(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY(PCT)
`
`(19) World Intellectual Property Organization { APi
`International Bureau
`
`se) ANTAL UMARUT
`
`(43) International Publication Date
`1 November 2007 (01.11.2007)
`
`(10) International Publication Number
`WO 2007/123424 Al
`
`
`
`(51) International Patent Classification:
`C1IB 7/00 (2006.01)
`A23L 1/48 (2006.01)
`C1IB 1/10 (2006.01)
`
`(21) International Application Number:
`PCT/NZ2007/000087
`
`(22) International Filing Date:
`
`20 April 2007 (20.04.2007)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`(30) Priority Data:
`546681
`
`English
`
`English
`
`20 April 2006 (20.04.2006) NZ
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BH, BR, BW,BY, BZ, CA, CH,
`CN, CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES,
`FI, GB, GD, GE, GH, GM, GT, HN, HR, HU,ID,IL, IN,
`IS, JP, KE, KG, KM,KN,KP, KR, KZ, LA, LC, LK, LR,
`LS, LT, LU, LY, MA, MD, MG, MK, MN, MW,MX, MY,
`MZ, NA, NG,NI, NO, NZ, OM,PG, PH, PL, PT, RO, RS,
`RU, SC, SD, SE, SG, SK, SL, SM, SV, SY, TJ, TM, TN,
`TR,TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM,KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU,TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK,EE,ES, FI,
`FR, GB, GR,HU,IE,IS, IT, LT, LU, LV, MC, MT, NL,PL,
`PT, RO,SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM,
`GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`Published:
`
`(71) Applicants and
`(72) Inventors: CATCHPOLE, Owen John, [NZ/NZ]; C/-
`Industrial Research Limited, Gracefield Research Centre,
`Gracefield Road, Lower Hutt (NZ). TALLON,Stephen
`John, [NZ/NZ]; C/- Industrial Research Limited, Grace-
`field Research Centre, Gracefield Road, Lower Hutt (NZ).
`—_with international search report
`
`(74) Agents: ADAMS,Matthew, D et al.; A J Park, 6th Floor
`Huddart Parker Building, Post Office Square, PO Box 949,
`Wellington, 6015 (NZ).
`
`For two-letter codes and other abbreviations, refer to the "Guid-
`ance Notes on Codes and Abbreviations" appearing at the begin-
`ning of each regular issue of the PCT Gazette.
`
`(54) Title: PROCESS FOR SEPARATING LIPID MATERIALS
`
`Flow Meter
`
`
`
`invention
`The present
`(57) Abstract:
`relates
`to processes
`for
`separating a
`feed material
`into soluble and insoluble
`
`Cold Trap
`
`Raffinate
`
`First Separator
`
`Second Separator
`
`Co-solvent
`Supply
`
`
`
`7/123424AXTINIANNA
`
`Lipid Feed
`components, a_feedby contacting
`
`
`
`Valve 1
`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
`Carbon Dioxide
`plasmalogens,
`at
`least 0.5 % by mass
`Supply
`
`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
`CO,,
`and a co-solvent comprising one
`or more C,-C3; monohydric alcohols, and
`water, wherein the co-solvent makes up at least 10% by mass of the CO,, 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 CO,, and a
`co-solvent comprising one or more C,-C; monohydric alcohols, and water, wherein the co-solvent makes up at least 10% by mass
`
`s of the CO,, and the water content of the co-solvent is 0 to 40% by mass.
`
`RIMFROST EXHIBIT 1009
`
`RIMFROST EXHIBIT 1009 page 0000
`
`page 0000
`
`

`

`WO2007/123424
`
`PCT/NZ2007/000087
`
`PRODUCT AND PROCESS
`
`FIELD OF INVENTION
`
`This invention relates to a separation process. Moreparticularly 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
`
`10
`
`15
`
`Phospholipids are a major componentof 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.
`
`20
`
`Both phospholipids and gangliosides are involvedin cell signalling events leadingto, 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 ofthese
`
`componentsis the bovine milk fat globule membrane (MFGM) which is known to contain
`useful quantities of sphingomyelin, ceramides, gangliosides, and phosphatidylserine.
`
`25
`
`Another source of these componentsis the green-shell mussel, which is known to contain
`
`useful quantities of plasmalogens, alkylacylphospholipids and ceramide
`
`aminoethylphosphonate
`
`30
`
`Both phospholipids and gangliosides have been implicated in conferring a numberof health
`benefits including brain health, skin health, eczema treatment, anti-infection, woundhealing,
`gut microbiota modifications, anti-cancer activity, alleviation of arthritis, improvement of
`
`RIMFROST EXHIBIT 1009=page 0001
`
`RIMFROST EXHIBIT 1009 page 0001
`
`

`

`WO 2007/123424
`PCT/NZ2007/000087
`cardiovascular health, and treatment of metabolic syndromes. They can also be used in
`sports nutrition.
`
`Cardiolipin is an important componentof the inner mitochondrial membrane. It is typically
`present in metabolically active cells of the heart and skeletal muscle. It serves as an
`insulator andstabilises the activity ofprotein complexes important to the electron transport
`chain.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Existing methodsfor isolation ofthese compoundsrely on the use of chromatographic
`techniques, which are slow andcostly 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
`alcoholas solvents. Kearnset al [3] describe a process for purification of egg yolk derived
`PC from PE using mixtures ofacetonitrile, hydrocarbons, and fluorocarbons. Again, these
`solvents are undesirable for nutritional or pharmaceuticaluse.
`
`Supercritical fluid extraction processes using CO, are becomingincreasingly popular
`because of a numberofprocessing and consumerbenefits. CO, can be easily removed from
`the final product by reducing the pressure, whereupon the CO>reverts to a gaseousstate,
`giving a completely solvent free product. The extract is considered to be more ‘natural’ than
`extracts produced using other solvents, and the use of CO, 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 ofmany compoundsin
`COyis low, and only neutral lipids can be extracted.
`
`It is known that the use of CO, with organic co-solvents such as ethanol allows extraction of
`some phosphatidyl choline and to a muchlesser extent phosphatidyl ethanolamine. For
`example, Teberikler et al [4] describe a process for extraction of PC from a soybean lecithin.
`Using 10% ethanol in CO, at 60°C they found that PC waseasily 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. Montanariet al [5] describe a process for extracting
`phospholipids from soybean flakes. After first extracting neutral lipids using only CO} at 320
`bar, they found that using 10 % ethanol co-solventat pressures of 194 to 689 bar resulted in
`
`RIMFROST EXHIBIT 1009=page 0002
`
`RIMFROST EXHIBIT 1009 page 0002
`
`

`

`WO 2007/123424
`
`PCT/NZ2007/000087
`
`some extraction of PC, PE, PI, and phosphatidic acid (PA). PC is selectively extracted under
`some conditions, but at higher temperatures and pressures someextraction 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.Tayloret al [6] describe a process in which soybean flakesare
`first extracted using only CO>2, followed by CO, 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.
`
`10
`
`15
`
`20
`
`Tanaka and Sakaki [7] describe a method for extracting phospholipids from waste tuna
`shavings using CO) and ethanol as a co-solvent. They describe extraction of DHA-
`containing phospholipids using 5 % ethanol in CO, and by presoakingthe tuna flakes in
`straight ethanol and then extracting using CO2. The phospholipids obtainedin this process
`are not specified and nofractionation of the different phospholipids is described. In addition,
`the phospholipids fraction makes upa relatively small proportion ofthe total processed
`material, requiring use of large pressure vessels to produce a small yield of phospholipids.
`
`Bulleyet al [8] describe extraction of frozen egg yolks using CO, and 3 % ethanol, and CO
`with up to 5 % methanol. Higherrates 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 phospholipidsis 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 purposeof 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
`
`priorart or form part of the common general knowledgein the art.
`
`It is an object of this invention to provide a process for producing a productthat contains
`desirable levels of particular phospholipids and/or gangliosides and/or cerebrosides,orat
`least to offer the public a useful choice.
`
`RIMFROST EXHIBIT 1009
`
`RIMFROST EXHIBIT 1009 page 0003
`
`page 0003
`
`

`

`WO2007/123424
`
`PCT/NZ2007/000087
`
`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 moreof:
`
`5
`
`(i) at least 1% by mass phosphatidyl serine
`
`(i1) 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 masscardiolipin
`
`10
`
`(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 C;-C3 monohydric alcohols, and water
`
`wherein the co-solvent makes up at least 10% by mass of the COz2, and the water content
`
`15
`
`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
`
`20
`
`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.
`
`4
`
`RIMFROST EXHIBIT 1009
`
`RIMFROST EXHIBIT 1009 page 0004
`
`page 0004
`
`

`

`WO2007/123424
`
`PCT/NZ2007/000087
`
`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. Mostpreferably the feed material
`comprises greater than 2% gangliosides.
`
`Alternatively the feed material comprises greater than 0.5% acylalkyphospholipids and/or
`plasmalogens. Morepreferably the feed material comprises greater than 2%
`acylalkyphospholipids and/or plasmalogens. Mostpreferably the feed material comprises
`greater than 10% acylalkyphospholipids and/or plasmalogens.
`|
`
`10
`
`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.
`
`15
`
`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,
`
`20
`
`(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 CO.
`
`20
`
`(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 andthefirst solvent
`
`RIMFROST EXHIBIT 1009
`
`RIMFROST EXHIBIT 1009 page 0005
`
`page 0005
`
`

`

`WO 2007/123424
`
`PCT/NZ2007/000087
`
`(ce) providing a second solvent comprising:
`
`(i)
`
`supercritical or near-critical CO2, and
`
`(ii) a co-solvent comprising one or more C;-C3 monohydric alcohols, and water
`
`wherein the co-solvent makes up at least 10% by massof the CO», and the water content
`of the co-solvent is 0 to 40% by mass
`
`5
`
`(f) contacting the first insoluble components andthe 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.
`
`10
`
`Preferably the first solvent comprises a mixture of supercritical or near-critical COand less
`than 10% C,-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% phosphatidylserine.
`
`Alternatively the feed material comprises greater than 1% sphingomyelin. Preferably the
`feed material comprises greater than 5% sphingomyelin. Morepreferably 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%
`acylallcyphospholipids and/or plasmalogens. More preferably the feed material comprises
`greater than 10% acylalkyphospholipids and/or plasmalogens.
`
`15
`
`20
`
`25
`
`RIMFROST EXHIBIT 1009=page 0006
`
`RIMFROST EXHIBIT 1009 page 0006
`
`

`

`WO 2007/123424
`
`PCT/NZ2007/000087
`
`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.
`
`10
`
`Preferably the feed material is derived from dairy material, soy material, eggs, animaltissue,
`animal organ or animal blood. Morepreferably the feed material is selected from: a
`composition comprising dairy lipids, a composition comprising egg lipids, and a
`
`composition comprising marinelipids.
`
`15
`
`Mostpreferably 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.
`
`20
`
`Preferably, the feed material is in solid form. Whensolid, the feed material may be
`
`cryomilled before contact with the solvent.
`
`The solvent of the present invention preferably comprises:
`
`(a) an alcoholselected from: methanol, ethanol, n-propanol, isopropanol and
`
`mixtures thereof; and
`
`25
`
`(b) 0 —40% v/v water
`
`More preferably the solvent comprises between 0 and 20% w/v water. Most preferably the
`solvent comprises between 1 and 10% v/v water.
`
`Preferably the alcohol is ethanol.
`
`RIMFROST EXHIBIT 1009
`
`RIMFROST EXHIBIT 1009 page 0007
`
`page 0007
`
`

`

`WO 2007/123424
`
`PCT/NZ2007/000087
`
`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 massfraction 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
`
`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
`
`10
`
`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.
`
`15
`
`Preferably the COz 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 batchesofsolvent.
`
`The lipid and solvent streams may be fed continuously.
`
`20
`
`Optionally, the feed material and co-solvent streams may be mixedprior to contacting with
`
`CO>.
`
`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
`
`25
`
`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 numberofsteps,
`
`there will be multiple “extract” products.
`
`RIMFROST EXHIBIT 1009
`
`RIMFROST EXHIBIT 1009 page 0008
`
`page 0008
`
`

`

`WO 2007/123424
`
`PCT/NZ2007/000087
`
`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 phosphatidylserine 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% phosphatidylserine.
`
`Preferably the product contains more gangliosides than the feed material. More preferably
`the product comprises greater than 2% gangliosides. Even morepreferably the product
`comprises greater than 4% gangliosides. Most preferably the product comprises greater than
`6% gangliosides.
`
`Preferably the product contains morecardiolipin than the feed material. More preferably the
`product comprises greater than 5% cardiolipin. Even morepreferably 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 morepreferably the product comprises
`greater than 10% acylalkyphospholipids and/or plasmalogens. Mostpreferably the product
`comprises greater than 25% acylalkyphospholipids and/or plasmalogens.
`
`Preferably the product contains more aminoethylphosphonate and/orother 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/orother
`phosphonolipids,
`
`10
`
`15
`
`20
`
`25
`
`RIMFROST EXHIBIT 1009
`
`RIMFROST EXHIBIT 1009 page 0009
`
`page 0009
`
`

`

`WO 2007/123424
`
`PCT/NZ2007/000087
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`The invention may be more fully understood by having reference to the accompanying
`
`drawings wherein:
`
`Figure 1 is scheme drawingillustrating a preferred processofthe current invention.
`Figure 2 is a scheme drawing illustrating a second preferred process of the current
`
`invention
`Figure 3 is a scheme drawingillustrating a third preferred processof the current invention
`Figure 4 is a scheme drawingillustrating a fourth preferred process of the current invention
`
`10
`
`ABBREVIATIONS AND ACRONYMS
`In this specification the following are the meaningsof the abbreviations or acronyms used.
`
`“CL” means cardiolipin
`
`“PC” means phosphatidyl! choline
`
`“PT” means phosphatidyl inositol
`
`15
`
`“PS” means phosphatidyl serine
`
`“PE” means phosphatidyl ethanolamine
`
`“PA” means phosphatidic acid
`
`“PL” means plasmalogen
`
`“Pp” means phosphonolipid
`
`20
`
`“ALP” means alkylacylphospholipid
`
`“SM” means sphingomyelin
`
`“CAEP” means ceramide aminoethylphosphonate
`
`“GS” means ganglioside
`
`“N/D” meansnot detected
`
`25
`
`“CO,” means carbon dioxide
`
`10
`
`RIMFROST EXHIBIT 1009
`
`RIMFROST EXHIBIT 1009 page 0010
`
`page 0010
`
`

`

`WO2007/123424
`
`PCT/NZ2007/000087
`
`GENERAL DESCRIPTION OF THE INVENTION
`
`Asdiscussedin the Background,it is known that supercritical CO» with up to 12.5% ethanol
`as a co-solvent can extract the phospholipids PC, and to a muchlesser 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 C)-C3 monohydric alcohol co-solvent, and
`that SM, ALP, PL and PPare soluble. Thereforeit is possible to separate the soluble
`phospholipids from the insoluble phospholipids and gangliosides to achieve fractions
`enriched in one orother of the desired components.
`
`10
`
`There are a numberoffactors 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
`
`20
`
`25
`
`30
`
`It is advantageousto start with a feed material containing at least 5 % by massoflipids, and
`ideally at least 2 % by mass of phospholipids, particularly PS, SM, CL, ALP, PL, PP, CAEP
`and/or gangliosides.
`
`The feed material can be processed using pure CO, before the co-solventis introduced to
`remove muchorall of neutral lipids. This reduces the neutral lipid content in the CO+co-
`solvent extract leading to an extract enriched in soluble phospholipids and/or gangliosides.
`
`The form of the feed material dependson the sourceofthelipids andits lipid composition.
`For exampledairy lipid extracts high in phospholipids may be substantially solid even at
`elevated temperatures. Egg yolk and marinelipids in comparison have a lower melting point.
`Thepresence of neutrallipids 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 morefluid by the
`inclusion of some ethanolor water.
`
`11
`RIMFROST EXHIBIT 1009=page 0011
`
`RIMFROST EXHIBIT 1009 page 0011
`
`

`

`WO 2007/123424
`PCT/NZ2007/000087
`Changing the processing conditions oftemperature, pressure, co-solvent concentration, and
`total solvent usage, influences the amount ofmaterial extracted, the purity ofthe 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 PS, GS and CL
`from the residualfraction. A high purity product may be achieved, but with a reduced yield,
`Conversely the enrichmentof soluble lipids will be greater if smaller amounts ofthe other
`lipids are co-extracted, but the total yield will be lower. Processing economics, and the
`relative values of the products, will determine wherethis balancelies. A further option to -
`obtain multiple enrichedfractionsis to carry out extractions under progressively more
`favourable extraction conditions, such as increasing the temperature.
`
`We have foundthat co-solvent concentrations below about 10% producevery little extract of
`phospholipids and/or gangliosides. At higher concentrationsthe rate of material extracted
`increases rapidly. We have found the co-solvent concentrationsofat least 20%, and more
`preferably 30% achieve high levels ofextraction ofPC, PE, SM, ALP, PL, PP and PI, while
`the lipids PS, CL and GS remainvirtually insoluble.
`
`Every substance hasits own “critical” point at which the liquid and vapour state ofthe
`substance becomeidentical. Abovebut closeto thecritical point of a substance,the
`substanceis in a fluid state that has properties of both liquids and gases. Thefluid 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 abovethecritical
`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 thecritical
`temperature. The term “near-critical” as used herein encompasses both “supercritical” and
`“subcritical” regions, and refers to pressures and temperatures near thecritical 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”.
`Wheninterpreting each statementin this specification that includes the term “comprising”,
`features other than that or those prefaced by the term mayalso be present. Related terms
`such as “comprise” and “comprises”are to be interpreted in the same manner.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`12
`RIMFROST EXHIBIT 1009=page 0012
`
`RIMFROST EXHIBIT 1009 page 0012
`
`

`

`WO2007/123424
`PCT/NZ2007/000087
`Theinvention consists in the foregoing and also envisages constructions of which the
`
`following gives examples only.
`
`EXAMPLES
`
`The experimental process is described, with referenceto figure 1, as follows.
`
`A measured massof feed material containinglipids to be fractionated was placed in basket
`BK1 with a poroussintered steel plate on the bottom. Basket BK1 was placed in a 300 mL
`extraction vessel EX1. The apparatus was suspended in heated water bath WB1 and
`maintained at a constant temperature throughuse ofa thermostat andelectric heater.
`
`In the continuous extraction modeof operation, liquid CO, from supply bottle B1 was
`pumped using pump P1 into extraction vessel EX1 until the pressure reached the desired
`operating pressure, after which valve V1 wasoperated to maintain a constant pressure in the
`extraction vessel. After passing through valve V1, the pressure was reduced to the supply
`cylinder pressure of 40 to 60 bar, which caused the CO?to be converted to a lower density
`fluid andloseits solvent strength. Precipitated material was captured in separation vessel
`SEP1, and the CO,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
`material was collected periodically from separator SEP1 by opening valve V2. The
`extraction was optionally carried out using CO. only until all of the compounds soluble in
`CO,only, such as neutral lipids, were extracted. When no further extract was produced by
`CO, extraction, ethanol co-solvent with or without added water was added to the CO,at the
`desired flow ratio from supply bottle B2 using pump P2. Ethanol and further extracted
`material were separated from the CO, in separator SEP1 andperiodically removed through
`valve V2. After the desired amountofethanol had been addedthe ethanol flow was stopped
`and the CO; flow continued alone until all the ethanol had been recovered from the system.
`The remaining CO, 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 modeof operation CO> alone wasoptionally passed continuously
`through the apparatus, as for the continuous flow mode of operation, until all CO. alone
`extractable material was removed. The CO.flow was then stopped and valve V1 closed to
`maintain the pressure. Approximately 14