(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`I lllll llllllll II llllll lllll lllll lllll llll I II Ill lllll lllll lllll 111111111111111111111111111111111
`
`( 43) International Publication Date
`1 November 2007 (01.11.2007)
`
`PCT
`
`(51) International Patent Classification:
`CllB 7100 (2006.01)
`A23L 1148 (2006.01)
`CllB 1110 (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:
`
`English
`
`English
`
`(30) Priority Data:
`546681
`
`20 April 2006 (20.04.2006) NZ
`
`(71) Applicants and
`(72) Inventors: CATCHPOLE, Owen John, [NZ/NZ]; C/(cid:173)
`Industrial Research Limited, Gracefield Research Centre,
`Gracefield Road, Lower Hutt (NZ). TALLON, Stephen
`John, [NZ/NZ]; Cl- Industrial Research Limited, Grace(cid:173)
`field Research Centre, Gracefield Road, Lower Hutt (NZ).
`
`(10) International Publication Number
`WO 2007/123424 Al
`(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,
`Fl, 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, Fl,
`FR, GB, GR, HU, IE, IS, IT, LT, LU, LV, MC, MT, NL, PL,
`PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM,
`GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`with international search report
`
`(74) Agents: ADAMS, Matthew, D et al.; A J Park, 6th Floor
`Huddart Parker Building, Post Office Square, P 0 Box 949,
`Wellington, 6015 (NZ).
`
`For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
`~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
`
`(54) Title: PROCESS FOR SEPARATING LIPID MATERIALS
`
`Flow Meter
`
`Valve2
`
`(57) Abstract:
`invention
`The present
`to processes
`for separating a
`relates
`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
`C02, 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 C02, 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,
`M comprising contacting a feed material and a first solvent and subsequently separating the first solvent containing the first soluble
`,....i components from the first insoluble components, wherein the feed material comprises one or more of: at least 1 % by mass
`i::::: phosphatidyl serine, at least 1 % by mass sphingomyelin, at least 0.3 % by mass acylalkylphospholipids and/or plasmalogens, at
`Q
`least 0.5 % by mass aminoethylphosphonate and/or other phosphonolipids, at least 1 % by mass cardiolipin, or at least 0.3% by
`0 mass gangliosides; and wherein the first solvent comprises supercritical or near-critical C02• The process then provides contacting
`M
`the first insoluble components with a second solvent and subsequently separating the second solvent containing the second soluble
`0 components from the second insoluble components, wherein the second solvent comprises supercritical or near- critical C02, 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 C02, and the water content of the co-solvent is 0 to 40% by mass.
`
`---iiiiiiii
`iiiiiiii ---
`!!!!!!!! -
`
`-
`
`iiiiiiii
`!!!!!!!!
`
`!!!!!!!!
`iiiiiiii
`
`iiiiiiii ----
`
`Carbon Dioxide
`Supply
`
`Co-solvent
`Supply
`
`Cold Trap
`
`Ftrst Separator
`
`Sec.ond Separator
`
`RIMFROST EXHIBIT 1009 page 0000
`
`

`

`WO 2007/123424
`
`PCT /NZ2007 /000087
`
`PRODUCT AND PROCESS
`
`FIELD OF INVENTION
`
`5
`
`This invention relates to a separation process. More particularly it relates to a process for
`
`separating lipid materials containing phospholipids and/or glycolipids, including for example
`
`phosphatidyl serine, gangliosides, cardiolipin, sphingomyelin, plasmalogens,
`
`alkylacylphospholipids, phosphonolipids, cerebrosides or a combination thereof.
`
`BACKGROUND
`
`10
`
`Phospholipids are a major component of all biological membranes, and include
`
`phosphoglycerides (phosphatidyl choline (PC), phosphatidyl ethanolamine (PE),
`
`phosphatidyl inositol (Pl), cardiolipin (CL), phosphatidyl serine (PS)), plasmalogens (PL),
`
`phosphonolipids (PP), alkylacylphospholipids (ALP); and sphingolipids such as
`
`15
`
`sphingomyelin (SM) and ceramide aminoethylphosphonate (CAEP).
`
`Gangliosides are glycolipid components in the cell plasma membrane, which modulate cell
`
`signal transductions events. They are implicated as being important in immunology and
`
`neurodegenerative disorders. Cerebrosides are impmiant components in animal muscle and
`
`nerve cell membranes.
`
`20 Both phospholipids and gangliosides are involved in cell signalling events leading to, for
`
`example, cell death (apoptosis), cell growth, cell proliferation, and cell differentiation.
`
`Reasonable levels of some of these components can be found in milk, soy products, eggs,
`
`animal glands and organs, marine animals, plants and other sources. A source of these
`
`components is the bovine milk fat globule membrane (MFGM) which is known to contain
`
`25
`
`useful quantities of sphingomyelin, ceramides, gangliosides, and phosphatidyl serine.
`
`Another source of these components is the green-shell mussel, which is lmown to contain
`
`useful quantities of plasmalogens, alkylacylphospholipids and ceramide
`
`aminoethylphosphonate
`
`Both phospholipids and gangliosides have been implicated in conferring a number of health
`
`30
`
`benefits including brain health, skin health, eczema treatment, anti-infection, wound healing,
`
`gut microbiota modifications, anti-cancer activity, alleviation of arthritis, improvement of
`
`1
`
`RIMFROST EXHIBIT 1009 page 0001
`
`

`

`WO 2007/123424
`cardiovascular health, and treatment of metabolic syndromes. They can also be used in
`
`PCT /NZ2007 /000087
`
`sports nutrition.
`
`Cardiolipin is an important component of the inner mitochondrial membrane. It is typically
`
`present in metabolically active cells of the heart and skeletal muscle. It serves as an
`
`5
`
`insulator and stabilises the activity of protein complexes important to the electron transport
`
`chain.
`
`Existing methods for isolation of these compounds rely on the use of chromatographic
`
`techniques, which are slow and costly processes to operate. These techniques can also
`
`require the use of solvents that are unsuitable and/or undesirable in products for nutritional
`
`10
`
`or human use. For example, Palacios and Wang [ 1] describe a process for extraction of
`
`phospholipids from egg yolks using acetone and ethanol extractions, followed by a
`
`methanol/chloroform separation. Kang and Row [2] describe a liquid chromatography
`
`process for separation of soybean derived PC from PE and PI. This process may be
`
`expensive to carry out on an industrial scale, and also uses hexane, methanol, and isopropyl
`
`15
`
`alcohol as solvents. Kearns et al [3] describe a process for purification of egg yolk derived
`
`PC from PE using mixtures of acetonitrile, hydrocarbons, and fluorocarbons. Again, these
`
`solvents are undesirable for nutritional or pharmaceutical use.
`
`Supercritical fluid extraction processes using C02 are becoming increasingly popular
`
`because of a number of processing and consumer benefits. C02 can be easily removed from
`
`20
`
`the final product by reducing the pressure, whereupon the C02 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 C02 in place of conventional organic
`
`solvents also confers environmental benefits through reduced organic solvent use. The
`
`disadvantage of supercritical C02 processing is that the solubility of many compounds in
`
`25 C02 is low, and only neutral lipids can be extracted.
`
`It is known that the use of C02 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 C02 at 60°C they found that PC was easily extracted, while PE and PI
`30 were extracted to a very low extent. Extraction at 12.5 % ethanol at 80°C gave a four-fold
`
`increase in solubility of PC. Montanari et al [5] describe a process for extracting
`
`phospholipids from soybean flakes. After first extracting neutral lipids using only C02 at 320
`
`bar, they found that using 10 % ethanol co-solvent at pressures of 194 to 689 bar resulted in
`
`2
`
`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 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
`
`5
`
`fatty acids to be degraded. Taylor et al [6] describe a process in which soybean flakes are
`
`first extracted using only C02, followed by C02 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
`
`Tanaka and Sakaki [7] describe a method for extracting phospholipids from waste tuna
`
`shavings using C02 and ethanol as a co-solvent. They describe extraction ofDHA(cid:173)
`containing phospholipids using 5 % ethanol in C02, and by presoaking the tuna flakes in
`straight ethanol and then extracting using C02. The phospholipids obtained in this process
`
`are not specified and no fractionation of the different phospholipids is described. In addition,
`
`15
`
`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 C02 and 3 % ethanol, and C02
`with up to 5 % methanol. Higher rates of triglyceride extraction were obtained with the use
`
`of the co-solvent. Extraction of small amounts of phospholipids, up to 17% concentration in
`
`20
`
`the extract, was also achieved. Fractionation of the phospholipids is not described.
`
`In this specification where reference has been made to patent specifications, other external
`
`documents, or other sources of information, this is generally for the purpose of providing a
`
`context for discussing the features of the invention. Unless specifically stated otherwise,
`
`reference to such external documents or such sources of information is not to be construed as
`
`25
`
`an admission that such documents or such sources of information, in any jurisdiction, are
`
`prior art or form part of the common general knowledge in the art.
`
`It is an object of this invention to provide a process for producing a product that contains
`
`desirable levels of particular phospholipids and/or gangliosides and/or cerebrosides, or at
`
`least to offer the public a useful choice.
`
`3
`
`RIMFROST EXHIBIT 1009 page 0003
`
`

`

`WO 2007/123424
`
`SUMMARY OF INVENTION
`
`PCT /NZ2007 /000087
`
`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 9f:
`
`5
`
`(i) at least 1 % by mass phosphatidyl serine
`
`(ii) at least 1 % by mass sphingomyelin
`
`(iii) at least 0.3 % by mass acylalkylphospholipids and/or plasmalogens
`
`(iv)at least 0.5 % by mass arninoethylphosphonate and/or other phosphonolipids
`
`(v) at least 1 % by mass cardiolipin
`
`10
`
`(vi) at least 0.3% by mass gangliosides
`
`(b) providing a solvent comprising:
`
`(i) supercritical or near-critical C02, 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 C02, and the water content
`of the co-solvent is 0 to 40 % by mass
`
`15
`
`( 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 page 0004
`
`

`

`WO 2007/123424
`Alternatively the feed material comprises greater than 1 % cardiolipin. More preferably the
`
`PCT /NZ2007 /000087
`
`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
`
`5
`
`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
`
`10
`
`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.
`
`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 C02
`
`25
`
`( 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) optiona!ly separating the first soluble components and the first solvent
`
`5
`
`RIMFROST EXHIBIT 1009 page 0005
`
`

`

`WO 2007/123424
`( e) providing a second solvent comprising:
`
`PCT /NZ2007 /000087
`
`(i) supercritical or near-critical C02, 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 C02, and the water content
`of the co-solvent is 0 to 40% by mass
`
`5
`
`(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.
`
`10
`
`Preferably the first solvent comprises a mixture of supercritical or near-critical C02 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.
`
`15 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
`
`20
`
`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
`
`25
`
`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.
`
`6
`
`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.
`
`5
`
`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
`
`10
`
`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.
`
`15 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.
`
`20
`
`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
`
`25
`
`(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.
`
`7
`
`RIMFROST EXHIBIT 1009 page 0007
`
`

`

`PCT /NZ2007 /000087
`WO 2007/123424
`Preferably the solvent used in the process of the present invention comprises 95% aqueous
`
`ethanol.
`
`Preferably the mass fraction of the co-solvent in C02 is between 5% and 60%. More
`
`preferably the mass fraction is between 20% and 50%. Most preferably the mass fraction is
`
`5
`
`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 C02 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.
`
`20 Optionally, the feed material and co-solvent streams may be mixed prior to contacting with
`
`COz.
`
`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 number of steps,
`
`there will be multiple "extract" products.
`
`8
`
`RIMFROST EXHIBIT 1009 page 0008
`
`

`

`WO 2007/123424
`Preferably the product contains more sphingomyelin than the feed material. More preferably
`
`PCT /NZ2007 /000087
`
`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.
`
`5
`
`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
`
`10
`
`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
`
`15
`
`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
`
`20
`
`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
`
`25
`
`comprises greater than 10% aminoethylphosphonate and/or other phosphonolipids. Most
`
`preferably the product comprises greater than 25% aminoethylphosphonate and/or other
`
`phosphonolipids.
`
`9
`
`RIMFROST EXHIBIT 1009 page 0009
`
`

`

`WO 2007/123424
`
`BRIEF DESCRIPTION OF THE DRAWINGS
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`PCT /NZ2007 /000087
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`The invention may be more fully understood by having reference to the accompanying
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`drawings wherein:
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`Figure 1 is scheme drawing illustrating a preferred process of the current invention.
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`Figure 2 is a scheme drawing illustrating a second preferred process of the current
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`invention
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`Figure 3 is a scheme drawing illustrating a third preferred process of the current invention
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`Figure 4 is a scheme drawing illustrating a fourth preferred process of the current invention
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`10 ABBREVIATIONS AND ACRONYMS
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`In this specification the following are the meanings of the abbreviations or acronyms used.
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`"CL" means cardiolipin
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`"PC" means phosphatidyl choline
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`"PI" means phosphatidyl inositol
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`"PS" means phosphatidyl serine
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`"PE" means phosphatidyl ethanolamine
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`"PA" means phosphatidic acid
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`"PL" means plasmalogen
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`"PP" means phosphonolipid
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`"ALP" means alkylacylphospholipid
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`"SM" means sphingomyelin
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`"CAEP" means ceramide aminoethylphosphonate
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`"GS" means ganglioside
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`"N/D" means not detected
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`"C02" means carbon dioxide
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`RIMFROST EXHIBIT 1009 page 0010
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`WO 2007/123424
`GENERAL DESCRIPTION OF THE INVENTION
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`PCT /NZ2007 /000087
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`As discussed in the Background, it is known that supercritical C02 with up to 12.5% ethanol
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`as a co-solvent can extract the phospholipids PC, and to a much lesser extent, PE and PI
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`from soy or egg. Surprisingly, we have found that the phospholipids PS, CAEP and CL; and
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`gangliosides are virtually insoluble in C02 and a C1-C3 monohydric alcohol co-solvent, and
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`that SM, ALP, PL and PP are soluble. Therefore it is possible to separate the soluble
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`phospholipids from the insoluble phospholipids and gangliosides to achieve fractions
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`enriched in one or other of the desired components.
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`There are a number of factors affecting the operation of the process:
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`• Feed material and feed preparation
`• Extraction temperature and pressure
`• Co-solvent concentration
`• Total solvent throughput
`• Solvent flow rate and contacting conditions
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`It is advantageous to start with a feed material containing at least 5 % by mass of lipids, and
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`ideally at least 2 % by mass of phospholipids, particularly PS, SM, CL, ALP, PL, PP, CAEP
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`and/or gangliosides.
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`The feed material can be processed using pure C02 before the co-solvent is introduced to
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`remove much or all of neutral iipids. This reduces the neutral lipid content in the C02+co-
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`solvent extract leading to an extract enriched in soluble phospholipids and/or gangliosides.
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`The form of the feed material depends on the source of the lipids and its lipid composition.
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`For example dairy lipid extracts high in phospholipids may be substantially solid even at
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`elevated temperatures. Egg yolk and marine lipids in comparison have a lower melting point.
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`The presence of neutral lipids also tends to produce a more fluid feed material. To promote
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`good contacting it may be beneficial to prepare the feed material. Solid materials containing
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`lipids may be able to be cryomilled. Lipid feed materials can also be made more fluid by the
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`inclusion of some ethanol or water.
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`RIMFROST EXHIBIT 1009 page 0011
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`WO 2007/123424
`Changing the processing conditions of temperature, pressure, co-solvent concentration, and
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`PCT /NZ2007 /000087
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`total solvent usage, influences the amount of material extracted, the purity of the final
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`product, and the recovery (or efficiency) of the process. For example, the virtually insoluble
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`lipids such as PS, GS, CAEP and CL, have very slight solubilities so that excessive use of
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`solvent, or very favourable extraction conditions, can result in small losses of PS, GS and CL
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`from the residual fraction. A high purity product may be achieved, but with a reduced yield.
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`Conversely the enrichment of soluble lipids will be greater if smaller amounts of the other
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`lipids are co-extracted, but the total yield will be lower. Processing economics, and the
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`relative values of the products, will determine where this balance lies. A further option to
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`obtain multiple enriched fractions is to carry out extractions under progressively more
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`favourable extraction conditions, such as increasing the temperature.
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`We have found that co-solvent concentrations below about 10% produce very little extract of
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`phospholipids and/or gangliosides. At higher concentrations the rate of material extracted
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`increases rapidly. We have found the co-solvent concentrations of at least 20%, and more
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`preferably 30% achieve high levels of extraction of PC, PE, SM, ALP, PL, PP and PI, while
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`the lipids PS, CL and GS remain virtually insoluble.
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`Every substance has its own "critical" point at which the liquid and vapour state of the
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`substance become identical. Above but close to the critical point of a substance, the
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`substance is in a fluid state that has properties of both liquids and gases. The fluid has a
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`density similar to a liquid, and viscosity and diffusivity similar to a gas. The term
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`"supercritical" as used herein refers to the pressure-temperature region above the critical
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`point of a substance. The term "subcritical" as used herein refers to the pressure-temperature
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`region equal to or above the vapour pressure for the liquid, but below the critical
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`temperature. The term "near-critical" as used herein encompasses both "supercritical" and
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`"subcritical" regions, and refers to pressures and temperatures near the critical point.
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`Percentages unless otherwise indicated are on a w/w solids basis.
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`The term "comprising" as used in this specification means "consisting at least in part of'.
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`When interpreting each statement in this specification that includes the term "comprising'',
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`features other than that or those prefaced by the term may also be present. Related terms
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`such as "comprise" and "comprises" are to be interpreted in the same manner.
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`RIMFROST EXHIBIT 1009 page 0012
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`WO 2007/123424
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`PCT /NZ2007 /000087
`The invention consists in the foregoing and also envisages constructions of which the
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`following gives examples only.
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`EXAMPLES
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`The experimental process is described, with reference to figure 1, as follows.
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`A measured mass of feed material containing lipids to be fractionated was placed in basket
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`BKl with a porous sintered steel plate on the bottom. Basket BKl was placed in a 300 mL
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`extraction vessel EXl. The apparatus was suspended in heated water bath WB 1 and
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`10 maintained at a constant temperature through use of a thermostat and electric heater.
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`In the continuous extraction mode of operation, liquid C02 from supply bottle B 1 was
`pumped using pump Pl into extraction vessel EXl until the pressure reached the desired
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`operating pressure, after which valve Vl was operated to maintain a constant pressure in the
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`extraction vessel. After passing through valve Vl, the pressure was reduced to the supply
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`cylinder pressure of 40 to 60 bar, which caused the C02 to be converted to a lower density
`fluid and lose its solvent strength. Precipitated material was captured in separation vessel
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`SEPl, and the C02 exited from the top of separator SEPl and was recycled back to the feed
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`pump through coriolis mass flow meter FMl and cold trap CTI operated at -5°C. Extracted
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`20 material was collected periodically from separator SEPl by opening valve V2. The
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`extraction was optionally carried out using C02 only until all of the compounds soluble in
`C02 only, such as neutral lipids, were extracted. When no further extract was produced by
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`C02 extraction, ethanol co-solvent with or without added water was added to the C0