WO2007123424A1

WO2007123424A1 - Process for separating lipid materials

Info

Publication number: WO2007123424A1
Application number: PCT/NZ2007/000087
Authority: WO
Inventors: Owen John Catchpole; Stephen John Tallon
Original assignee: Owen John Catchpole; Stephen John Tallon
Priority date: 2006-04-20
Filing date: 2007-04-20
Publication date: 2007-11-01
Also published as: AU2007241642A1; TW200811280A; AU2007241642B2

Abstract

The present invention 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 present invention 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.

Description

PRODUCT AND PROCESS

FIELD OF INVENTION

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, alkylacylphospholipids, 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 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 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 and gangliosides have been implicated in conferring a number of health benefits including brain health, skin health, eczema treatment, anti-infection, wound healing, gut microbiota modifications, anti-cancer activity, alleviation of arthritis, improvement of 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 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 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 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 the final product by reducing the pressure, whereupon the 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 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 ethanolamine. 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 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 some extraction of PC, PE, PI, and phosphatid acid (PA). PC is selectively extracted under some conditions, but at higher temperatures and pressures some extraction of PE and PI was achieved. The pressures required to achieve good extraction were impractically high for industrial application, and the high temperatures used (80°C) could cause polyunsaturated fatty acids to be degraded. Taylor et al [6] describe a process in which soybean flakes are first extracted using only CO₂, 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 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 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. 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 aminoethylphosphonate and/or other phosphonolipids

(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 CO_2, 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 CO₂, 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) optionally separating the soluble components and the solvent.

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 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% 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% aminoethylphosphonate and/or other phosphonolipids. More preferably the feed material comprises greater than 5% aminoethylphosphonate and/or other phosphonolipids. Most preferably the feed material comprises greater than 20% aminoethylphosphonate and/or other phosphonolipids.

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

(iv) at least 0.5 % by mass aminoethylphosphonate and/or other phosphonolipids

(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 CO_2, and

wherein the co-solvent makes up at least 10% by mass of the CO_2, and the water content of the co-solvent is 0 to 40% by mass

(f) contacting the first insoluble components and the second solvent and subsequently separating the second solvent containing the second soluble components from the second insoluble components

(g) 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 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. 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 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% aminoethylphosphonate and/or other phosphonolipids. Preferably the feed material comprises greater than 5% aminoethylphosphonate and/or other phosphonolipids. More preferably the feed material comprises greater than 20% aminoethylphosphonate and/or other phosphonolipids.

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 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, 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 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 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 55⁰C and 65°C. Most 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 preferably the separating pressure is between 40 bar and 60 bar.

Preferably the co-solvent is recycled for further use.

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 "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 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 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 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 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% acylalkyphospholipids and/or plasmalogens. Most preferably the product comprises greater than 25% acylalkyphospholipids and/or plasmalogens.

Preferably the product contains more aminoethylphosphonate and/or other phosphonolipids than the feed material. More preferably the product comprises greater than 5% aminoethylphosphonate and/or other phosphonolipids. Even more preferably the product comprises greater than 10% aminoethylphosphonate and/or other phosphonolipids. Most preferably the product comprises greater than 25% aminoethylphosphonate and/or other phosphonolipids. BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be more folly understood by having reference to the accompanying drawings wherein:

Figure 1 is scheme drawing illustrating a preferred process of the current invention. 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

"PS" means phosphatidyl serine

"PE" means phosphatidyl ethanolamine

"PA" means phosphatidic acid

"PL" means plasmalogen

"PP" means phosphonolipid

"ALP" means alkylacylphospholipid

"SM" means sphingomyelin

"CAEP" means ceramide aminoethylphosphonate

"GS" means ganglioside

"N/D" means not detected

"CO₂" means carbon dioxide 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 from soy or egg. Surprisingly, we have found that the phospholipids PS, CAEP and CL; and gangliosides are virtually insoluble in CO₂ and 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 ^■ 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 ideally at least 2 % by mass of phospholipids, particularly PS, SM₅ CL, ALP, PL, PP, CAEP and/or gangliosides.

The feed material can be processed using pure CO₂ before the co-solvent is introduced to remove much or all of neutral lipids. This reduces the neutral lipid content in the CO₂+co- 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. 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. 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 solvent, or very favourable extraction conditions, can result in small losses of PS₃ 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 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 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 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 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 that 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. 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 I₅ as follows.

A measured mass of feed material containing lipids to be fractionated was placed in basket BKl with a porous sintered steel plate on the bottom. Basket BKl was placed in a 300 mL extraction vessel EXl . The apparatus was suspended in heated water bath WBl and 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 Bl was pumped using pump Pl into extraction vessel EXl until the pressure reached the desired operating pressure, after which valve Vl was operated to maintain a constant pressure in the extraction vessel. After passing through valve Vl , 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 SEPl, and the CO₂ exited from the top of separator SEPl and was recycled back to the feed pump through coriolis mass flow meter FMl and cold trap CTl operated at -5⁰C. Extracted material was collected periodically from separator SEPl 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 material were separated from the CO₂ in separator SEPl 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 BKl 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 Vl closed to maintain the pressure. Approximately 14Og of ethanol was pumped from supply bottle B2 through pump P2 into extraction vessel EXl . The system was left for 15 minutes to allow the system to equilibrate, after which time the CO₂ flow was started and valve Vl opened to maintain a constant pressure and allow ethanol and dissolved compounds to flow through to separator SEPl . This process was repeated twice more, after which the CO₂ was vented and the residual material in basket BKl was removed and dried under vacuum.

Extract and residue fractions were analysed for phospholipid content and profile by P- NMR. The phospholipid mass fractions reported here are for phosphatidylcholine (PC), phosphatidylinositol (PI)₅ phosphatidylethanolamine (PE), plasmalogens (PL)₃ 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).

4Og 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. The '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 in CO₂ of 25%. The total ethanol and water added was 88Og. 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 the extract phase indicating very low solubility in CO₂ and ethanol, and almost complete recovery of phosphatidylserine in the residue phase. Table 1

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 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 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

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 continuous extraction mode of operation at 60⁰C 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 96Og. The composition of extract 2 and residual fractions are shown in Table 3. The concentration of phosphatidylserine in the residue fraction is higher than found in example 1 and example 2 at 20.7 %. The 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

Example 4: Fractionation of dairy lipid extract A, 40⁰C

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 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 extract is higher. The concentration of PS in the residue, at 12.4 %, is lower than examples 1 to 3.

Table 4

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 6O⁰C without the prior CO₂ only extraction step. The ethanol (95% aqueous ethanol) mass fraction in CO₂ was 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 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

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 the batch extraction mode of operation at 300 bar and 6O⁰C. A total of 22% of the feed mass was extracted in three sequential extractions each consisting of 14Og of ethanol (95% aqueous ethanol) in 30OmL 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 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

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 high fat whey protein concentrate processes disclosed in PCT international applications PCT/NZ2004/000014 (published as WO WO2004/066744).

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 300 bar and 60⁰C. 52% of the feed mass was extracted using CO₂ alone (extract 1). Only 3% of the feed lipid was further extracted using 46Og 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 case, both the PS and SM are enriched in the residue.

Table 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 mode of operation at 300 bar and 6O⁰C. 51% of the feed mass was extracted using CO₂ alone (extract 1). A further 7% of the feed material was extracted using 76Og 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 Table 8

Example 9: Fractionation of dairy lipid extract A, ethanol mass 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 60°C, 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 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 15Og of ethanol in 485Og 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 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 CO₂ alone. A further 49% of the feed material was extracted using ethanol and CO₂ with a total ethanol flow of 64Og. Phospholipid profiles for the extract and residual fractions are shown in Table 9. In this example, the phosphatidylserine levels in the residual material are substantially enriched compared with non-detectable levels in the feed material. Table 9

Example 11: Fractionation of egg yolk phospholipid extract

This example relates to fractionation of an egg yolk phospholipid fraction with phospholipid profile shown in Table 9. In this example 4Og of the feed material was extracted using the continuous extraction mode of operation at 300 bar and 60⁰C, 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 flow of 80Og. 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

Example 12: Fractionation of Hoki head lipid extract

This example relates to fractionation of a Hoki head lipid extract with phospholipid profile 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 CO₂ alone. A further 72% of the feed material was extracted using ethanol and CO₂ with a total ethanol flow of 94Og. Phospholipid profiles for the extract and residual fractions are shown in Table 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 alkylacylphospliolipid, is completely extracted. Table 11

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 4Og of the feed material was extracted using the continuous extraction mode of operation at 300 bar and 60⁰C, and 95% aqueous ethanol at a concentration of 33% in CO₂. No lipid was extracted using CO₂ alone. 79% of the feed material was extracted using ethanol and CO₂ with a total ethanol flow of 96Og. 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

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 CO₂ with a total propanol mass of 81Og. Phospholipid profiles for the extract and residual fractions are shown in Table 13. The phosphatidylserine levels in the 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 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

Example 15: Fractionation of soy lecithin

This example relates to fractionation of a soy lecithin (Healtheries Lecithin natural dietary 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 6O⁰C₅ 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 52Og. Phospholipid profiles for the extract and 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

Example 16: Continuous fractionation of egg yolk lipids

This example relates to fractionation of an egg yolk lipid extract containing 15% phospholipids and the balance mostly neutral lipids by HPLC analysis. The phospholipid fraction contains 55% PC, 29% PE, and 14% PI . The feed lipid was pumped into the top of a 1OL 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 60⁰C. An extract phase was continuously taken from the top of the contacting vessel, and a raffinate phase 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 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 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 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 alkylacylphosphatidylcholine (AAPC), a type of alkylacylphospholipid, is partially extracted.

Table 15

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 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 of 650 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 CO₂ of 11 %. The CO₂ and ethanol extract phase was passed 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, whilst alkylacylphosphatidylethanolamine (AAPE), another type of alkylacylphospholipid, is not extracted to any great degree. Table 16

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 %. The powder was extracted with this mixture for 90 minutes (7.82 kg ethanol). The CO₂ and 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 of 262.2 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 enriched in sphingomyelin, whilst the residue is enriched in phosphatidylserine.

Table 17

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, JJ., Tremblay, P.A., Robey, RJ., 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 5. Montanari, L., Fantozzi, P., Snyder, J.3VL, 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, L, 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 CO2 and alcohol entrainers, J supercritical fluids, 5, 1992.

Claims

WHAT WE CLAIM IS:

1. A process for separating a feed material into soluble and insoluble components, comprising

5 (e) 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 aminoethylphosphonate and/or other phosphonolipids

10 (v) at least 1% by mass cardiolipin

(vi) at least 0.3% by mass gangliosides

(f) providing a solvent comprising:

(i) supercritical or near-critical CO₂, and

15 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

(g) contacting the feed material and the solvent and subsequently separating the solvent containing the soluble components from the insoluble components

(h) optionally separating the soluble components and the solvent.

20.

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

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

4. The process of claim 1 wherein the feed material comprises greater than 5% 25 phosphatidyl serine.

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

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

7. The process of claim 1 wherein the feed material comprises greater than 15% sphingomyelin.

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

9. The process of claim 1 wherein the feed material comprises greater than 2% cardiolipin.

10. The process of claim 1 wherein the feed material comprises greater than 5% cardiolipin.

11. The process of claim 1 wherein the feed material comprises greater than 0.3% gangliosides.

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

13. The process of claim 1 wherein the feed material comprises greater than 2% gangliosides.

14. The process of claim 1 wherein the feed material comprises greater than 0.5% acylalkyphospholipids and/or plasmalogens.

15. The process of claim 1 wherein the feed material comprises greater than 2% acylalkyphospholipids and/or plasmalogens.

16. The process of claim 1 wherein the feed material comprises greater than 10% acylalkyphospholipids and/or plasmalogens.

17. The process of claim 1 wherein the feed material comprises greater than 0.5% aminoethylphosphonate and/or other phosphonolipids.

18. The process of claim 1 wherein the feed material comprises greater than 5% aminoethylphosphonate and/or other phosphonolipids.

19. The process of claim 1 wherein the feed material comprises greater than 20% aminoethylphosphonate and/or other phosphonolipids.

20. A process for separating a feed material into soluble and insoluble components, comprising

(h) 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 aminoethylphosphonate and/or other phosphonolipids

(v) at least 1% by mass cardiolipin, or

(vi) at least 0.3% by mass gangliosides

(i) providing a first solvent comprising supercritical or near-critical CO₂

(j) contacting the feed material and the first solvent and subsequently separating the first solvent containing the first soluble components from the first insoluble components

(k) optionally separating the first soluble components and the first solvent

(1) providing a second solvent comprising:

(iii) supercritical or near-critical CO_2> and

(iv)a co-solvent comprising one or more Ci-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

(m) contacting the first insoluble components and the second solvent and subsequently separating the second solvent containing the second soluble components from the second insoluble components

(n) 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% Ci-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 claim 20 or claim 21 wherein the feed material comprises greater than 2% phosphatidyl serine.

24. The process of claim 20 or claim 21 wherein the feed material comprises greater than 5% phosphatidyl serine.

25. The process of claim 20 or claim 21 wherein the feed material comprises greater than 1% sphingomyelin.

26. The process of claim 20 or claim 21 wherein the feed material comprises greater than 5% sphingomyelin.

27. The process of claim 20 or claim 21 wherein the feed material comprises greater than 15% sphingomyelin.

28. The process of claim 20 or claim 21 wherein the feed material comprises greater than 1% cardiolipin.

29. The process of claim 20 or claim 21 wherein the feed material comprises greater than 2% cardiolipin.

30. The process of claim 20 or claim 21 wherein the feed material comprises greater than 5% cardiolipin.

31. The process of claim 20 or claim 21 wherein the feed material comprises greater than 0.3% gangliosides.

32. The process of claim 20 or claim 21 wherein the feed material comprises greater than 1% gangliosides.

33. The process of claim 20 or claim 21 wherein the feed material comprises greater than 2% gangliosides.

34. The process of claim 20 or claim 21 wherein the feed material comprises greater than 0.5% acylalkyphospholipids and/or plasmalogens.

35. The process of claim 20 or claim 21 wherein the feed material comprises greater than 2% acylalkyphospholipids and/or plasmalogens.

36. The process of claim 20 or claim 21 wherein the feed material comprises greater than 10% acylalkyphospholipids and/or plasmalogens.

37. The process of claim 20 or claim 21 wherein the feed material comprises greater than 0.5% aminoethylphosphonate and/or other phosphonolipids.

38. The process of claim 20 or claim 21 wherein the feed material comprises greater than 5% aminoethylphosphonate and/or other phosphonolipids.

39. The process of claim 20 or claim 21 wherein the feed material comprises greater than 20% aminoethylphosphonate and/or other phosphonolipids.

40. The process of any one of claim 1 to 39 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, 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 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, and a composition comprising marine lipids.

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.

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.

49. The process of claim 48 wherein the feed material comprises at least:

(a) 1% phosphatidyl serine,

(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 to 50 wherein the feed material has been genetically modified.

52. The process of any one of claims 1 to 51 wherein the feed material is in solid form.

53. The process of claim 52 wherein the feed material is cryomilled before contact with a solvent.

54. The process of any one of claims 1 to 53 wherein the co-solvent comprises:

(b) 0-40% by mass water.

55. The process of claim 54 wherein the co-solvent comprises between 0 and 20% by mass water.

56. The process of claim 54 wherein the co-solvent comprises between 1 and 10% by mass water.

57. The process of any one of claims 54 to 56 wherein the alcohol is ethanol.

58. The process of any one of claims 1 to 57 wherein the co-solvent is 95% aqueous ethanol.

59. The process of any one of claims 1 to 58 wherein the mass fraction of the co-solvent in CO₂ is between 5% and 60%.

60. The process of claim 59 wherein the mass fraction is between 20% and 50%.

61. The process of claim 59 wherein the mass fraction is between 25% and 30%.

62. The process of any one of claims 1 to 61 wherein the contacting temperature between the feed material and solvent is between 10°C and 8O⁰C.

63. The process of claim 62 wherein the contacting temperature is between 55°C and 65°C.

64. The process of any one of claims 1 to 63 wherein the contacting pressure is between 100 bar and 500 bar.

65. The process of claim 64 wherein the contacting pressure is between 200 bar and 300 bar.

66. The process of any one of claims 1 to 65 wherein the ratio of the co-solvent to feed material is in the range 10:1 to 200:1.

67. The process of claim 66 wherein the ratio of the co-solvent to feed material is in the range 15:1 to 50:1.

68. The process of any one of claims 1 to 67 wherein the separating pressure is between atmospheric pressure and 90 bar.

69. The process of claim 68 wherein the separating pressure is between 40 bar and 60 bar.

70. The process of any one of claims 1 to 69 wherein the co-solvent is recycled for further use.

71. The process of any one of claims 1 to 70 wherein the CO₂ is recycled for further use.

72. The process of any one of claims 1 to 71 wherein the co-solvent is removed by evaporation under vacuum.

73. The process of any one of claims 1 to 72 wherein the feed material is contacted with a continuous flow of solvent.

74. The process of any one of claims 1 to 72 wherein the feed material is contacted with one or more batches of solvent.

75. The process of any one of claims 1 to 73 wherein the lipid and solvent streams are fed continuously.

76. The process of any one of claims 1 to 75 wherein the feed material and co-solvent streams are mixed prior to contacting with CO₂.

77. A product produced by the process of any one of claims 1 to 76.

78. The product of claim 77 wherein the product contains more sphingomyelin than the feed material.

79. The product of claim 77 wherein the product comprises greater than 3% sphingomyelin.

80. The product of claim 77 wherein the product comprises greater than 10% sphingomyelin.

81. The product of claim 77 wherein the product comprises greater than 15% sphingomyelin.

82. The product of claim 77 wherein the product contains more phosphatidyl serine than the feed material.

83. The product of claim 77 wherein the product comprises greater than 5% phosphatidyl serine.

84. The product of claim 77 wherein the product comprises greater than 30% phosphatidyl serine.

85. The product of claim 77 wherein the product comprises greater than 70% phosphatidyl serine.

86. The product of claim 77 wherein the product contains more gangliosides than the feed material.

87. The product of claim 77 wherein the product comprises greater than 2% gangliosides.

88. The product of claim 77 wherein the product comprises greater than 4% gangliosides.

89. The product of claim 77 wherein the product comprises greater than 6% gangliosides.

90. The product of claim 77 wherein the product contains more cardiolipin than the feed material.

91. The product of claim 77 wherein the product comprises greater than 5% cardiolipin.

92. The product of claim 77 wherein the product comprises greater than 10% cardiolipin.

93. The product of claim 77 wherein the product comprises greater than 25% cardiolipin.

94. The product of claim 77 wherein the product contains more acylalkyphospholipids and/or plasmalogens than the feed material.

95. The product of claim 77 wherein the product comprises greater than 5% acylalkyphospholipids and/or plasmalogens.

96. The product of claim 77 wherein the product comprises greater than 10% acylalkyphospholipids and/or plasmalogens.

97. The product of claim 77 wherein the product comprises greater than 25% acylalkyphospholipids and/or plasmalogens.

98. The product of claim 77 wherein the product contains more aminoethylphosphonate and/or other phosphonolipids than the feed material.

99. The product of claim 77 wherein the product comprises greater than 5% aminoethylphosphonate and/or other phosphonolipids.

100. The product of claim 77 wherein the product comprises greater than 10% aminoethylphosphoriate and/or other phosphonolipids.

101. The product of claim 77 wherein the product comprises greater than 25% aminoethylphosphonate and/or other phosphonolipids.