available under
`Document made
`Patent Cooperation Treaty (PCT)
`
`the
`
`International application number: PCT/NZ2007/000087
`
`International filing date:
`
`20 April 2007 (20.04.2007)
`
`Documenttype:
`
`Certified copy of priority document
`
`Documentdetails:
`
`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 17.1(a) or (b)
`
`
`
`World Intellectual Property Organization (WIPO) - Geneva, Switzerland
`Organisation Mondiale de la Propriété Intellectuelle (OMPI) - Genéve, Suisse
`RIMFROST EXHIBIT 1120 Page 0001
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`
`
`. Eee
`MinistryofECONOMIC (%)
`Development
`Manati Ohanga
`7
`—_
`-
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`
`
`
`
`PCT/NZ2007/000087
`
`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.
`
`| hereby certify that annexedis a true copyof the Provisional Specification as filed on
`20 April 2006 with an application for Letters Patent number 546681 made by
`OWEN JOHN CATCHPOLEand STEPHEN JOHN TALLON.
`
`Dated 17 August 2007.
`
`Lhd
`
`Commissioner of Patents, Trade Marks and Designs
`
`Neville Harris
`
`>0 tox 2RAMEEROSEHEBEDbd0x1Page-8002
`RIMFROST EXHIBIT 1120 Page 0002
`Freephone: 0508 4 IPONZ (0508 447 669), International callers dial: +64 3 962 2607, Fax: +64 4 978 3691, www.iponz.govi.nz
`
`

`

`_
`
`
`@
`
`4.
`
`24668 4
`
`NEW ZEALAND
`
`PATENTSACT,1953
`
`PROVISIONAL SPECIFICATION
`
`PRODUCT AND PROCESS
`
`statement:
`
`We, OWEN JOHN CATCHPOLE and STEPHEN JOHN TALLON, both New Zealand
`citizens c/~ Industrial Research Limited, Gracefield Research Centre, Gracefield Road, Lower
`Hutt, New Zealand, do hereby declare this invention to be described in the following
`| INTELLECTUAL PROPERTY |
`OFFICE OF NZ.
`“20 APR 2006
`Page 0003
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`

`

`
`@
`
`PRODUCTANDPROCESS
`
`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
`
`15
`
`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 importantin 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 foundin 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 knownto contain useful quantities
`of sphingomyelin, ceramides, gangliosides, and phosphatidyl serine.
`
`Both phospholipids and gangliosides have been implicated in conferring a numberofhealth
`benefits including brain health, sports nutrition, skin health, eczema treatment, anti-infection,
`wound healing, gut microbiota modifications, anti-canceractivity, alleviation ofarthritis,
`improvement of cardiovascular health, and treatment of metabolic syndromes.
`
`Cardiolipin is an important componentofthe inner mitochondrial membrane. It is typically
`present in metabolically active cells ofthe heart and skeletal muscle. It serves as an insulator
`and stabilises the activity ofprotein complexes important to the electron transport chain.
`
`25
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`30
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`
`@.....methodsforisolationofthesecompoundsrelyontheuseofchromatographic
`
`techniques, which are slow andcostly 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 ofphospholipids from egg yolks
`using acetone and ethanolextractions, 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 expensiveto carry out on an industrial scale, and also
`uses hexane, methanol, and IPA as solvents. Kearnsetal [3] describe a process for
`purification ofegg yolkderived PC from PE using mixtures ofacetonitrile, hydrocarbons, and
`fluorocarbons. Again, these solvents are undesirable for nutritional or pharmaceutical use.
`
`10
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`15
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`20
`
`25
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`30
`
`Supercritical fluid extraction processes using CO, are becoming increasingly popular because
`of a number ofprocessing 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 ihe use of CO;in place of conventional organic solvents
`also conveys environmental benefits through reduced organic solvent use. The disadvantage
`of supercritical CO, processingis that the solubility ofmany compoundsis low, and only
`neutral lipids are extracted.
`
`It is knownthat the use of organic co-solvents such as ethanolallows extraction of some polar
`compounds. For example, Teberikler et al [4] describe a process for extraction ofPC 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-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 ofthe
`insoluble phospholipids is obtained. The pressures required to achieve good extraction were
`impractically high for industrial application, and the high temperaturesused (80°C) could
`cause polyunsaturatedfatty acids to be degraded. Taylor et al [6] describe a process in which
`soybean flakesare first extracted using only COz, followed by CO2 with 15% ethanol at 80°C
`and 665 bar. A mixture ofphospholipidsis obtained which were fractionated by alumina
`column. Again, the temperatures and pressures are too high for practical application. In these
`
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`
`
`@....soybeanderivedstartingmaterialsdonotcontaindetectablelevelsofSM, CL,GS
`
`or PS.
`
`Tanaka and Sakaki [7] describe a method for extracting phospholipids from waste tuna
`shavings using CO, and ethanolas a co-solvent. They describe extraction of DHA containing
`phospholipids using 5 % ethanolin COz, and by presoakingin straight ethanol and extracting
`using CO. The phospholipids obtained in this process are not specified and no fractionation
`ofthe different phospholipids is described. In addition, the phospholipid fraction makes up a
`relatively small proportion ofthe 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 CO. and 3 % ethanol, and CO,
`with up to 5 % methanol. Higher rates oftriglyceride 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 processfor fractionation ofneutrallipids (oil) from phospholipids
`(lecithin) by counter — current contacting with propaneat 40 bar and 60°C to 80°C. No
`fractionation of the phospholipid phase is described.
`
`It is an object ofthis invention to provide a process for producing a product that contains
`desirable levels of particular complex lipids and/orat least to offer the public a useful choice.
`
`Os
`
`SUMMARYOF INVENTION
`
`Ina 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 CO2 and at
`least 10% C)-C3 monohydric alcohol
`
`25
`
`(c) contacting the starting material and the solvent and subsequently separating the
`solvent from the insoluble material
`
`(d) optionally separating the soluble pnaterial and the solvent.
`
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` a©f
`
`In a second aspectthe invention provides a process for separating a starting material,
`comprising
`
`(a) providing a starting material comprising at least 1% sphingomyelin
`
`(b) providing a solvent comprising a mixture of supercritical or near-critical CO2 and at
`least 10% C,-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
`
`(a) providing a starting material comprising at least 1% cardiolipin
`
`(b) providing a solvent comprising a mixture of supercritical or near-critical CO2 and at
`least 10% C)-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.
`
`5
`
`C
`
`10
`
`15
`
`C
`
`In a fourth aspect the invention provides a process for separating a starting material,
`comprising
`
`20
`
`\
`
`(a) providinga starting material comprising at least 0.3% gangliosides
`
`(b) providing a solvent comprising a mixture of supercritical or near-critical CO2 and at
`least 10% C,-C3 monohydric alcohol
`
`> RIMFROST EXHIBIT 1120 Page 0007
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`

` @ (c) contactingthestartingmaterialandthesolventandsubsequentlyseparatingthe
`
`solvent from the insoluble material
`
`(d) optionally separating the soluble material and the solvent.
`
`5
`
`10
`
`15
`
`C
`
`Ina 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 CO? and at
`least 10% C)-C3 monohydric alcohol
`
`(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,
`comprising
`
`(a) providingastartingmaterial comprising atleast 1%phosphatidyl serine
`
`(b) providing a first solvent comprising supercritical or near-critical CO2
`
`(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 CO2
`and at least 10% C,-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
`
`© RIMFROST EXHIBIT 1120 Page 0008
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`

`

` @ (g) optionallyseparatingthesolublematerialandthesecondsolvent.
`
`Preferably the first solvent comprises a mixture of supercritical or near-critical CO2 and less
`than 10% C,-C3 monohydric alcohol.
`
`5
`
`Inaseventh aspect the invention provides a process for separating a starting material,
`comprising
`
`(a) providing a starting material comprising at least 1% sphingomyelin
`
`(b) providinga first solvent comprising supercritical or neat-critical CO2
`(c) contacting the starting material and the first solvent and subsequently separating the
`first solvent from the insoluble material
`
`C
`10
`
`(d) optionally separating the soluble material and the first solvent
`(e) providing a second solvent comprising a mixture of supercritical or near-critical CO2
`and at least 10% C,-C3 monhydric alcohol
`
`(f) contacting the insoluble material producedin 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 COz and less
`than 10% C)-C3 monohydric alcohol.
`
`20
`
` Inaeighth aspect the invention provides a process for separating a starting material,
`comprising
`
`(a) providing a starting material comprising at least 1% cardiolipin
`
`(b) providinga first solvent comprising supercritical or near-critical CO2
`
`7 RIMFROST EXHIBIT 1120 Page 0009
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`

`@ (c) contactingthestartingmaterialandthefirstsolventandsubsequentlyseparatingthe
`
`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 CO2
`and at least 10% C-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
`
`(g) optionally separating the soluble material and the secondsolvent.
`
`st)
`
`Preferably the first solvent comprises a mixture of supercritical or near-critical CO2 and less
`than 10% C)-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
`
`20
`
`(b) providinga first solvent comprising supercritical or near-critical CO
`(c) contacting the starting material and thefirst solvent and subsequently separating the
`first solvent from the insoluble material
`
`(d) optionally separating the soluble material and thefirst solvent
`(e) providing a secondsolvent comprising a mixture of supercritical or near-critical CO2
`and at least 10% C)-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
`
`(g) optionally separating the soluble material and the second solvent.
`
`Preferably the first solvent comprises a mixture of supercritical or near-critical COz and less
`than 10% C)-C3 monohydric alcohol.
`
`25
`
`8 RIMFROST EXHIBIT 1120 Page 0010
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`

`

`nan
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`4i
`
`
`
`In a tenth aspect the invention provides a process for separating a starting material,
`comprising
`
`(h) providinga starting material comprising at least 10% phosphatidyl choline
`
`5
`
`(i) providinga first solvent comprising supercritical or near-critical CO2
`
`(j) 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 thefirst solvent
`
`(1) providing a second solvent comprising a mixture of supercritical or near-critical CO2
`and at least 10% C)-C3 monhydric alcohol
`
`10
`
`(m)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 CO2 andless
`than 10% C;-C3 monohydric alcohol.
`
`15
`
`Preferably the starting material contains greater than 1% PS. Morepreferably the starting
`material contains more than 2% PS. Mostpreferably the starting material contains greater
`
`than 5% PS.
`
`20
`
`25
`
`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
`greater than 15% SM.
`
`° RIMFROST EXHIBIT1120 Page 0011
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`

`

`\@......thestartingmaterialcomprisesgreaterthan 1% cardiolipin. Morepreferablythe
`
`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. Morepreferably the starting material is a
`composition comprising milk fat globule membrane, egg lipids, marine lipids, or animal
`tissue or organ lipids.
`
`Mostpreferably the starting material is derived from bovine milk.
`
`Thestarting material may have been genetically modified.
`
`Morepreferably the starting material, whenin solid form, is prepared to maximise the surface
`area using conventional techniques. More preferably the starting material is prepared by
`cryomilling.
`
`15
`
`It will be appreciated by those skilled in the art that the CO2 may be substituted by other
`suitable solvents such as tetrafluoroethane, nitrous oxide, sulphur hexafluoride, ethane and
`
`ethylene.
`
`20
`
`Preferably the alcohol co-solventis selected from solvents methanol, ethanol, n-propanol or
`isopropanol and mixtures thereof comprising 0 — 40% water, preferably 0 — 20% water.
`
`Morepreferably the alcohol co-solventis ethanol.
`
`Optionally the alcohol co-solvent comprises 1-10% water. Preferably the alcohol co-solvent
`is 95% aqueousethanol.
`
`25
`
`Preferably the mass fraction ofthe alcohol co-solvent in CO» is between 5% and 60%. More
`preferably the massfraction is between 20% and 50%. Most preferably the massfraction is
`between 25% and 30%.
`
`‘0 RIMFROSTEXHIBIT 1120 Page 0012
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`é...thecontactingtemperaturebetweenthestartingmaterialandsolventisbetween
`
`10°C and 80°C. Mostpreferably 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 300bar.
`
`Preferably the ratio of the alcohol co-solventto starting material is in the range 10:1 to 200:1.
`Mostpreferably the ratio ofthe alcohol co-solventto 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 CO. 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 CO2 and C;-C3 alcohol).
`
`In another embodimentthe starting material is contacted with one or more batches of solvent
`
`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
`supercritical COp.
`
`20
`
`In another embodiment,the starting material is contacted with the solventfirst at a lower
`temperature, followed by contact at one or more higher temperatures.
`
`The invention also provides products produced by the processesof the invention:
`
`(1)
`
`the CO,soluble material produced in step (c) ofthe second aspectof the present
`invention;
`
`25
`
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`

`@ (2)
`
`theinsolublematerialremainingaftercontactwiththeCOandalcoholsolvent
`
`(the “residue’’), and
`
`(3)
`
`the material contained in the CO> and alcoholsolvent 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/orcardiolipin 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
`4% gangliosides, most preferably greater than 6% gangliosides.
`
`Alternatively the residue contains greater than 5% cardiolipin, more preferably greater than
`10% cardiolipin, most preferably greater than 25% cardiolipin.
`
`15
`
`Preferably the extract contains a greater percentage of sphingomyelin than that ofthe starting
`material. More preferably the extract contains greater than 3% sphingomyelin, more
`preferably greater than 10% sphingomyelin and mostpreferably greater than 15%
`
`sphingomyelin.
`
`20
`
`25
`
`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 moresaid 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
`
`forth.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`12 RIMFROST EXHIBIT 1120 Page 0014
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`

`

`a .
`
`@ry
`
`The invention may be more fully understood by having reference to the accompanying
`
`drawings wherein:
`
`5
`
`Figure 1 is scheme drawingillustrating a preferred process ofthe current invention.
`
`Figure 2 is a scheme drawingillustrating a second preferred process of the current invention
`
`Figure 3 is a scheme drawingillustrating a third preferred process of the current invention
`
`Figure 4 is a scheme drawingillustrating a fourth preferred processof the current invention
`
`10
`
`c
`
`15
`
`ABBREVIATIONS OR ACRONYMS
`
`In this specification the following are the meanings of the abbreviations or acronymsused.
`
`“PL” means phospholipids
`
`20
`
`“CL” meanscardiolipin
`
`C _ “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
`
`13 RIMFROST EXHIBIT 1120 Page 0015
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`

`

`
`
`6...meansnotdetected
`
`“CO,” means carbon dioxide
`
`14 RIMFROST EXHIBIT 1120 Page 0016
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`

`
`
`Davis.DESCRIPTIONOFTHEINVENTION
`
`It is known that supercritical COwith 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 CO2 and a
`
`C,-C;3 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.
`
`10
`
`- There are a numberoffactors affecting the operation of the process:
`
`=
`
`Feed material and feed preparation
`
`« Extraction temperature and pressure
`
`* Co-solvent concentration
`
`15
`
`= Total solvent throughput
`
`=
`
`Solvent flow rate and contacting conditions
`
`It is advantageousto start with a feed material containing a high proportionoflipids, and
`ideally a high proportion of complex lipids, particularly PS, SM, CL, and gangliosides. The
`
`20
`
`starting material can be processed using pure CO, before the co-solvent is introduced to
`
`remove muchorall of lower value neutral lipids. This reduces the neutral lipid content in the
`
`CO2+co-solvent extract leading to an extract enriched in soluble complex lipids.
`
`25
`
`The form of the starting material depends onthe sourceofthe lipids andits lipid composition.
`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 morefluid starting material. To promote
`
`good contacting it may be beneficial to prepare the starting material. Solid lipid materials may
`
`be able tobe cryomilled for example. Lipid feed materials can also be made morefluid by the
`inclusion of some ethanol or water.
`
`30
`
`Changing the processing conditions of temperature, pressure, co-solvent concentration, and
`
`total solvent usage, influences the amount of material extracted, the purity of the final
`
`product, and the recovery (or efficiency) of the process. For example, the ‘insoluble’ lipids
`
`15 RIMFROST EXHIBIT 1120 Page 0017
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`
`

`

` @.asPS, GS andCL,haveveryslightsolubilitiessothatexcessiveuseofsolvent, orvery
`
`favourable extraction conditions, results in losses of PS, GS and CL from theresidual
`fraction. A high purity product maybe achieved, but with a reduced yield. Conversely the
`enrichment of soluble lipids will be greater if smaller amounts ofthe otherlipids are co-
`extracted, but the total yield will be lower. Processing economics, and the relative values of
`the products, will determine where this balance lies. A further option to obtain multiple
`enriched fractionsis to carry out extractions underprogressively more favourable extraction
`
`conditions, such as increasing the temperature.
`
`10
`
`15
`
`20
`
`Wehave foundthat co-solvent concentrations below about 10% produceverylittle complex
`lipid extract. At higher concentrations the rate of material extracted increases rapidly. We
`have found the co-solvent concentrations of at least 20%, and morepreferably 30% achieve
`high levels of extraction of PC, PE, SM andPI, while the lipids PS, CL and GS remain
`virtually insoluble.
`
`Percentages unless otherwise indicated are on a w/w solidsbasis.
`
`Theterm “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
`features, prefaced by that term in each statement, all need to be present but other features can
`
`also be present.
`
`The invention consists in the foregoing and also envisages constructions of which the
`
`following gives examples only.
`
`16 RIMFROST EXHIBIT 1120 Page 0018
`RIMFROST EXHIBIT 1120 Page 0018
`
`

`

`Deoosiss:
`
`The experimentalprocessis described, with referenceto figure 1, as follows.
`
`A measured massoflipid material to be fractionated was placed in basket BK1 with a porous
`sintered steel plate on the bottom. Basket BK1 was placed in a 300 mL extraction vessel EX1.
`The apparatus was suspendedin heated water bath WB1 and maintained at a constant
`temperature through use of a thermostat and electric heater.
`
`In the continuous extraction modeof operation, liquid CO2 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 was operated to maintain a constant pressure in the
`extraction vessel. After passing throughvalve 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 andlose its solvent strength. Precipitated material was captured in separation vessel
`SEP1, and the COexited from the top of separator SEP1 and was recycled back to the feed
`pumpthrough coriolis mass flow meter FM1and cold trap CT1 operatedat -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 compoundssoluble in CO? only,
`such as neutral lipids, were extracted. When no further extract was produced by CO2
`extraction, ethanol co-solvent with or without added water was addedto the COat the
`desired flow ratio from supply bottle B2 using pump P2. Ethanol and further extracted
`material was separated from the COin separator SEP1 andperiodically removed through
`valve V2. After the desired amount of ethanol had been added the ethanol flow wasstopped
`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. Theextract fraction was evaporated to dryness by rotary evaporation.
`
`In the batch extraction mode ofoperation CO, alone wasoptionally passed continuously
`through the apparatus, as for the continuous flow modeof operation, until all CO2 alone
`extractable material was removed. The CO,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 EX1. The system wasleft for a period of 15 minutes
`to allow the system to equilibrate, after which time the CO. flow wasstarted and valve V1
`
`10
`
`Oy
`
`15
`
`20
`
`25
`
`30
`
`'7 RIMFROST EXHIBIT 1120 Page 0019
`RIMFROST EXHIBIT 1120 Page 0019
`
`

`

`
`
`@.....tomaintainaconstantpressureandallowethanolanddissolvedcompoundstoflow
`
`through to separator SEP1. This process was repeated twice more, after which the CO, was
`
`vented and the residual material in basket BK 1 was removed and dried under vacuum.
`
`5
`
`Extract and residue fractions were analysed for phospholipid content by ?'P-NMR. The
`phospholipid mass fractions reported here are for phosphatidylcholine (PC),
`
`phosphatidylinositol (PI), phosphatidylethanolamine (PE), sphingomyelin (SM), and
`. phosphatidylserine (PS), and cardiolipin (CL).
`
`10
`
`The process optionillustrated 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 extractA, ethanol mass
`
`fraction 25%
`
`15
`
`20
`
`40g of dairy lipid extract A, with composition shown in Table 1 (feed), was extracted using
`the continuousextraction 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 COQ, only extraction stage. A further 31% of the feed material was extracted using 95%
`aqueous ethanolat a concentration in CO2 of 25%. Thetotal ethanol and water added was
`
`880g. The composition of the fraction extracted with CO, 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 CO, and ethanol, while the residual
`fraction is substantially enrichedin phospatidylserine (PS). Phospatidylserine levels are
`virtually undetectable in the extract phase indicating very low solubility in CO, and ethanol,
`
`25
`
`and almost complete recovery of phosphatidylserine in the residue phase.
`
`Table 1
`
`
`
`Yield
`
`paneoffeed
`
`30
`
`: RIMFROSTEXHIBIT 1120 Page 0020
`RIMFROST EXHIBIT 1120 Page 0020
`
`

`

`‘1Occ 2: Fractionation ofdairylipidextractA, ethanolmass
`
`fraction 31%
`
`41g of dairy lipid extract A, with composition as for example 1 was extracted using the
`continuous extraction modeof operation at 60°C and 300 bar as for example 1, using 95%
`aqueous ethanol at a concentration in CO? of 31%. 33% ofthe feed material was extracted.
`
`Thetotal ethanol and water added was 1150g. The compositionof the residualfraction is
`shownin Table 2. The higher ethanol concentration gives a more complete extraction oflipids
`and the concentration of phosphatidylserine in the residue fraction is higher than found in
`example 1.
`
`10
`
`Table 2
`
`
`
`“Other
`
`Other compounds
`
`PhospmHp a
`
`
`
`15
`
`Example 3: Fractionation ofdairy lipid extract A, ethanol mass
`fraction 43%
`
`40gof dairy lipid extract A, with composition as for example 1 was extracted using the
`continuous extraction modeofopera