Document made
`
`available under
`
`the
`
`Patent Cooperation Treaty (PCT)
`
`International application number: PCT/NZ2007/000087
`
`International filing date:
`
`20 April 2007 (20.04.2007)
`
`Document type:
`
`Certified copy of priority document
`
`Document details:
`
`Country/Office: NZ
`Number:
`546681
`
`Filing date:
`
`20 April 2006 (20.04.2006)
`
`Date of receipt at the International Bureau:
`
`17 September 2007 (17.09.2007)
`
`Remark:
`
`Priority document submitted or transmitted to the International Bureau in
`compliance with Rule l7.l(a) or (b)
`
`
`
`World Intellectual Property Organization (WIPO) - Geneva, Switzerland
`Organisation Mondiale de la Propriété Intellectuelle (OMPI) - Geneve, Suisse
`
`RIMFROST EXHIBIT 1120 Page 0001
`RIMFROST EXHIBIT 1120 Page 0001
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`
`Ministry nfEconomlc @1
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`
`
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`_
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`was x
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`
`
`Development
`Manatfl élxanga
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`PCT/NZZOO7/000087
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`CERTIFICATE
`
`This certificate is issued in support of an application for Patent registration in a country
`
`outside New Zealand pursuant to the Patents Act 1953 and the Regulations thereunder.
`
`I hereby certify that annexed is a true copy of the Provisional Specification as filed on
`20 April 2006 with an application for Letters Patent number 546681 made by
`
`OWEN JOHN CATCHPOLE and STEPHEN JOHN TALLON.
`
`Dated 17 August 2007.
`
`MM;
`
`Commissioner of Patents, Trade Marks and Designs
`
`Neville Harris
`
`205 Victona Street, Wellin ton
`RIMFROST EXHIBIT 1120 Page 0002
`PO Box 924MMERQISflgtE1XI-IIIBLEd1r1fl9xBageuWGOZ
`Freephone: 0508 4 IPONZ [0508 447 669],Intemationa1 callers dial: +64 3 962 2607, Fax: +64 4 978 3691, www.iponz..govtn2
`
`

`

`
`
`54658?
`
`NEW ZEALAND
`
`PATENTS ACT, 1953
`
`PROVISIONAL SPECIFICATION
`
`PRODUCT AND PROCESS
`
`We, OWEN JOHN CATCHPOLE and STEPHEN JOHN TALLON, both New Zealand
`Industrial Research Limited, Gracefield Research Centre, Gracefield Road, Lower
`citizens c/-
`Hutt, New Zealand, do hereby declare this invention to be described in the following
`
`statement:
`
`‘
`
`‘
`
`VI
`
`‘ INTELLECWAL PROPERTY
`QFFICE OF NZ.
`ZUAPR 2005
`RIMFROST EXHIBIT 1120 Page 0003
`Page 0003
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`1
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`

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`.
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`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, cerebrosides, sphingomyelin, or a combination
`
`thereof.
`
`BACKGROUND
`
`10
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`15
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`25
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`30
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`Phospholipids are a major component of all biological membranes, and include
`phosphoglycerides (phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol,
`cardiolipin and phosphatidyl serine); and sphingolipids such as sphingomyelin,
`
`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, cell differentiation.
`
`Reasonable levels 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 usefiil quantities
`of sphingomyelin, ceramides, gangliosides, and phosphatidyl serine.
`
`Both phospholipids and gangliosides have been implicated in conferring a number of health
`benefits including brain health, sports nutrition, 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.
`
`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.
`
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`.{istingmethodsfor isolationofthese compoundsrely ontheuse ofchromatographic
`
`techniques, which are slow and costly processes to operate, or 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 IPA 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 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 conveys environmental benefits through reduced organic solvent use. The disadvantage
`of supercritical C02 processing is that the solubility of many compounds is low, and only
`
`neutral lipids are extracted.
`
`It is known that the use of organic co—~solvents such as ethanol allows extraction of some polar
`compounds. For example, Teberikler et al [4] describe a process for extraction of PC from a
`soybean lecithin Using 10% ethanolin C02 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—foldincrease 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 some extraction of PC, PE, PI, and PA. PC is selectively extracted under some
`
`conditions, but at higher temperatures and pressures a higher degree of extraction of the
`insoluble phospholipidsis obtained. 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 a1 [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
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`.mnkshthe soybeanderivedstartingmaterials donotcontaindetectable levelsofSM, CL, GS
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`
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`or PS.
`
`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 of DHA containing
`phospholipids using 5 % ethanol in C02, and by presoaking in straight ethanol and extracting
`using C02. The phospholipids obtained in this process are not specified and no fractionation
`of the different phospholipids is described. In addition, the phospholipid 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 a1 [8] describe extraction of frozen egg yolks using C02 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 was not described.
`
`15
`
`Heidlas [9] describes a process for fractionation of neutral lipids (oil) from phospholipids
`(lecithin) by counter —- current contacting with propane at 40 bar and 60°C to 80°C. No
`fractionation of the phospholipid phase is described.
`
`It is an object of this invention to provide a process for producing a product that contains
`desirable levels of particular complex lipids and/or at least to offer the public a useful choice.
`
`Q;
`
`SUMMARY OF INVENTION
`
`In a first aspect the invention provides a process for separating a starting material, comprising
`
`(a) providing a starting material comprising at least 1% phosphatidyl serine
`
`»
`
`(b) providing a solvent comprising a mixture of supercritical or near-critical C02 and at
`
`25
`
`least 10% Cl-C3 monohydric alcohol
`
`(c) contacting the starting material and the solvent and subsequently separating the
`
`solvent from the insoluble material
`
`(d) optionally separating the soluble material and the solvent.
`
`RIMFROST EXHIBIT 1120 Page 0006
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`_ /-
`
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`/.t
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`In a second aspect the invention provides a process for separating a starting material,
`
`comprising
`
`(a) providing a starting material comprising at least 1% sphingomyelin
`
`5
`
`(b) providing a solvent comprising a mixture of supercritical or near-critical C02 and at
`least 10% C1—C3 monohydric alcohol
`
`(c) contacting the starting material and the solvent and subsequently separating the
`solvent from the insoluble material
`
`(d) optionally separating the soluble material and the solvent.
`
`In a third aspect the invention provides a process for separating a starting material, comprising
`
`1(a) providing a starting material comprising at least 1% cardiolipin
`
`(b) providing a solvent comprising a mixture of supercritical or near-critical C02 and at
`least 10% C1—C3 monohydric alcohol
`
`(c) contacting the starting material and the solvent and subsequently separating the
`solvent from the insoluble material
`
`(d) optionally separating the soluble material and the solvent.
`
`C
`
`10
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`15
`
`C.
`
`In a fourth aspect the invention provides a process for separating a starting material,
`
`20
`
`comprising
`
`\
`
`(a) providing a starting material comprising at least 0.3% gangliosides
`
`(b) providing a solvent comprising a mixture of supercritical or near-
`
`critical C02 and at
`
`least 10% C1-C3 monohydric alcohol
`
`RIMFROST EXHIBIT 1120 Page 0007
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` . (c) contactingthe startingmaterial andthe solvent and subsequently separatingthe
`
`solvent from the insoluble material
`
`(d) optionally separating the soluble material and the solvent.
`
`5
`
`In a fifth aspect the invention provides a process for separating a starting material, comprising
`
`(e) providing a starting material comprising at least 10% phosphatidylcholine
`
`(f) providing a solvent comprising a mixture of supercritical or near—critical C02 and at
`least 10% C1-C3 monohydric alcohol
`
`C
`10
`
`(g) contacting the starting material and the solvent and subsequently separating the
`solvent from the insoluble material
`
`(h) optionally separating the soluble material and the solvent.
`
`In a sixth aspect the invention provides a process for separating a starting material,
`
`1 5
`
`comprising
`
`C
`
`(a) providing a starting material comprising at least 1% phosphatidyl serine
`
`(b) providing a first solvent comprising supercritical or near—critical C02
`
`(c) contacting the starting material and the first solvent and subsequently separating the
`first solvent from the insoluble material
`
`20
`
`(d) optionally separating the soluble material and the first solvent
`
`(e) providing a second solvent comprising a mixture of supercritical or near—critical C02
`and at least 10% C1—C3 monhydric alcohol
`
`(1) contacting the insoluble material produced in step (c) and the second solvent and
`subsequently separating the second solvent from the insoluble material
`
`RIMFROST EXHIBIT 1120 Page 0008
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` . (g) optionally separatingthe solublematerial andthe second solvent.
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`Preferably the first solvent comprises a mixture of supercritical or near-critical C02 and less
`
`than 10% C1-C3 monohydric alcohol.
`
`5
`
`In a seventh aspect the invention provides a process for separating a starting material,
`
`comprising
`
`(a) providing a starting material comprising at least 1% sphingomyelin
`
`(b) providing a first solvent comprising supercritical or near-critical C02
`
`C
`10
`
`(c) contacting the starting material and the first solvent and subsequently separating the
`first solvent from the insoluble material
`
`((1) optionally separating the soluble material and the first solvent
`
`(6) providing a second solvent comprising a mixture of supercritical or near-critical C02
`and at least 10% C1-C3 monhydric alcohol
`
`(f) contacting the insoluble material produced in step (c) and the second solvent and
`subsequently separating the second solvent from the insoluble material
`
`15
`
`(g) optionally separating the soluble material and the second solvent.
`
`C Preferably the first solvent comprises a mixture of supercritical or near-critical C02 and less
`
`than 10% C1_-C3 monohydric alcohol.
`
`20
`
`In a eighth aspect the invention provides a process for separating a starting material,
`
`comprising
`
`(a) providing a starting material comprising at least 1% cardiolipin
`
`(b) providing a first solvent comprising supercritical or near—critical C02
`
`RIMFROST EXHIBIT 1120 Page 0009
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`

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`. (c) contactingthestartingmaterial andthe firstsolventand subsequently separatingthe
`
`first solvent from the insoluble material
`
`(d) optionally separating the soluble material and the first solvent
`
`(e) providing a second solvent comprising a mixture of supercritical or near—critical C02
`and at least 10% C1—C3 monhydric alcohol
`
`5
`
`(i) contacting the insoluble material produced in step (c) and the second solvent and
`subsequently separating the second solvent from the insoluble material
`
`(g) optionally separating the soluble material and the second solvent.
`
`C‘
`
`10
`
`Preferably the first solvent comprises a mixture ofsupercritical or near-critical C02 and less
`
`than 10% C1—C3 monohydric alcohol.
`
`In a ninth aspect the invention provides a process for separating a starting material,
`
`comprising
`
`(a) providing a starting material comprising at least 0.3% gangliosides
`
`15
`
`(b) providing a first solvent comprising supercritical or near-critical C02
`
`(0) contacting the starting material and the first solvent and subsequently separating the
`first solvent from the insoluble material
`(d) optionally separating the soluble material and the first solvent
`
`(e) providing a second solvent comprising a mixture of supercritical or near—critical C02
`and at least 10% C1-C3 monhydric alcohol
`
`C
`
`20
`
`(t) contacting the insoluble material produced in step (c) and the second solvent and
`subsequently separating the second solvent from the insoluble material
`
`(g) optionally separating the soluble material and the second solvent.
`
`Preferably the first solvent comprises a mixture of supercritical or near—critical C02 and less
`
`25
`
`than 10% C1-C3 monohydric alcohol.
`
`RIMFROST EXHIBIT 1120 Page 0010
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`/4_
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`/(
`
`In a tenth aspect the invention provides a process for separating a starting material,
`
`comprising
`
`(h) providing a starting material comprising at least 10% phosphatidyl choline
`
`(i) providing a first solvent comprising supercritical or near-critical C02
`
`0) contacting the starting material and the first solvent and subsequently separating the
`
`first solvent from the insoluble material
`
`(k) optionally separating the soluble material and the first solvent
`
`(1) providing a second solvent comprising a mixture of supercritical or near-critical C02
`
`10
`
`and at least 10% C1—C3 monhydric alcohol
`
`(In) contacting the insoluble material produced in step (c) and the second solvent and
`subsequently separating the second solvent from the insoluble material
`
`(n) optionally separating the soluble material and the second solvent.
`
`Preferably the first solvent comprises a mixture of supercritical or near-critical C02 and less
`
`15
`
`than 10% C1—C3 monohydric alcohol.
`
`Preferably the starting material contains greater than 1% PS. More preferably the starting
`material contains more than 2% PS. Most preferably the starting material contains greater
`
`than 5% PS.
`
`20
`
`Alternatively the starting material contains greater than 0.3% gangliosides. More preferably
`the starting material contains greater than 1% gangliosides. Most preferably the starting
`
`material contains greater than 2% gangliosides.
`
`Alternatively the starting material comprises greater than 1% SM. More preferably the
`starting material contains greater than 5% SM. Most preferably the starting material contains
`
`25
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`greater than 15% SM.
`
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`

`

`/Qltemativelythe starting material comprises greaterthan 1% cardiolipin. More preferablythe
`
`starting material contains greater than 2% cardiolipin. Most preferably the starting material
`
`contains greater than 5% cardiolipin.
`
`Alternatively the starting material comprises greater than 15% phosphatidyl choline. More
`
`preferably the starting material contains greater than 20% phosphatidyl choline.
`
`Preferably the starting material is derived from dairy, soy, eggs, animal tissue or organs,
`
`marine animals, other plants, or bacterial sources. More preferably the starting material is a
`
`composition comprising milk fat globule membrane, egg lipids, marine lipids, or animal
`
`tissue or organ lipids.
`
`Most preferably the starting material is derived from bovine milk.
`
`The starting material may have been genetically modified.
`
`More preferably the starting material, when in solid form, is prepared to maximise the surface
`
`area using conventional techniques. More preferably the starting material is prepared by
`
`15
`
`cryomilling.
`
`It will be appreciated by those skilled in the art that the C02 may be substituted by other
`
`suitable solvents such as tetrafluoroethane, nitrous oxide, sulphur hexafluoride, ethane and
`
`ethylene.
`
`20
`
`Preferably the alcohol co—solvent is selected from solvents methanol, ethanol, n—propanol or
`
`isopropanol and mixtures thereof comprising 0 — 40% water, preferably 0 — 20% water.
`
`More preferably the alcohol co—solvent is ethanol.
`
`Optionally the alcohol co-solvent comprises 1-10% water. Preferably the alcohol co-solvent
`
`is 95% aqueous ethanol.
`
`25
`
`Preferably the mass fraction of the alcohol 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
`
`between 25% and 30%.
`
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`

`Qeferablythe contactingtemperaturebetweenthe startingmaterial andsolventisbetween
`
`10°C and 80°C. Most preferably the contacting temperature is between 55°C and 65°C.
`
`Preferably the contacting pressure is between 100 bar and 500 bar. Most preferably the
`
`pressure is between 200 bar and 300 bar.
`
`Preferably the ratio of the alcohol co—solvent to starting material is in the range 10:1 to 200:1.
`Most preferably the ratio of the alcohol co—solvent to starting material is in the range 15:1 to
`
`50:1.
`
`Preferably the separating pressure is between atmospheric pressure and 90 bar. Most
`
`preferably the separating pressure is between 40 bar and 60 bar.
`
`Preferably the separation is carried out in two stages, so that separation of most of the
`
`dissolved lipids occurs in the first separator, and the co—solvent in the second separator.
`
`Optionally the co-solvent can be recycled for further use.
`
`Preferably the separated C02 phase is recycled.
`
`Optionally the alcohol is removed by evaporation under vacuum.
`
`15
`
`In one embodiment, the starting material is contacted with a continuous flow of solvent
`
`(supercritical C02 and C1—C3 alcohol).
`
`In another embodiment the starting material is contacted with one or more batches of solvent
`
`C,
`
`In another embodiment, both the lipid and solvent streams are fed continuously
`
`In a further embodiment, the lipid and co—solvent streams are mixed prior to contacting with
`
`20
`
`supercritical C02.
`
`In another embodiment, the starting material is contacted with the solvent first at a lower
`
`temperature, followed by contact at one or more higher temperatures.
`
`The invention also provides products produced by the processes of the invention:
`
`(1)
`
`the CO2 soluble material produced in step (c) of the second aspect of the present
`
`25
`
`invention;
`
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`

`

`. (2)
`
`the insoluble material remaining after contactwiththe C02 and alcohol solvent
`
`(the “residue”), and
`
`(3)
`
`the material contained in the C02 and alcohol solvent after contact with the
`
`starting material (the “extract”). Where the extracted material is removed
`
`continuously, or semi-continuously, or where there are multiple stages to the
`
`process, there will be multiple “extract” products.
`
`Preferably the residue contains a greater percentage of phosphatidyl serine, and/or
`
`gangliosides and/or cardiolipin than that of the starting material.
`
`More preferably the residue contains greater than 5% PS, more preferably greater than 30%
`
`PS, most preferably greater than 70% PS.
`
`Alternatively the residue contains greater than 2% gangliosides, more preferably greater than
`
`14% gangliosides, most preferably greater than 6% gangliosides.
`
`Alternatively the residue contains greater than 5% cardiolipin, more preferably greater than
`
`15
`
`10% cardiolipin, most preferably greater than 25% cardiolipin.
`
`Preferably the extract contains a greater percentage of sphingomyelin than that of the starting
`
`material. More preferably the extract contains greater than 3% sphingomyelin, more
`
`preferably greater than 10% sphingomyelin and most preferably greater than 15%
`
`sphingomyelin.
`
`20
`
`This invention may also be said broadly to consist in the parts, elements and features referred
`
`to or indicated in the specification of the application, individually or collectively, and any or
`
`all combinations of any two or more said parts, elements or features, and where specific
`
`integers are mentioned herein that have known equivalents in the art to which this invention
`relates, such known equivalents are deemed to be incorporated herein as if individually set
`
`25
`
`forth.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`RIMFROST EXHIBIT 1120 Page 0014
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`

`

`,/
`
`‘
`
`/./
`
`The invention may be more fully understood by having reference to the accompanying
`
`drawings wherein:
`
`5
`
`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
`
`1 O
`
`C
`
`Figure 4 is a scheme drawing illustrating a fourth preferred process ofthe current invention
`
`15
`
`ABBREVIATIONS OR ACRONYMS
`
`In this specification the following are the meanings of the abbreviations or acronyms used.
`
`“PL” means phospholipids
`
`20
`
`“CL” means cardiolipin
`
`C A “PC” means phosphatidyl choline
`
`“Pl” means phosphatidyl inoSitol
`
`“PS” means phosphatidyl serine
`
`“PE” means phosphatidyl ethanolamine
`
`25
`
`“SM” means sphingomyelin
`
`“DHSM” means dihydrosphingomyelin
`
`“GS” means ganglioside
`
`RIMFROST EXHIBIT 1120 Page 0015
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`

`

`
`.WD” means not detected
`
`“C02” means carbon dioxide
`
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`

`
`
`.ENERAL DESCRIPTION OF THE INVENTION
`
`It is known that supercritical C02 with ethanol as a co-solvent up to 12.5% can extract the
`
`phospholipids PC, and to a lesser extent, PE and PI from soy or egg. Surprisingly, we have
`
`found that the phospholipids PS and CL; and gangliosides are Virtually insoluble in C02 and a
`
`C1-C3 alcohol co-solvent, and that SM is soluble. Therefore it is possible to separate the
`
`soluble phospholipids from the insoluble phospholipids to achieve fractions enriched in one or
`
`other of the desired components.
`
`. There are a number of factors affecting the operation of the process:
`
`I
`
`Feed material and feed preparation
`
`I Extraction temperature and pressure
`
`I Co-solvent concentration
`
`I Total solvent throughput
`
`I
`
`Solvent flow rate and contacting conditions
`
`It is advantageous to start with a feed material containing a high proportion of lipids, and
`
`ideally a high proportion of complex lipids, particularly PS, SM, CL, and gangliosides. The
`
`starting material can be processed using pure C02 before the co-solvent is introduced to
`
`remove much or all of lower value neutral lipids. This reduces the neutral lipid content in the
`
`C02+co-solvent extract leading to an extract enriched in soluble complex lipids.
`
`10
`
`I
`
`15
`
`20
`
`The form of the starting material depends on the source of the lipids and its lipid composition.
`
`25
`
`For example dairy lipid extracts high in phospholipids may be substantially solid even at
`
`elevated temperatures.- Egg yolk lipids in comparison have a lower melting point. The
`
`presence of neutral lipids also tends to produce a more fluid starting material. To promote
`
`good contacting it may be beneficial to prepare the starting material. Solid lipid materials may
`
`be able to be cryomilled for example. Lipid feed materials can also be made more fluid by the
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`30
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`inclusion of some ethanol or water.
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`Changing the processing conditions of temperature, pressure, co-solvent concentration, and
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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 ‘insoluble’ lipids
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`RIMFROST EXHIBIT 1120 Page 0017
`15 RIMFROST EXHIBIT 1120 Page 0017
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`.fich as PS, GS and CL, have very slight solubilities so that excessive use ofsolvent, orvery
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`favourable extraction conditions, results in losses of PS, GS and CL from the residual
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`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-
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`5
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`extracted, but the total yield will be lower. Processing economics, and the relative values of
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`the products, will determine where this balance lies. A further option to obtain multiple
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`enriched fractions is to carry out extractions under progressively more favourable extraction
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`conditions, such as increasing the temperature.
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`10 We have found that co—solvent concentrations below about 10% produce very little complex
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`lipid extract. 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 and PI, while the lipids PS, CL and GS remain
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`virtually insoluble.
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`rt
`”
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`l 5
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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’, that
`
`is to say when interpreting statements in this specification which include that term, the
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`20
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`features, prefaced by that term in each statement, all need to be present but other features can
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`also be present.
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`The invention consists in the foregoing and also envisages constructions of which the
`following gives examples only.
`
`C.
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`RIMFROST EXHIBIT 1120 Page 0018
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`.XAMPLES
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`The experimental process is described, with reference to figure 1, as follows.
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`A measured mass of lipid material to be fractionated was placed in basket BKl with a porous
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`sintered steel plate on the bottom. Basket BKl was placed in a 300 mL extraction vessel EXl.
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`The apparatus was suspended in heated water bath WBl and maintained at a constant
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`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 B1 was
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`10
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`pumped using pump Pl into extraction vessel EXl until the pressure reached the desired
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`operating pressure, after which valve V1 was operated to maintain a constant pressure in the
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`extraction vessel. After passing through’valve V1, 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
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`fluid and lose its solvent strength. Precipitated material was captured in separation vessel
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`15
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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 CTl operated at —5°C. Extracted
`material was collected periodically from separator SEPl by opening valve V2. The extraction
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`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 C02
`
`2O
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`extraction, ethanol co—solvent with or without added water was added to the C02 at the
`
`desired flow ratio from supply bottle B2 using pump P2. Ethanol and further extracted
`
`material was separated from the C02 in separator SEPl and periodically removed through
`valve V2. After the desired amount of ethanol had been added the ethanol flow was-stopped
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`and the C02 flow continued alone until all the ethanol had been recovered from the system.
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`25
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`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 C02 alone was optionally passed continuously
`
`through the apparatus, as for the continuous flow mode of operation, until all CO2 alone
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`30
`
`extractable material was removed. The C02 flow was then stopped and valve V1 closed to
`
`maintain the pressure. Approximately 140g of ethanol was pumped from supply bottle B2
`
`through pump P2 into extraction vessel EXl. The system was left for a period of 15 minutes
`
`to allow the system to equilibrate, after which time the C02 flow was started and valve V1
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`K“penedto maintain a constant pressure and allowethanol and dissolvedcompounds to flow
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`through to separator SEPl. This process was repeated twice more, after which the C02 was
`
`vented and the residual material in basket BK] was removed and dried under vacuum.
`
`5
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`Extract and residue fractions were analysed for phospholipid content by 31P-NMR. The
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`phospholipid mass fractions reported here are for phosphatidylcholine (PC),
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`phosphatidylinositol (PI), phosphatidylethanolamine (PE), sphingomyelin (SM), and
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`‘phosphatidylserine (PS), and cardiolipin (CL).
`
`10
`
`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%
`~
`
`1 5
`
`40g of dairy lipid extract A, with composition shown in Table 1 (feed), was extracted using
`the continuous extraction mode of operation at 60°C and 300 bar. The ‘other compounds’
`
`consist mainly of neutral lipids. 44% of the feed material was extracted as neutral lipid from
`
`the C02 only extraction stage. A further 31% of the feed material was extracted using 95%
`
`20
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`aqueous ethanol at a concentration in C02 of 25%. The total ethanol and water added was
`
`880g. The composition of the fraction extracted with C02 and ethanol, and the composition of
`
`the residual fraction are shown in Table 1. The extract is enriched in phosphatidylcholine (PC)
`C and sphingomyelin (SM) which are more soluble in C02 and ethanol, while the residual
`
`fraction is substantially enriched in phospatidylserine (PS). Phospatidylserine levels are
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`25
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`Virtually undetectable in the extract phase indicating very low solubility in C02 and ethanol,
`
`and almost complete recovery of phosphatidylserine in the residue phase.
`
`Table 1
`
`
`
`30
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`18 RIMFROST EXHIBIT 1120 Page 0020
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`‘Qxample 2: Fractionation ofdairylipid extractA, ethanol mass
`
`fraction 31%
`
`41 g 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 95%
`
`aqueous ethanol at a concentration in C02 of 31%. 33% of the feed material was extracted.
`
`The total ethanol and water added was 1150g. The composition of the r