`phase is a semi-solid liquid of higher density than the liquid phase containing neutrallipids.
`It is
`postulated that DME can form a weak association with complexlipids (especially phospholipids)
`similar to that formed between water and phospholipids. The so-called DME-hydrated complex
`lipids in the gum-like phase can easily be separated from the neutrallipids.
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`The use of heat during the recovery of the extract, the ratio of neutral to complexlipids in the
`lipid mixture, and the composition of the neutral lipids are important factors for promoting the
`formation of DME-hydrated complexlipids.
`If the total lipid mixture contains around 50 — 90 %
`neutrallipids without high levels of free fatty acids and/or partial glycerides, and the lipid mixture
`is liquid at room temperature, the process of recovery of the extract, and subsequent degassing
`of DME from the extract by pressure loss and/or heating can give rise to the formation of the
`complex. Separation of the gum-like and liquid phases is accelerated by the use of heating
`and/or centrifuging. The DME-hydrated complex lipid phase thus obtainedstill contains some
`neutral
`lipids, but
`the neutral
`lipid phase is free from complex lipids.
`This discovery is
`particularly applicable to egglipids, and fish headlipids.
`
`Liquid DME can be used to extract both the neutral and complex lipids from both wet or dry
`biomass, giving a mixed extract after separation from the DME. When the biomassis wet,
`waier will also be extracted, and is separated from the lipid by conventional means, such as
`evaporation under vacuum, membrane separation, or phase separation especially by
`centrifuging. There is then the option of further extraction of the mixed extract using near-critical
`carbon dioxide to separate and recover the neutral lipids to give an extract further enrichedin
`complexlipids that contain HUFAs. The complexlipids are not hydrated and do not require
`further processing to remove water or DME.
`
`The plant or animal material may be extracted with near-critical carbon dioxide to remove
`neutral lipids before the extraction with liquid DME. This order of processing steps also enables
`an extract enriched in complex lipids to be obtained.
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`Preferably the near-critical carbon dioxide pressure is at least 73.2 bar and the temperatureis in
`the range 304.2 to 373 K (supercritical region); or the carbon dioxide pressureis greater than or
`equal to the vapour pressure, and the temperature is in the range 273 to 304.1 K (subcritical
`region). More preferably, the carbon dioxide pressure is at least 250 bar, and the temperature
`in the range 313 to 353 K.
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`A key elementof certain embodimentsof the invention is the drying or partial drying of the plant
`or animal material prior to extraction with liquid DME. Plant and animal materials typically have
`a water content of 60-80% by weight of the total material. Removal of at least some of that
`waiter prior to extraction has the practical advantage that for a fixed volume of the material, the
`yields of lipid are larger because the amount of water has been reduced. There is therefore
`either a reduced need for large volume processing apparatus, or a greater throughput andlipid
`yield for a fixed volume processing plant. However, the process is also applicable to wet
`biomass, which can be advantageousin avoiding drying costs, and deactivating enzymes that
`can degradelipids or result in their encapsulation within the dry biomass matrix that prevents
`their extraction.
`
`is advantageous to dry the plant or animal
`The applicant has also importantly found that it
`maierial but not remove the water altogether. When the water content of the material to be
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`extracted is reduced to belowalevel of 30 % by weight of the total material, then the processof
`the invention can be performed without significant degradation or denaturation of enzymes and
`other proteins present
`in the material. The residue of plant or animal material following
`extraction may therefore be particularly useful
`in various applications such as nutritional
`supplements that are enriched in proteins and reduced in fat, for example body building
`products such as defatted bovine liver; as a source of enzymes such as proteases, lipases,
`transglutaminases. Degradation of the enzymes would limit the usefulness of the residue.
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`Complex lipids vary widely in their polarity, and soit is difficult to find a solvent or solvent
`mixture in which the majority of phospholipids present in a plant or animal tissue can be
`extracted.
`It is even moredifficult to find a solvent system which does not denature the residual
`proteins and/or complex non-lipid molecules during the extraction process, so that the defatted
`residual material can be used to extract non-lipid components such as enzymes, or use the
`defatted residual material as a nutraceutical. Surprisingly, the applicant has found that liquid
`DME used at a temperat