NZ571454A

NZ571454A - A process for supercritical extraction and separation of lipid materials

Info

Publication number: NZ571454A
Application number: NZ571454A
Authority: NZ
Inventors: Owen John Catchpole; Stephen John Tallon
Original assignee: Ind Res Ltd
Priority date: 2006-04-20
Filing date: 2006-04-20
Publication date: 2010-02-26

Abstract

The disclosure relates to processes for separating a feed material into soluble and insoluble components, by contacting a feed material and a solvent and subsequently separating the solvent containing the soluble components from the insoluble components, wherein the feed material comprises one or more of: at least 1% by mass phosphatidyl serine, at least 1% by mass sphingomyelin, at least 0.3 % by mass acylalkylphospholipids and/or plasmalogens, at least 0.5 % by mass aminoethylphosphonate and/or other phosphonolipids, at least 1% by mass cardiolipin, and at least 0.3% by mass gangliosides; and wherein the solvent comprises: supercritical or near-critical CO2, and a co-solvent comprising one or more C1-C3 monohydric alcohols, and water, wherein the co-solvent makes up at least 10% by mass of the CO2, and the water content of the co-solvent is 0 to 40 % by mass. The disclosure also relates to processes for separating a feed material into soluble and insoluble components, comprising contacting a feed material and a first solvent and subsequently separating the first solvent containing the first soluble components from the first insoluble components, wherein the feed material comprises one or more of: at least 1 % by mass phosphatidyl serine, at least 1% by mass sphingomyelin, at least 0.3 % by mass acylalkylphospholipids and/or plasmalogens, at least 0.5 % by mass aminoethylphosphonate and/or other phosphonolipids, at least 1% by mass cardiolipin, or at least 0.3% by mass gangliosides; and wherein the first solvent comprises supercritical or near-critical CO2. The process then provides contacting the first insoluble components with a second solvent and subsequently separating the second solvent containing the second soluble components from the second insoluble components, wherein the second solvent comprises supercritical or near- critical CO2, and a co-solvent comprising one or more C1-C3 monohydric alcohols, and water, wherein the co-solvent makes up at least 10% by mass of the CO2, and the water content of the co-solvent is 0 to 40% by mass. (62) Divided Out of 546681

Description

571454 RECEIVED at IPONZ on 29 January 2010 NEW ZEALAND PATENTS ACT, 1953 No: Divided out of NZ 546681 Date: 20 April 2006 COMPLETE SPECIFICATION LIPID EXTRACTION PRODUCT AND PROCESS We, OWEN JOHN CATCHPOLE and STEPHEN JOHN TALLON, both New Zealand citizens, c/- Industrial Research limited, Gracefield Research Centre, Gracefield Road, Lower Hutt, New Zealand, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: 571454 FIELD OF INVENTION This application is a divisional of NZ 546681 5 This invention relates to a separation process. More particularly it relates to a process for separating lipid materials containing phospholipids and/or glycolipids, including for example phosphatidyl serine, gangliosides, cardiolipin, sphingomyelin, plasmalogens, atkylacylphospholipids, phosphonolipids, cerebrosides or a combination thereof.

BACKGROUND Phospholipids are a major component of all biological membranes, and include phosphoglycerides (phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI), cardiolipin (CL), phosphatidyl serine (PS)), plasmalogens (PL), phosphonolipids (PP), alkylacylphospholipids (ALP); and sphingolipids such as 15 sphingomyelin (SM) and ceramide aminoethylphosphonate (CAEP).

Gangliosides are glycolipid components in the cell plasma membrane, which modulate cell signal transductions events. They are implicated as being important in immunology and neurodegenerative disorders. Cerebrosides are important components in animal muscle and nerve cell membranes.

Both phospholipids and gangliosides are involved in cell signalling events leading to, for example, cell death (apoptosis), cell growth, cell proliferation, and cell differentiation.

Reasonable levels of some of these components can be found in milk, soy products, eggs, animal glands and organs, marine animals, plants and other sources. A source of these components is the bovine milk fat globule membrane (MFGM) which is known to contain 25 useful quantities of sphingomyelin, ceramides, gangliosides, and phosphatidyl serine.

Another source of these components is the green-shell mussel, which is known to contain useful quantities of plasmalogens, alkylacylphospholipids and ceramide aminoethylphosphonate Both phospholipids aiid gangliosides have been implicated in conferring a number of health 30 benefits including brain health, skin health, eczema treatment, anti-infection, wound healing, gut microbiota modifications, anti-cancer activity, alleviation of arthritis, improvement of 1 571454 cardiovascular health, and treatment of metabolic syndromes. They can also be used in sports nutrition.

Cardiolipin is an important component of the inner mitochondrial membrane. It is typically present in metabolically active cells of the heart and skeletal muscle. It serves as an 5 insulator and stabilises the activity of protein complexes important to the electron transport chain.

Existing methods for isolation of these compounds rely on the use of chromatographic techniques, which are slow and costly processes to operate. These techniques can also require the use of solvents that are unsuitable and/or undesirable in products for nutritional 10 or human use. For example, Palacios and Wang [1] describe a process for extraction of phospholipids from egg yolks using acetone and ethanol extractions, followed by a methanol/chloroform separation. Kang and Row [2] describe a liquid chromatography process for separation of soybean derived PC from PE and PI. This process may be expensive to carry out on an industrial scale, and also uses hexane, methanol, and isopropyl 15 alcohol as solvents. Kearns et al [3] describe a process for purification of egg yolk derived PC from PE using mixtures of acetonitrile, hydrocarbons, and fluorocarbons. Again, these solvents are undesirable for nutritional or pharmaceutical use.

Supercritical fluid extraction processes using CO2 are becoming increasingly popular because of a number of processing and consumer benefits. CO2 can be easily removed from 20 die final product by reducing the pressure, whereupon die CO2 reverts to a gaseous state, giving a completely solvent free product. The extract is considered to be more 'natural' than extracts produced using other solvents, and the use of CO2 in place of conventional organic solvents also confers environmental benefits through reduced organic solvent use. The disadvantage of supercritical CO2 processing is that the solubility of many compounds in 25 CO2 is low, and only neutral lipids can be extracted.

It is known that the use of CO2 with organic co-solvents such as ethanol allows extraction of some phosphatidyl choline and to a much lesser extent phosphatidyl ethaaolamine. For example, Teberikler et al [4] describe a process for extraction of PC from a soybean lecithin. Using 10% ethanol in CO2 at 60°C they found that PC was easily extracted, while PE and PI 30 were extracted to a very low extent. Extraction at 12.5 % ethanol at 80°C gave a four-fold increase in solubility of PC. Montanari et al [5] describe a process for extracting phospholipids from soybean flakes. After first extracting neutral lipids using only CO2 at 320 bar, they found that using 10 % ethanol co-solvent at pressures of 194 to 689 bar resulted in 2 571454 some extraction of PC, PE, PI, and phosphatidic acid (PA). PC is selectively extracted undei some conditions, but at higher temperatures and pressures some extraction of PE and PI was achieved. The pressures required to achieve good extraction were unpractically high for industrial application, and the high temperatures used (80°C) could cause polyunsaturated 5 fatty acids to be degraded. Taylor et al [6] describe a process in which soybean flakes are first extracted using only C02, followed by CO2 with 15% ethanol at 80°C and 665 bar. A mixture of phospholipids is obtained which were fractionated by alumina column. Again, the temperatures and pressures are too high for practical application. In these works, the soybean-derived feed materials do not contain detectable levels of SM, CL, GS or PS.

Tanaka and Sakaki [7] describe a method for extracting phospholipids from waste tuna shavings using CO2 and ethanol as a co-solvent. They describe extraction of DHA-containing phospholipids using 5 % ethanol in CO2, and by presoaking the tuna flakes in straight ethanol and then extracting using CO2. The phospholipids obtained in this process are not specified and no fractionation of the different phospholipids is described. In addition, the phospholipids fraction makes up a relatively small proportion of the total processed material, requiring use of large pressure vessels to produce a small yield of phospholipids.

Bulley et al [8] describe extraction of frozen egg yolks using CO2 and 3 % ethanol, and CO2 with up to 5 % methanol. Higher rates of triglyceride extraction were obtained with the use of the co-solvent. Extraction of small amounts of phospholipids, up to 17% concentration in 20 the extract, was also achieved. Fractionation of the phospholipids is not described.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents or such sources of information is not to be construed as 25 an admission that such documents or such sources of information, in any jurisdiction, are prior art or form part of the common general knowledge in the art.

It is an object of this invention to provide a process for producing a product that contains desirable levels of particular phospholipids and/or gangliosides and/or cerebrosides, or at least to offer the public a useful choice. 3 RECEIVED at IPONZ on 29 January 2010 571454 SUMMARY OF INVENTION Accordingly the present invention provides a process for separating a feed material into soluble and insoluble components, comprising: (a) providing a feed material comprising one or more of: (i) at least 1% by mass phosphatidyl serine (ii) at least 1% by mass sphingomyelin (iii) at least 0.3 % by mass acylalkylphospholipids and/or plasmalogens (iv)at least 0.5 % by mass ceramide aminoethylphosphonate and/or derivatives thereof (v) at least 1% by mass cardiolipin (vi) at least 0.3% by mass gangliosides (b) providing a solvent comprising: (i) supercritical or near-critical CO2, and (ii) a co-solvent comprising one or more C1-C3 monohydric alcohols, and water wherein the co-solvent makes up at least 10% by mass of the solvent, and the water content of the co-solvent is 0 to 40 % by mass (c) contacting the feed material and the solvent and subsequently separating the solvent containing the soluble components from the insoluble components (d) separating the solvent containing the soluble components from the insoluble components, the soluble components comprising one or more of sphingomyelin, choline-based acylphospholipids, choline-based plasmalogens, and the insoluble components comprising one or more of phosphatidyl serine, non-choline based acylalkylphospholipids, non-choline based plasmalogens, ceramide aminoethylphosphonate, cardiolipin and gangliosides, and (e) optionally separating the soluble components and the solvent. 4 571454 RECEIVED at IPONZ on 29 January 2010 Preferably the feed material comprises greater than 1% phosphatidyl serine. More preferably the feed material comprises greater than 2% phosphatidyl serine. Most preferably the feed material comprises greater than 5% phosphatidyl serine.

Alternatively the feed material comprises greater than 1% sphingomyelin. More preferably 5 the feed material comprises greater than 5% sphingomyelin. Most preferably the feed material comprises greater than 15% sphingomyelin.

Alternatively the feed material comprises greater than 1% cardiolipin. More preferably the feed material comprises greater than 2% cardiolipin. Most preferably the feed material comprises greater than 5% cardiolipin.

Alternatively the feed material comprises greater than 0.3% gangliosides. More preferably the feed material comprises greater than 1% gangliosides. Most preferably the feed material comprises greater than 2% gangliosides.

Alternatively the feed material comprises greater than 0.5% acylalkyphospholipids and/or plasmalogens. More preferably the feed material comprises greater than 2% 15 acylalkyphospholipids and/or plasmalogens. Most preferably the feed material comprises greater than 10% acylalkyphospholipids and/or plasmalogens.

Alternatively the feed material comprises greater than 0.5% ceramide aminoethylphosphonate and/or derivatives thereof. More preferably the feed material comprises greater than 5% ceramide aminoethylphosphonate and/or derivatives thereof. Most preferably the feed 20 material comprises greater than 20% ceramide aminoethylphosphonate and/or derivatives thereof.

The present invention also provides a process for separating a feed material into soluble and insoluble components, comprising (a) providing a feed material comprising one or more of: (i) at least 1% by mass phosphatidyl serine, (ii) at least 1% by mass sphingomyelin, (iii) at least 0.3 % by mass acylalkylphospholipids and/or plasmalogens 571454 RECEIVED at IPONZ on 29 January 2010 (iv) at least 0.5 % by mass ceramide aminoethylphosphonate and/or derivatives thereof (v) at least 1% by mass cardiolipin, or (vi) at least 0.3% by mass gangliosides (b) providing a first solvent comprising supercritical or near-critical CO2 (c) contacting the feed material and the first solvent and subsequently separating the first solvent containing the first soluble components from the first insoluble components (d) optionally separating the first soluble components and the first solvent (e) providing a second solvent comprising: (i) supercritical or near-critical CO2, and (ii) a co-solvent comprising one or more C1-C3 monohydric alcohols, and water wherein the co-solvent makes up at least 10% by mass of the solvent and the water content of the co-solvent is 0 to 40% by mass (f) contacting the first insoluble components and the second solvent (g) separating the second solvent containing the second soluble components from the second insoluble components, the second soluble components comprising one or more of sphingomyelin, choline-based acylphospholipids, choline-based plasmalogens, and the second insoluble components comprising one or more of phosphatidyl serine, non-choline based acylalkyl phospholipids, non-choline based plasmalogens, ceramide 20 aminoethylphosphonate, cardiolipin and gangliosides. (h) optionally separating the second soluble components and the second solvent.

Preferably the first solvent comprises a mixture of supercritical or near-critical CO2 and less than 10% C1-C3 monohydric alcohol.

The feed material preferably comprises greater than 1% phosphatidyl serine. More preferably 25 the feed material comprises greater than 2% phosphatidyl serine. Most preferably the feed material comprises greater than 5% phosphatidyl serine. 6 RECEIVED at IPONZ on 29 January 2010 571454 Alternatively the feed material comprises greater than 1% sphingomyelin. Preferably the feed material comprises greater than 5% sphingomyelin. More preferably the feed material comprises greater than 15% sphingomyelin.

Alternatively the feed material comprises greater than 1% cardiolipin. Preferably the feed 5 material comprises greater than 2% cardiolipin. More preferably the feed material comprises greater than 5% cardiolipin.

Alternatively the feed material comprises greater than 0.3% gangliosides. Preferably the feed material comprises greater than 1% gangliosides. More preferably the feed material comprises greater than 2% gangliosides.

Alternatively the feed material comprises greater than 0.5% acylalkyphospholipids and/or plasmalogens. Preferably the feed material comprises greater than 2% acylalkyphospholipids and/or plasmalogens. More preferably the feed material comprises greater than 10% acylalkyphospholipids and/or plasmalogens.

Alternatively the feed material comprises greater than 0.5% ceramide aminoethylphosphonate 15 and/or derivatives thereof. Preferably the feed material comprises greater than 5% aminoethylphosphonate and/or derivatives thereof. More preferably the feed material comprises greater than 20% ceramide aminoethylphosphonate and/or derivatives thereof.

The feed material of the present invention may be derived from terrestrial animals, marine animals, terrestrial plants, marine plants, or micro-organisms such as microalgae, yeast and 20 bacteria. Preferably the feed material is derived from sheep, goat, pig, mouse, water buffalo, camel, yak, horse, donkey, llama, bovine or human.

Optionally the feed material is selected from: tissue, a tissue fraction, organ, an organ fraction, milk, a milk fraction, colostrum, a colostrum fraction, blood and a blood fraction.

Preferably the feed material is derived from dairy material, soy material, eggs, animal tissue, 25 animal organ or animal blood. More preferably the feed material is selected from: a composition comprising dairy lipids, a composition comprising egg lipids, and a composition comprising marine lipids.

Most preferably the feed material used in the process of the present invention is a bovine milk fraction. Preferably the feed material is selected from: buttermilk, a buttermilk fraction, beta 7 571454 RECEIVED at IPONZ on 29 January 2010 serum, a beta serum fraction, butter serum, a butter serum fraction, whey, a whey fraction, colostrum, and a colostrum fraction.

The feed material may comprise milk fat globule membrane.

Preferably, the feed material is in solid form. When solid, the feed material may be 5 cryomilled before contact with the solvent.

The solvent of the present invention preferably comprises: (a) an alcohol selected from: methanol, ethanol, n-propanol, isopropanol and mixtures thereof; and (b) 0 ~ 40% v/v water More preferably the solvent comprises between 0 and 20% v/v water. Most preferably the solvent comprises between 1 and 10% v/v water.

Preferably the alcohol is ethanol.

Preferably the solvent used in the process of the present invention comprises 95% aqueous ethanol.

Preferably the mass fraction of the co-solvent in CO2 is between 5% and 60%. More preferably the mass fraction is between 20% and 50%. Most preferably the mass fraction is between 25% and 30%.

Preferably the contacting temperature between the feed material and solvent is between 10°C and 80°C. More preferably the contacting temperature is between 55°C and 65°C. Most 20 preferably the contacting pressure is between 100 bar and 500 bar.

Preferably the contacting pressure is between 200 bar and 300 bar. More preferably the ratio of the co-solvent to feed material is in the range 10:1 to 200:1. Most preferably the ratio of the co-solvent to feed material is in the range 15:1 to 50:1.

Preferably the separating pressure is between atmospheric pressure and 90 bar. More 25 preferably the separating pressure is between 40 bar and 60 bar.

Preferably the co-solvent is recycled for further use. 8 571454 RECEIVED at IPONZ on 29 January 2010 Preferably the CO2 is recycled for further use.

The co-solvent may be removed by evaporation under vacuum.

Preferably the feed material is contacted with a continuous flow of solvent.

Preferably the feed material is contacted with one or more batches of solvent.

The lipid and solvent streams may be fed continuously.

Optionally, the feed material and co-solvent streams may be mixed prior to contacting with co2.

The invention also provides products produced by the process of the invention, both the insoluble components remaining after contact with the solvent (also referred to herein as the 10 "residue"); and the soluble components that are dissolved in the solvent after contact with the feed material (also referred to herein as the "extract"). Where the feed material is contacted with more than one batch of solvent, or the solvent is cooled in a number of steps, there will be multiple "extract" products.

Preferably the product contains more sphingomyelin than the feed material. More preferably 15 the product comprises greater than 3% sphingomyelin. Even more preferably the product comprises greater than 10% sphingomyelin. Most preferably the product comprises greater than 15% sphingomyelin.

Preferably the product contains more phosphatidyl serine than the feed material. More preferably the product comprises greater than 5% phosphatidyl serine. Even more preferably 20 the product comprises greater than 30% phosphatidyl serine. Most preferably the product comprises greater than 70% phosphatidyl serine.

Preferably the product contains more gangliosides than the feed material. More preferably the product comprises greater than 2% gangliosides. Even more preferably the product comprises 9 571454 RECEIVED at IPONZ on 29 January 2010 greater than 4% gangliosides. Most preferably the product comprises greater than 6% gangliosides.

Preferably the product contains more cardiolipin than the feed material. More preferably the product comprises greater than 5% cardiolipin. Even more preferably the product comprises 5 greater than 10% cardiolipin. Most preferably the product comprises greater than 25% cardiolipin.

Preferably the product contains more acylalkyphospholipids and/or plasmalogens than the feed material. More preferably the product comprises greater than 5% acylalkyphospholipids and/or plasmalogens. Even more preferably the product comprises greater than 10% 10 acylalkyphospholipids and/or plasmalogens. Most preferably the product comprises greater than 25% acylalkyphospholipids and/or plasmalogens.

Preferably the product contains more ceramide aminoethylphosphonate and/or derivatives thereof than the feed material. More preferably the product comprises greater than 5% ceramide aminoethylphosphonate and/or derivatives thereof. Even more preferably the 15 product comprises greater than 10% ceramide aminoethylphosphonate and/or derivatives thereof. Most preferably the product comprises greater than 25% ceramide aminoethylphosphonate and/or derivatives thereof. 9a 571454 BRIEF DESCRIPTION OF THE DRAWINGS The invention may be more fully understood by having reference to the accompanying drawings wherein: Figure 1 is scheme drawing illustrating a preferred process of the current invention. 5 Figure 2 is a scheme drawing illustrating a second preferred process of the current invention Figure 3 is a scheme drawing illustrating a third preferred process of the current invention Figure 4 is a scheme drawing illustrating a fourth preferred process of the current invention ABBREVIATIONS AND ACRONYMS In this specification the following are the meanings of the abbreviations or acronyms used.

"CL" means cardiolipin "PC" means phosphatidyl choline "PI" means phosphatidyl inositol 15 "PS" means phosphatidyl serine "PE" means phosphatidyl ethanolamine "PA" means phosphatide acid "PL" means plasmalogen "PP" means phosphonolipid 20 "ALP" means alkylacylphospholipid "SM" means sphingomyelin "CAEP" means ceramide aminoethylphosphonate "GS" means ganglioside "N/D" means not detected 25 "CO2" means carbon dioxide 571454 GENERAL DESCRIPTION OF THE INVENTION As discussed in the Background, it is known that supercritical CO2 with up to 12.5% ethanol as a co-solvent can extract the phospholipids PC, and to a much lesser extent, PE and PI 5 from soy or egg. Surprisingly, we have found that the phospholipids PS, CAEP and CL; and gangliosides are virtually insoluble in C02and a C1-C3 monohydric alcohol co-solvent, and that SM, ALP, PL and PP are soluble. Therefore it is possible to separate the soluble phospholipids from the insoluble phospholipids and gangliosides to achieve fractions enriched in one or other of the desired components.

There are a number of factors affecting the operation of the process: ■ Feed material and feed preparation ■ Extraction temperature and pressure 15 ■ Co-solvent concentration * Total solvent throughput ■ Solvent flow rate and contacting conditions It is advantageous to start with a feed material containing at least 5 % by mass of lipids, and 20 ideally at least 2 % by mass of phospholipids, particularly PS, SM, CL, ALP, PL, PP, CAEP and/or gangliosides.

Hie feed material can be processed using pure CO2 before the co-solvent is introduced to remove much or all of neutral iipids. This reduces the neutral lipid content in the CO2+CO-25 solvent extract leading to an extract enriched in soluble phospholipids and/or gangliosides.

The form of the feed material depends on the source of the lipids and its lipid composition. For example dairy lipid extracts high in phospholipids may be substantially solid even at elevated temperatures. Egg yolk and marine lipids in comparison have a lower melting point. 3 0 The presence of neutral lipids also tends to produce a more fluid feed material. To promote good contacting it may be beneficial to prepare the feed material. Solid materials containing lipids may be able to be cryomilled. Lipid feed materials can also be made more fluid by the inclusion of some ethanol or water. 11 571454 Changing the processing conditions of temperature, pressure, co-solvent concentration, and total solvent usage, influences the amount of material extracted, the purity of the final product, and the recovery (or efficiency) of the process. For example, the virtually insoluble lipids such as PS, GS, CAEP and CL, have very slight solubilities so that excessive use of 5 solvent, or very favourable extraction conditions, can result in small losses of PS, GS and CL from the residual fraction. A high purity product may be achieved, but with a reduced yield. Conversely the enrichment of soluble lipids will be greater if smaller amounts of the other lipids are co-extracted, but the total yield will be lower. Processing economics, and the relative values of the products, will determine where this balance lies. A further option to 10 obtain multiple enriched fractions is to carry out extractions under progressively more favourable extraction conditions, such as increasing the temperature.

We have found that co-solvent concentrations below about 10% produce very little extract of phospholipids and/or gangliosides. At higher concentrations the rate of material extracted 15 increases rapidly. We have found the co-solvent concentrations of at least 20%, and more preferably 30% achieve high levels of extraction of PC, PE, SM, ALP, PL, PP and PI, while the lipids PS, CL and GS remain virtually insoluble.

Every substance has its own "critical" point at which the liquid and vapour state of the 20 substance become identical. Above but close to the critical point of a substance, the substance is 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. The term "supercritical" as used herein refers to the pressure-temperature region above the critical point of a substance. The term "subcritical" as used herein refers to the pressure-temperature 25 region equal to or above the vapour pressure for the liquid, but below the critical temperature. The term "near-critical" as used herein encompasses both "supercritical" and "subcritical" regions, and refers to pressures and temperatures near the critical point.

Percentages unless otherwise indicated are on a w/w solids basis.

The term "comprising" as used in this specification means "consisting at least in part of'. When interpreting each statement in this specification (hat includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner. 12 571454 The invention consists in the foregoing and also envisages constructions of which the following gives examples only.

EXAMPLES The experimental process is described, with reference to figure 1, as follows.

A measured mass of feed material containing lipids to be fractionated was placed in basket BK1 with a porous sintered steel plate on the bottom. Basket BfCI was placed in a 300 mL extraction vessel EX1. The apparatus was suspended in heated water bath WB1 and 10 maintained at a constant temperature through use of a thermostat and electric heater.

In the continuous extraction mode of operation, liquid CO2 from supply bottle B1 was pumped using pump PI into extraction vessel EX1 until the pressure reached the desired operating pressure, after which valve V1 was operated to maintain a constant pressure in the 15 extraction vessel. After passing through valve V1, the pressure was reduced to the supply cylinder pressure of 40 to 60 bar, which caused the CO2 to be converted to a lower density fluid and lose its solvent strength. Precipitated material was captured in separation vessel SEP1, and the CO2 exited from the top of separator SEP1 and was recycled back to the feed pump through coriolis mass flow meter FM1 and cold trap CT1 operated at -5°C, Extracted 20 material was collected periodically from separator SEP1 by opening valve V2. The extraction was optionally carried out using CO2 only until all of the compounds soluble in CO2 only, such as neutral lipids, were extracted. When no further extract was produced by CO2 extraction, ethanol co-solvent with or without added water was added to the CO2 at the desired flow ratio from supply bottle B2 using pump P2. Ethanol and further extracted 25 material were separated from the CO2 in separator SEP1 and periodically removed through valve V2. After the desired amount of ethanol had been added the ethanol flow was stopped and the CO2 flow continued alone until all the ethanol had been recovered from the system. The remaining CO2 was vented and the residual material in basket BK1 was removed and dried under vacuum. The extract fraction was evaporated to dryness by rotary evaporation, In the batch extraction mode of operation CO2 alone was optionally passed continuously through the apparatus, as for the continuous flow mode of operation, until all CO2 alone extractable material was removed. The CO2 flow was then stopped and valve VI closed to maintain the pressure. Approximately 140g of ethanol was pumped from supply bottle B2 13 571454 through pump P2 into extraction vessel EX1. The system was left for 15 minutes to allow the system to equilibrate, after which time the CO2 flow was started and valve VI opened to maintain a constant pressure and allow ethanol and dissolved compounds to flow through to separator SEP1. This process was repeated twice more, after which the C02 was vented and 5 the residual material in basket BK1 was removed and dried under vacuum.

Extract and residue fractions were analysed for phospholipid content and profile by 31P-NMR. The phospholipid mass fractions reported here are for phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylethanolamine (PE), plasmalogens (PL), 10 phosphonolipids (PP), alkylacylphospholipids (ALP), sphingomyelin (SM), ceramide aminoethylphosphonate (CAEP), phosphatidylserine (PS), and cardiolipin (CL).

The process option illustrated in Figure 1 is for a batch process while the processing options illustrated in Figures 2-4 are for a continuous flow process.

Example 1: Fractionation of dairy lipid extract A, ethanol mass fraction 25% Lipid extract A is a total lipid extract obtained by a processes disclosed in PCT international applications PCT/NZ2005/000262 (published as WO 2006/041316). 40g of dairy lipid extract A, with composition shown in Table 1 (feed), was extracted using the continuous extraction mode of operation at 60°C and 300 bar. Hie 'other compounds' consist mainly of neutral lipids. 44% of the feed material was extracted (extract 1) using CO2 only. This extract contained no phospholipids, and was entirely neutral lipids. A further 31% of the feed material (extract 2) was extracted using 95% aqueous ethanol at a concentration 25 in CO2 of 25%. The total ethanol and water added was 880g. The composition of the fraction extracted with CO2 and ethanol (extract 2), and the composition of the residual fraction are shown in Table 1. The extract is enriched in phosphatidylcholine (PC) and sphingomyelin (SM) which are more soluble in CO2 and ethanol, while the residual fraction is substantially enriched in phosphatidylserine (PS). Phosphatidylserine levels are virtually undetectable in 30 the extract phase indicating very low solubility in CO2 and ethanol, and almost complete recovery of phosphatidylserine in the residue phase. 14 571454 Table 1 Yield % of feed Composition. % PC PI PS PE SM Other Phospholipids Other compounds Feed 11.2 2.8 4.3 13.2 7.8 2.2 58.3 Extract 2 31 28.2 0.0 0.2 14.4 .4 4.9 37.0 Residue 6.5 .5 .6 .8 .2 3.6 22.8 Example 2: Fractionation of dairy lipid extract A, ethanol mass fraction 31% 41g of dairy lipid extract A, with composition as for example 1 was extracted using the 10 continuous extraction mode of operation at 60°C and 300 bar as for example 1, using firstly CO2 alone to extract 50 % of the feed material (extract 1), which is neutral lipids only, and then using 95% aqueous ethanol at a concentration in CO2 of 31%. 33% of the feed material was extracted (extract 2). The total ethanol and water added was 1150g. The composition of the residual fraction is shown in Table 2. The higher ethanol concentration gives a more 15 complete extraction of lipids and the concentration of phosphatidylserine in the residue fraction is higher than found in example 1 at 19.3 %.

Table 2 Yield % of feed Composition, % PC PI PS PE SM Other Phospholipids Other compounds Feed 11.2 2.8 4.3 13.2 7.8 2.2 58.3 Extract 2 33 - - - _ - - - Residue 17 4.4 12.6 19.3 27.1 8.5 2.5 .5 Example 3: Fractionation of dairy lipid extract A, ethanol mass fraction 43% 40g of dairy lipid extract A, with composition as for example 1 was extracted using the 25 continuous extraction mode of operation at 60°C and 300 bar as for example 1, using firstly CO2 alone to extract 41 % of the feed material (extract 1), which is neutral lipids only, and then using 95% aqueous ethanol at a concentration in CO2 of 43% to extract 32 % of the feed (extract 2). The total ethanol and water added was 960g. The composition of extract 2 and residual fractions are shown in Table 3. The concentration of phosphatidylserine in the 30 residue fraction is higher than found in example 1 and example 2 at 20.7 %. The 571454 concentration of SM in the extract, at 12.5 % by mass, is enriched relative to the feed, at 7.8 % by mass, even though it also contains a high level of neutral lipids.

Table 3 Yield % of feed Composition, % PC PI PS PE SM Other Phospholipids Other compounds Feed 11.2 2.8 4.3 13.2 7.8 2.2 58.3 Extract 2 32 21.1 0.0 0.5 13.3 12.5 3.5 49.1 Residue 27 4.2 13.6 .7 26.7 7.8 1.9 .0 Example 4: Fractionation of dairy lipid extract A, 40°C 39g of dairy lipid extract A, with composition as for example 1 was extracted using the continuous extraction mode of operation at 300 bar using firstly CO2 alone to extract 54 % of 10 the feed material (extract 1), which is neutral lipids only, and then using 95% aqueous ethanol at a concentration in CO2 of 30 % to extract 12 % of the feed (extract 2). The temperature in this example was 40°C. The total ethanol and water added was 975g. The composition of the extracted and residual fractions are shown in Table 5. The degree of extraction of SM is lower than for examples 1 to 3 at 60°C, but the concentration in the 15 extract is higher. The concentration of PS in the residue, at 12.4 %, is lower than examples 1 to 3.

Table 4 Yield % of feed Composition, % PC PI PS PE SM Other Phospholipids Other compounds Feed 11.2 2.8 4.3 13.2 7.8 2.2 58.3 Extract 2 12 27.9 0.0 0.3 16.7 .3 4.9 34.9 Residue 34 9.9 8.1 12.4 .3 122 3.6 28.5 Example 5: Fractionation of dairy phospholipid concentrate 40g of a dairy phospholipid concentrate with composition as shown in Table 5 (feed) was extracted using the continuous extraction mode of operation at 300 bar and 60°C without the prior CO2 only extraction step. The ethanol (95% aqueous ethanol) mass fraction in CO2 was 25 30%. The total ethanol and water added was 1026g. The composition of the extracted and residual fractions are shown in Table 5. Only 11% of the feed lipid was extracted, so the enrichment of phosphatidylserine in the residue is not significant, but the concentration did increase from 8% to 8.8%. The poor degree of extraction in this example is due to the 16 571454 physical properties of the solid feed material limiting mass transfer. In comparison, the dairy lipid extract in examples 1 through 4, is liquid at the processing temperature and better extraction rates are observed.

Different feed preparation methods and/or longer equilibration times and/or greater solvent quantities are expected to increase the amount of extractable material.

Table 5 Yield % of feed Composition, % PC PI PS PE SM Other Phospholipids Other compounds Peed .4 .3 8.0 21.6 .1 0.3 34.3 Extract 11 Residue 89 13.0 .9 8.8 21.4 .9 2.8 37.2 Example 6; Fractionation of dairy phospholipid concentrate using the batch extraction process 19g of a dairy phospholipid concentrate with composition as described in example 5 was extracted using die batch extraction mode of operation at 300 bar and 60°C. A total of 22% 15 of the feed mass was extracted in three sequential extractions each consisting of 140g of ethanol (95% aqueous ethanol) in 300mL of CO2. The composition of the extracted and final residual fractions are shown in Table 6. In this example 22% of the feed lipid was extracted, significantly higher than that obtained in the continuous extraction example (example 5) and using a lower total quantity of ethanol co-solvent. The phosphatidylserine concentration in 20 the residue has increased from 8% to 11.2%; and the sphingomyelin concentration in the extract has increased from 15.1 to 16.7 %. This example shows the increase in total extracted material by allowing a greater contacting time to more completely dissolve the soluble fraction.

Table 6 Yield % of feed Composition, % PC PI ' PS PE SM Other Phospholipids Other compounds Feed .4 .3 8.0 21.6 .1 0.3 34.3 Extract 22 32.4 0.5 0.4 17.7 16.7 4.7 27.5 Residue 78 13.6 7.4 11.2 26.6 13.3 2.9 .0 17 571454 Example 7: Fractionation of dairy lipid extract B, ethanol mass fraction 10% This example relates to extraction of dairy lipid extract B, a total lipid extract obtained from 5 high fat whey protein concentrate processes disclosed in PCT international applications PCT/NZ2004/000014 (published as WO W02004/066744). with composition shown in Table 7 (feed). The 'other compounds' listed include 2-3% gangliosides and about 3% lactose, both absent in dairy lipid extract A. In this example 42g of dairy lipid extract B was extracted using the continuous extraction mode of operation at 10 300 bar and 60°C. 52% of the feed mass was extracted using C02 alone (extract 1). Only 3% of the feed lipid was further extracted using 460g of 95% aqueous ethanol (extract 2), and the extract contained less than 10% phospholipids. The extraction of phospholipids does not occur to any significant extent for ethanol mass fractions of 10% or lower. The ethanol does however extract some additional neutral lipid that is not extracted using CO2 alone. In this 15 case, both the PS and SM are enriched in the residue.

Table 7 Yield % of feed Composition, % PC PI PS PE SM Other Phospholipids Other compounds Feed 7.4 2.5 3.9 .3 .7 1.3 69.0 Extract 2 3 4.5 0.0 0.0 1.6 1.0 0.3 92.6 Residue 45 .0 6.1 8.7 21.8 12.0 .9 .7 Example 8: Fractionation of dairy lipid extract B, ethanol mass fraction 30% In this example 40g of dairy lipid extract B was extracted using the continuous extraction 25 mode of operation at 300 bar and 60°C. 51 % of the feed mass was extracted using CO2 alone (extract 1). A further 7% of the feed material was extracted using 760g of 95% aqueous ethanol at a mass concentration of 30% in CO2 (extract 2). Phospholipid profiles for the extract and residual fractions are shown in Table 8. Both PS and SM are enriched in the residue 18 571454 Table 8 Yield % of feed Composition, % PC PI PS PE SM Other Phospholipids Other compounds Feed 7.4 2.5 3.9 .3 .7 1.3 69.0 Extract (2) 7 22.5 0.5 0.4 14.0 11.2 3.3 48.2 Residue 41 12.0 .5 8.5 .2 .0 2.4 41.5 Example 9: Fractionation of dairy lipid extract A, ethanol mass 5 fraction 3% This example shows that when the co-solvent concentration is below 10% by mass, no phospholipids are extracted.

In this example 27g of dairy lipid extract A, as described in example 1, was extracted using the continuous extraction mode of operation at 300 bar and 60CC, using 98% ethanol at 3 % by mass ratio with CO2, without the CO2 only extraction step. 62% of the feed mass was extracted. No detectable phospholipids were extracted. This extract represents 90% of the neutral lipid present in the feed material. The rate of extraction of neutral lipid from the feed 15 material was substantially faster using the ethanol co-solvent than using CO2 only. The extract material was substantially extracted using less than the total of 150g of ethanol in 4850g of CO2 used, while typically 10 kg of CO2 alone is required for extraction of neutral lipids, as in example 1.

Example 10: Fractionation of egg yolk lecithin This example relates to fractionation of a commercially available egg yolk lecithin, with phospholipid profile shown in Table 9. No phosphatidylserine was detected in the feed lipid, indicating concentration levels <0.5%. In this example 34g of the feed material was 25 extracted using the continuous extraction mode of operation at 300 bar and 60°C, and 95% aqueous ethanol at a concentration of 25%. 45% of the feed mass was extracted as neutral lipids using C02 alone. A further 49% of the feed material was extracted using ethanol and CO2 with a total ethanol flow of640g. Phospholipid profiles for the extract and residual fractions are shown in Table 9. In this example, the phosphatidylserine levels in the residual 30 material are substantially enriched compared with non-detectable levels in the feed material. 19 571454 Table 9 Yield Vo of feed Composition, % PC PI PS PE SM Other Phospholipids Other compounds Feed 56.4 N/D N/D 6.4 2.0 .7 29.4 Extract 49 43.5 N/D N/D 9.2 2.6 2.1 42.5 Residue 6 17.4 8.0 .9 19.1 3.8 3.8 42.0 Example 11: Fractionation of egg yoik phospholipid extract This example relates to fractionation of an egg yolk phospholipid fraction with phospholipid profile shown in Table 9. In this example 40g of the feed material was extracted using die continuous extraction mode of operation at 300 bar and 60°C, and 95% aqueous ethanol at a concentration of 28%. 50% of the feed mass was extracted as neutral lipids using CO2 alone. A further 46% of the feed material was extracted using ethanol and CO2 with a total ethanol 10 flow of 800g. Phospholipid profiles for the extract and residual fractions are shown in Table 10. In this example, the phosphatidylserine levels in the residual material are substantially enriched compared with levels in the feed material, while sphingomyelin is enriched in the extract relative to the feed.

Table 10 Yield % of feed Composition, % PC PI PS PE SM Other Phospholipids Other compounds Feed 21.2 0.6 0.4 .2 1.6 0.9 70.1 Extract 46 65.6 0.3 N/D 6.3 2.8 2.3 22.8 Residue 4 12.9 11.2 8.2 27.6 2.8 82 29.2 Example 12: Fractionation of Hoki head lipid extract This example relates to fractionation of a Hoki head lipid extract with phospholipid profile 20 shown in Table 11. In this example 25g of the feed material was extracted using the continuous extraction mode of operation at 300 bar and 60°C, and 95% aqueous ethanol at a concentration of 31%. 1% of the feed mass was extracted as neutral lipids using C02 alone. A further 72% of the feed material was extracted using ethanol and CO2 with a total ethanol flow of 940g. Phospholipid profiles for the extract and residual fractions are shown in Table 25 11. In this example, the phosphatidylserine levels in the residual material are substantially enriched compared with levels in the feed material. Some PS is also observed in the extract phase. The alkylacylphosphatidylcholine (AAPC), a type of alkylacylphospholipid, is completely extracted. 571454 Table 11 Yield Zo of feed Composition, % PC PI PS PE SM AAPC Other phosph Other compounds Feed 1 9.2 1.1 1.4 4.8 0.5 1.1 1.8 80.8 Extract 72 14.2 0.0 0.7 .3 0.5 1.6 0.6 71.2 Residue 27 14.3 7.1 7.6 13.9 0.0 0.0 6.2 47.7 Example 13: Fractionation of bovine heart lipid extract This example relates to fractionation of a bovine heart phospholipid lipid extract with phospholipid profile shown in Table 9. In this example 40g of the feed material was extracted using the continuous extraction mode of operation at 300 bar and 60°C, and 95% aqueous ethanol at a concentration of 33% in CO2. No lipid was extracted using CO2 alone. 10 79% of the feed material was extracted using ethanol and CO2 with a total ethanol flow of 960g. Phospholipid profiles for the extract and residual fractions are shown in Table 12. The phosphatidylserine levels in the residual material are substantially enriched compared with levels in the feed material. Cardiolipin is also significantly enriched in the residue.

Table 12 Yield wt% of feed Composition, wt% CL PC PI PS PE SM Other Phospholipid s Other compounds Feed 16.8 13.4 3.2 1.5 12.3 3.6 .3 33.9 Extract 79 8.2 18.6 0.8 0.4 8.6 3.5 13.1 46.7 Residue 21 42.2 2.8 14.1 4.7 23.4 12.8 0.0 Example 14: Fractionation of dairy lipid extract A with propan-2-ol co'solvent In this example 39g of the dairy lipid extract A, as described in example 1, was extracted using the continuous extraction mode of operation at 300 bar and 60°C, and 95% aqueous propan-2-ol at a mass concentration of 35% in CO2. 48% of the feed material was extracted as neutral lipids using CO2 alone. 23% of the feed material was further extracted using the propan-2-ol co-solvent and C02 with a total propanol mass of 81 Og. Phospholipid profiles for the extract and residual fractions are shown in Table 13. The phosphatidylserine levels in file residual material are substantially enriched, and the result is comparable to results for examples 1 and 2. A slightly lower total PS level is achieved than for example 2 using a 21 571454 comparable concentration of ethanol. The levels of PS observed in the extracted fraction is also higher suggesting the propan-2-ol is not as selective as ethanol. On this basis alone ethanol would be the preferred co-solvent.

Table 13 Yield % of feed . Composition, % PC PI PS PE SM Other Phospholipids Other compounds Feed 11.2 2.8 4.3 13.2 7.8 2.2 58.3 Extract 23 27.9 0.8 1.3 19.5 14.0 4.2 32.4 Residue 29 .7 8.6 13.0 23.8 .5 3.4 .0 Example 15: Fractionation of soy lecithin This example relates to fractionation of a soy lecithin (Healtheries Lecithin natural dietary 10 supplement, Healtheries of New Zealand Limited) with composition shown in Table 9. In this example 42g of feed material was extracted using the continuous extraction mode of operation at 300 bar and 60°C, and 95% aqueous ethanol at a concentration of 33% in CO2. No lipid was extracted using CO2 alone. 91% of the feed material was extracted using ethanol and CO2 with a total ethanol flow of 520g. Phospholipid profiles for the extract and 15 residual fractions are shown in Table 14. PC and PE are preferentially extracted and are significantly enriched in the extract There are no detectable levels of PS or SM in this example.

Table 14 Yield % of feed Composition, % PC PI PS PE SM Other Phospholipids Other compounds Feed 22.2 12.3 0.0 17.4 0.0 11.7 36.4 Extract 9 31.9 0.7 0.0 9.9 0.0 6.1 51.4 Residue 91 .7 13.2 0.0 18.4 0.0 12.4 .2 Example 16: Continuous fractionation of egg yolk lipids This example relates to fractionation of an egg yolk lipid extract containing 15% 25 phospholipids and the balance mostly neutral lipids by HPLC analysis. The phospholipid fraction contains 55% PC, 29% PE, and 14% PI. Hie feed lipid was pumped into the top of a 10L pressure vessel, and contacted with CO2 containing 8.7 % of 98% aqueous ethanol flowing upwards through the vessel at 300 bar pressure and temperature of 60°C. An extract phase was continuously taken from the top of the contacting vessel, and a raffinate phase 22 571454 was periodically withdrawn from the bottom of the vessel. The lipid feed rate was 1.5 kg/hr. The CO2+ co-solvent flow rate was 27 kg/hr.

The extract phase was predominantly neutral lipids but contained 20% of the phospholipids present in the feed stream. The phospholipids in the extract fraction consisted of between 5 70% and 100% PC, with the balance mostly PE. This represents a preferential extraction of PC over other phospholipids.

In a second experiment, feed lipid was premixed with 98% ethanol (with 2 % water) at a concentration of 10.2% lipid. This mixture was pumped into the top of the pressure vessel and contacted with CO2 in upflow. The overall concentration of ethanol in CO2 under steady 10 state processing conditions was 5.9%. In this case 50% of the mass of phospholipids in the feed were extracted. The composition of the extract phase consisted of between 60% and 70% PC, with the balance mostly PE. The presence of PI and other phospholipids in the extract was not detectable by the HPLC method used.

Example 17: Fractionation of green-lipped mussel lipid extract This example relates to fractionation of a green-lipped mussel lipid extract with phospholipid profile shown in Table 11. In this example 12.2g of the feed material was extracted using a batch stirred tank method at 250 bar and 60°C using CO2 and ethanol (containing 5 % water) at a concentration of 30.5 %. The lipid was placed in the stirred tank, CO2 was added to give 20 the desired pressure and then the 95 % ethanol was added in during constant stirring. 65 % of the feed material was then extracted using CO2 and ethanol after stirring for 1 hour by sampling the extract phase at constant pressure. Phospholipid profiles for the extract and residual fractions are shown in Table 15. In this example, the CAEP levels in the residual material are substantially enriched compared with levels in the feed material. The 25 alkylacylphosphatidylcholine (AAPC), a type of alkylacylphospholipid, is partially extracted.

Table 15 Yield % of feed Composition, % PC PI PS PE SM AAPC CAEP Other compounds Feed 1.89 0.0 0.0 1.60 0.0 0.8 2.0 922 Extract 65.3 1.97 0.0 0.0 0.0 0.0 0.9 0.71 96.0 Residue 34.7 3.77 0.0 0.0 3.67 0.0 1.3 3.22 84.0 23 571454 Example 18: Fractionation of krill lipids This example shows the fractionation of krill lipids from krill powder and demonstrates concentration of AAPC in the extract, and AAPE in the residue. 5619.9 g of freeze-dried krill powder containing 21.4 % lipid and corresponding phospholipids concentrations shown 5 in table 16 was extracted continuously with supercritical CO2 at 300 bar and 313 K until no further extract was obtained. This extract (extract 1) contained no phospholipids, and was substantially all neutral lipids. A total of650 g of this extract was obtained, and 66.41 kg of CO2 was used. The residual powder was then extracted with CO2 and absolute ethanol, using a mass ratio of ethanol to C02 of 11 %. The CO2 and ethanol extract phase was passed 10 through two sequential separators in which the pressure was 95 and 60 bar respectively. The bulk of the phospholipids-rich extract (extract 2) was obtained in the first separator, and the bulk of the co-solvent in the second separator (extract 3). The composition of extract 2 and residual powder are shown in table 16. The alkylacylphosphatidylcholine (AAPC), a type of alkylacylphospholipid, is highly enriched in the concentrated phospholipids-rich extract, 15 whilst alkylacylphosphatidylethanolamine (AAPE), another type of alkylacylphospholipid, is not extracted to any great degree.

Table 16 Yield Vo of feed Composition, % PC PI PS PE CL AAPC AAPE Other compounds Feed 6.6 0.0 0.0 0.4 0.1 0.6 0.1 78.6 Extract 2 4.3 39.8 0.0 0.0 0.3 0.2 4.6 0.2 53.7 Residue 79.2 3.6 0.0 0.0 0.3 02 0.5 0.1 93.4 Example 18: Fractionation of dairy lipids from beta-serum powder This example shows the fractionation of dairy lipids from beta-serum powder (a milk fat globular membrane concentrate powder) and demonstrates concentration of PS in the residual powder, and concentration of SM in the extract obtained using supercritical CO2 + ethanol. 5835.3 grams of beta-serum powder containing phospholipids in the concentrations shown in table 17, was extracted continuously with supercritical CO2 at 300 bar and 313 K until no further extract was obtained. This extract contained no phospholipids, and was substantially all neutral lipids. 1085.6 g of this extract (extract 1) was obtained using 94.42 kg of CO2.2906.3 grams of the residual powder was then re-extracted with CO2 and anhydrous ethanol at 300 bar and 323 K, using a mass ratio of ethanol to CO2 of 25 %. Hie powder was extracted with this mixture for 90 minutes (7.82 kg ethanol). The CO2 and 24 571454 ethanol extract phase was passed through two sequential separators in which the pressure was 100 (extract 2) and 54 bar (extract 3) respectively. The extract was split between both separators. A total of262.2 g of extract was obtained. The composition of the combined extract (extract 2 and 3) and residual powder are shown in table 17. The extract is highly 5 enriched in sphingomyelin, whilst the residue is enriched in phosphatidylserine.

Table 17 Yield % of feed Composition, % PC PI PS PE SM Other Phospholipids Other compounds Peed 4.9 1.5 2.3 .6 4.3 0.1 81.3 Extract 2+3 9.02 49.6 0.0 0.0 12.4 .1 0.7 7.1 Residue 71.14 0.3 2.0 3.0 3.0 0.5 0.1 91.1 INDUSTRIAL APPLICATION The present invention has utility in providing products with high levels of particular phospholipids and/or glycolipids including cardiolipin and phosphatidyl serine, and sphingomyelin. The described compositions and methods of the invention may be employed in a number of applications, including infant formulas, brain health, sports nutrition and dermatological compositions.

REFERENCES 1. Palacios, L.E., Wang, T., Extraction of egg-yolk lecithin, JAOCS 82,8,2005 2. Kang, D.H., Row, K.H., Fractionation of soybean phospholipids by preparative high-performance liquid chromotography with sorbents of various particle size, Journal of Chromatography A, 949,2002 3. Kearns, J.J., Tremblay, P.A., Robey, R.J., Sunder, S., Process for purification of phospholipids, US patent 4814111,1989 4. Teberikler, L., Koseoglu, S., Akgerman, A., Selective extraction of phosphatidylcholine from lecithin by supercritical carbon dioxide/ethanol mixture, JAOCS 78,2,2001 571454 . Montanari, L., Fantozzi, P., Snyder, J.M., King, J.W., Selective extraction of phospholipids from soybeans with supercritical carbon dioxide and ethanol, J Supercritical Fluids, 14,1999 6. Taylor, S.L., King, J.W., Montanari, L., Fantozzi, P., Blanco, M.A., Enrichment and fractionation of phospholipid concentrates by supercritical fluid extraction and chromatography, Ital J Food Sci, 1,12,2000 7. Tanaka, Y., Sakaki, I., Extraction of phospholipids from unused natural resources with supercritical carbon dioxide and an entrainer, Journal of oleo science, 54,11, 2005 8. Bulley, N.R., Labay, L., Arntfield, S.D., Extraction/Fractionation of egg yolk using supercritical C02 and alcohol entrainers, J supercritical fluids, 5,1992. 26 571454 RECEIVED at IPONZ on 29 January 2010

Claims

WHAT WE CLAIM IS:

1. A process for separating a feed material into soluble and insoluble components, comprising (a) providing a feed material comprising one or more of: 5 (i) at least 1% by mass phosphatidyl serine (ii) at least 1% by mass sphingomyelin (iii) at least 0.3 % by mass acylalkylphospholipids and/or plasmalogens (iv)at least 0.5 % by mass ceramide aminoethylphosphonate and/or derivatives thereof 10 (v) at least 1% by mass cardiolipin (vi) at least 0.3% by mass gangliosides (b) providing a solvent comprising: (i) supercritical or near-critical CO2, and (ii) a co-solvent comprising one or more C1-C3 monohydric alcohols, and water 15 wherein the co-solvent makes up at least 10% by mass of the solvent, and the water content of the co-solvent is 0 to 40 % by mass (c) contacting the feed material and the solvent (d) separating the solvent containing the soluble components from the insoluble components, the soluble components comprising one or more of sphingomyelin, 20 choline-based acylphospholipids, choline-based plasmalogens, and the insoluble components comprising one or more of phosphatidyl serine, non-choline based acylalkylphospholipids, non-choline based plasmalogens, ceramide aminoethylphosphonate, cardiolipin and gangliosides, and (e) optionally separating the soluble components and the solvent. 27 RECEIVED at IPONZ on 29 January 2010 571454

2. The process of claim 1 wherein the feed material comprises greater than 1% phosphatidyl serine.

3. The process of claim 1 or claim 2 wherein the feed material comprises greater than 2% phosphatidyl serine. 5

4. The process of any preceding claim wherein the feed material comprises greater than 5% phosphatidyl serine.

5. The process of any preceding claim wherein the feed material comprises greater than 1% sphingomyelin.

6. The process of any preceding claim wherein the feed material comprises greater than 5% 10 sphingomyelin.

7. The process of any preceding claim wherein the feed material comprises greater than 15% sphingomyelin.

8. The process of any preceding claim wherein the feed material comprises greater than 1% cardiolipin. 15

9. The process of any preceding claim wherein the feed material comprises greater than 2% cardiolipin.

10. The process of any preceding claim wherein the feed material comprises greater than 5% cardiolipin.

11. The process of any preceding claim wherein the feed material comprises greater than 20 0.3% gangliosides.

12. The process of any preceding claim wherein the feed material comprises greater than 1% gangliosides.

13. The process of any preceding claim wherein the feed material comprises greater than 2% gangliosides. 25 14. The process of any preceding claim wherein the feed material comprises greater than 0.5% acylalkyphospholipids and/or plasmalogens. 28

RECEIVED at IPONZ on 29 January 2010 571454

15. The process of any preceding claim wherein the feed material comprises greater than 2% acylalkyphospholipids and/or plasmalogens.

16. The process of any preceding claim wherein the feed material comprises greater than 10% acylalkyphospholipids and/or plasmalogens. 5

17. The process of any preceding claim wherein the feed material comprises greater than 0.5% ceramide aminoethylphosphonate and/or derivatives thereof.

18. The process of any preceding claim wherein the feed material comprises greater than 5% ceramide aminoethylphosphonate and/or derivatives thereof.

19. The process of any preceding claim wherein the feed material comprises greater than 20% 10 ceramide aminoethylphosphonate and/or derivatives thereof.

20. A process for separating a feed material into soluble and insoluble components, comprising (a) providing a feed material comprising one or more of: (i) at least 1% by mass phosphatidyl serine, 15 (ii) at least 1% by mass sphingomyelin, (iii) at least 0.3 % by mass acylalkylphospholipids and/or plasmalogens (iv) at least 0.5 % by mass ceramide aminoethylphosphonate and/or derivatives thereof (v) at least 1% by mass cardiolipin, or 20 (vi) at least 0.3% by mass gangliosides (b) providing a first solvent comprising supercritical or near-critical CO2 (c) contacting the feed material and the first solvent and subsequently separating the first solvent containing the first soluble components from the first insoluble components (d) optionally separating the first soluble components and the first solvent 29 RECEIVED at IPONZ on 29 January 2010 571454 (e) providing a second solvent comprising: (i) supercritical or near-critical CO2, and (ii) a co-solvent comprising one or more C1-C3 monohydric alcohols, and water wherein the co-solvent makes up at least 10% by mass of the solvent and the water content of the co-solvent is 0 to 40% by mass (f) contacting the first insoluble components and the second solvent (g) separating the second solvent containing the second soluble components from the second insoluble components, the second soluble components comprising one or more of sphingomyelin, choline-based acylphospholipids, choline-based plasmalogens, and the second insoluble components comprising one or more of phosphatidyl serine, non-choline based acylalkyl phospholipids, non-choline based plasmalogens, ceramide aminoethylphosphonate, cardiolipin and gangliosides. (h) optionally separating the second soluble components and the second solvent.

21. The process of claim 20 wherein the first solvent comprises a mixture of supercritical or near-critical CO2 and less than 10% C1-C3 monohydric alcohol.

22. The process of claim 20 or claim 21 wherein the feed material comprises greater than 1% phosphatidyl serine.

23. The process of any one of claims 20-22 wherein the feed material comprises greater than 2% phosphatidyl serine.

24. The process of any one of claims 20-23 wherein the feed material comprises greater than 5% phosphatidyl serine.

25. The process of any one of claims 20-24 wherein the feed material comprises greater than 1% sphingomyelin.

26. The process of any one of claims 20-25 wherein the feed material comprises greater than 5% sphingomyelin. 30 571454 RECEIVED at IPONZ on 29 January 2010

27. The process of any one of claims 20-26 wherein the feed material comprises greater than 15% sphingomyelin.

28. The process of any one of claims 20-27 wherein the feed material comprises greater than 1% cardiolipin. 5

29. The process of any one of claims 20-28 wherein the feed material comprises greater than 2% cardiolipin.

30. The process of any one of claims 20-29 wherein the feed material comprises greater than 5% cardiolipin.

31. The process of any one of claims 20-30 wherein the feed material comprises greater than 10 0.3% gangliosides.

32. The process of any one of claims 20-31 wherein the feed material comprises greater than 1% gangliosides.

33. The process of any one of claims 20-32 wherein the feed material comprises greater than 2% gangliosides. 15

34. The process of any one of claims 20-33 wherein the feed material comprises greater than 0.5% acylalkyphospholipids and/or plasmalogens.

35. The process of any one of claims 20-34 wherein the feed material comprises greater than 2% acylalkyphospholipids and/or plasmalogens.

36. The process of any one of claims 20-35 wherein the feed material comprises greater than 20 10% acylalkyphospholipids and/or plasmalogens.

37. The process of any one of claims 20-36 wherein the feed material comprises greater than 0.5% ceramide aminoethylphosphonate and/or derivatives thereof.

38. The process of any one of claims 20-37 wherein the feed material comprises greater than 5% ceramide aminoethylphosphonate and/or derivatives thereof. 25 39. The process of any one of claims 20-38 wherein the feed material comprises greater than ceramide 20% aminoethylphosphonate and/or derivatives thereof. 31 571454

RECEIVED at IPONZ on 29 January 2010

40. The process of any preceding claim wherein the feed material is derived from terrestrial animals, marine animals, terrestrial plants, marine plants, or micro-organisms such as microalgae, yeast and bacteria.

41. The process of claim 40 wherein the feed material is derived from sheep, goat, pig, mouse, 5 water buffalo, camel, yak, horse, donkey, llama, bovine or human.

42. The process of claim 40 or claim 41 wherein the feed material is selected from: tissue, a tissue fraction, organ, an organ fraction, milk, a milk fraction, colostrum, a colostrum fraction, blood and a blood fraction.

43. The process of claim 40 wherein the feed material is derived from dairy material, soy 10 material, eggs, animal tissue, animal organ or animal blood.

44. The process of claim 40 wherein the feed material is selected from: a composition comprising dairy lipids, a composition comprising egg lipids, a composition comprising soy lipids, a composition comprising marine lipids, and a composition comprising animal lipids. 15

45. The process of any one of claims 1 to 44 wherein the feed material is a bovine milk fraction.

46. The process of claim 45 wherein the feed material is selected from: buttermilk, a buttermilk fraction, beta serum, a beta serum fraction, butter serum, a butter serum fraction, whey, a whey fraction, colostrum, and a colostrum fraction. 20

47. The process of any one of claims 1 to 46 wherein the feed material comprises milk fat globule membrane.

48. The process of any one of claims 1 to 47 wherein the feed material comprises at least: (a) 1% phosphatidyl serine, and (b) 0.3% gangliosides. 25 49. The process of claim 48 wherein the feed material comprises at least: (a) 1% phosphatidyl serine, 32 571454

RECEIVED at IPONZ on 29 January 2010 (b) 1% sphingomyelin, and (c) 0.3% gangliosides.

50. The process of claim 48 wherein the feed material comprises at least: (a) 1% phosphatidyl serine, (b) 1% sphingomyelin, (c) 1% cardiolipin, and (d) 0.3% gangliosides.

51. The process of any one of claims 1-39 wherein the feed material comprises at least one of buttermilk, a buttermilk fraction, milk, a milk fraction, beta serum, a beta serum fraction, butter serum, a butter serum fraction, whey, a whey fraction, milk fat globule membrane, colostrum, and a colostrum fraction wherein the feed material comprises one or more of (i) at least 1% phosphatidyl serine (ii) at least 1% sphingomyelin, or (iii) at least 0.3% gangliosides.

52. The process of any one of claims 1-39 wherein the feed material comprises marine animals or marine plants and wherein the feed material comprises one or more of (i) at least 1% sphingomyelin, (ii) at least 0.3% acylalkylphospholipids and/or plasmalogens, (iii) at least 1% phosphatidyl serine, or (iv) at least 0.5% ceramide aminoethylphosphonate and/or derivatives thereof.

53. The process of any one of claims 1-39 wherein the feed material comprises terrestrial animals, animal tissue or tissue fraction; animal organ or organ fraction; animal blood or blood fraction wherein the feed material comprises one or more of (i) at least 1% phosphatidyl serine, (ii) at least 0.3% acylalkylphospholipids and/or plasmalogens, (iii) at least 1% sphingomyelin, 33 571454 RECEIVED at IPONZ on 29 January 2010 (iv) at least 1% cardiolipin, or (v) at least 0.3% gangliosides.

54. The process of any preceding claim wherein the feed material has been genetically modified.

55. The process of any one of claims 1 to 54 wherein the feed material is in solid form.

56. The process of claim 55 wherein the feed material is cryomilled before contact with a solvent.

57. The process of any one of claims 1 to 56 wherein the co-solvent comprises: (a) an alcohol selected from: methanol, ethanol, n-propanol, isopropanol and mixtures thereof; and (b) 0-40% by mass water.

58. The process of claim 57 wherein the co-solvent comprises between 0 and 20% by mass water.

59. The process of claim 57 wherein the co-solvent comprises between 1 and 10% by mass water.

60. The process of any one of claims 57 to 59 wherein the alcohol is ethanol.

61. The process of any one of claims 1 to 60 wherein the co-solvent is 95% aqueous ethanol.

62. The process of any one of claims 1 to 61 wherein the mass traction of the co-solvent in the solvent is between 5% and 60%.

63. The process of claim 62 wherein the mass fraction is between 30% and 50%.

64. The process of claim 62 wherein the mass fraction is between 20% and 30%.

65. The process of any one of claims 1 to 64 wherein the contacting temperature between the feed material and solvent is between 10°C and 80°C.

66. The process of claim 65 wherein the contacting temperature is between 55°C and 65°C. 34 571454 RECEIVED at IPONZ on 29 January 2010

67. The process of any one of claims 1 to 66 wherein the contacting pressure is between 100 bar and 500 bar.

68. The process of claim 67 wherein the contacting pressure is between 200 bar and 300 bar.

69. The process of any one of claims 1 to 68 wherein the ratio of the co-solvent to feed 5 material is in the range 10:1 w/w to 200:1 w/w.

70. The process of claim 69 wherein the ratio of the co-solvent to feed material is in the range 15:1 w/w to 50:1 w/w.

71. The process of any one of claims 1 to 70 wherein the separating pressure is between atmospheric pressure and 90 bar. 10

72. The process of claim 71 wherein the separating pressure is between 40 bar and 60 bar.

73. The process of any one of claims 1 to 72 wherein the co-solvent is recycled for further use without processing.

74. The process of any one of claims 1 to 73 wherein the CO2 is recycled for further use.

75. The process of any one of claims 1 to 74 wherein the co-solvent is removed by 15 evaporation under vacuum.

76. The process of any one of claims 1 to 75 wherein the feed material is contacted with a continuous flow of solvent.

77. The process of any one of claims 1 to 75 wherein the feed material is contacted with one or more batches of solvent. 20

78. The process of any one of claims 1 to 76 wherein the feed material and solvent streams are fed continuously.

79. The process of any one of claims 1 to 78 wherein the feed material and co-solvent streams are mixed prior to contacting with CO2.

80. A product produced by the process of any one of claims 1 to 79. 25 81. The product of claim 80 wherein the product contains more sphingomyelin than the feed material. 35 571454

RECEIVED at IPONZ on 29 January 2010

82. The product of claim 80 wherein the product comprises greater than 3% sphingomyelin.

83. The product of claim 80 wherein the product comprises greater than 10% sphingomyelin.

84. The product of claim 80 wherein the product comprises greater than 15% sphingomyelin.

85. The product of claim 80 wherein the product contains more phosphatidyl serine than the 5 feed material.

86. The product of claim 80 wherein the product comprises greater than 5% phosphatidyl serine.

87. The product of claim 80 wherein the product comprises greater than 30% phosphatidyl serine. 10

88. The product of claim 80 wherein the product comprises greater than 70% phosphatidyl serine.

89. The product of claim 80 wherein the product contains more gangliosides than the feed material.

90. The product of claim 80 wherein the product comprises greater than 2% gangliosides. 15

91. The product of claim 80 wherein the product comprises greater than 4% gangliosides.

92. The product of claim 80 wherein the product comprises greater than 6% gangliosides.

93. The product of claim 80 wherein the product contains more cardiolipin than the feed material.

94. The product of claim 80 wherein the product comprises greater than 5% cardiolipin. 20

95. The product of claim 80 wherein the product comprises greater than 10% cardiolipin.

96. The product of claim 80 wherein the product comprises greater than 25% cardiolipin.

97. The product of any claim 80 wherein the product contains more acylalkyphospholipids and/or plasmalogens than the feed material. 36 571454 RECEIVED at IPONZ on 29 January 2010

98. The product of claim 80 wherein the product comprises greater than 5% acylalkyphospholipids and/or plasmalogens.

99. The product of claim 80 wherein the product comprises greater than 10% acylalkyphospholipids and/or plasmalogens.

100. The product of claim 80 wherein the product comprises greater than 25% acylalkyphospholipids and/or plasmalogens.

101. The product of claim 80 wherein the product contains more ceramide aminoethylphosphonate and/or derivatives thereof than the feed material.

102. The product of claim 80 wherein the product comprises greater than 5% ceramide aminoethylphosphonate and/or derivatives thereof.

103. The product of claim 80 wherein the product comprises greater than 10% ceramide aminoethylphosphonate and/or derivatives thereof.

104. The product of claim 80 wherein the product comprises greater than 25% ceramide aminoethylphosphonate and/or derivatives thereof.

105. The product of claim 80 which is enriched in one or more of: (i) phosphatidyl serine (ii) sphingomyelin (iii) acylalkylphospholipids and/or plasmalogens (iv) ceramide aminoethylphosphonate and/or derivatives thereof (v) cardiolipin, or (vi) gangliosides by at least 50% relative to the feed material.

106. The product of claim 80 which is enriched in one or more of: (i) phosphatidyl serine (ii) sphingomyelin (iii) acylalkylphospholipids and/or plasmalogens (iv) ceramide aminoethylphosphonate and/or derivatives thereof 37 RECEIVED at IPONZ on 29 January 2010 571454 (vii) cardiolipin, or (viii) gangliosides by at least 100% relative to the feed material.

107. The product of claim 105 or 106 which comprises one or more of: (i) at least 30% phosphatidyl serine (ii) at least 10% sphingomyelin (iii) at least 10% acylalkylphospholipids and/or plasmalogens (vi) at least 10 % ceramide aminoethylphosphonate and/or derivatives thereof (vii) at least 5% cardiolipin, or (viii) at least 2% gangliosides.

108. A process according to claim 1 or claim 20, substantially as herein described with reference to any example thereof. 38