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`Preferably the solvent used in the process of the present invention comprises 95% aqueous
`
`ethanol.
`
`‘
`
`Preferably the mass fraction of the co-solvent in CO2 is between 5% and 60%. More
`
`preferably the mass fraction is between 20% and 50%. Most preferably the mass fraction is
`
`between 25% and 30%.
`
`-
`
`Preferably the contacting temperature between the feed material and solvent is between 10°C
`
`and 80°C. More preferably the contacting temperature is between 55°C and 65°C. Most
`
`preferably the contacting pressure is between 100 bar and 500 bar.
`
`Preferably the contacting pressure is between 200 bar and 300 bar. More preferably the ratio
`
`10
`
`of the co-solvent to feed material is in the range 10:1 to 200:1. Most preferably the ratio of
`
`the co-solvent to feed material is in the range 15:1 to 50:1.
`
`Preferably the separating pressure is between atmospheric pressure and 90 bar. More
`
`preferably the separating pressure is between 40 bar and 60 bar.
`
`Preferably the co-solvent is recycled for further use.
`
`15
`
`Preferably the COz is recycled for further use.
`
`The co-solvent may be removed by evaporation under vacuum.
`
`Preferably the feed material is contacted with a continuous flow of solvent.
`
`Preferably the feed material is contacted with one or more batchesof solvent.
`
`Thelipid and solvent streams may be fed continuously.
`
`20
`
`Optionally, the feed material and co-solvent streams may be mixed prior to contacting with
`
`CO.
`
`The invention also provides products produced by the process of the invention, both the
`insoluble components remaining after contact with the solvent (also referred to herein as the
`“residue”); and the soluble components that are dissolved in the solvent after contact with
`
`25
`
`the feed material (also referred to herein as the “extract”). Where the feed material is
`
`contacted with more than one batch of solvent, or the solvent is cooled in a numberof steps,
`
`there will be multiple “extract” products.
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`Preferably the product contains more sphingomyelin than the feed material. More preferably
`the product comprises greater than 3% sphingomyelin. Even more preferably the product
`comprises greater than 10% sphingomyelin. Most preferably the product comprisesgreater
`than 15% sphingomyelin.
`
`Preferably the product contains more phosphatidyl serine than the feed material. More
`preferably the product comprises greater than 5% phosphatidyl serine. Even more preferably
`the product comprises greater than 30% phosphatidyl serine. Most preferably the product
`comprises greater than 70% phosphatidylserine.
`
`Preferably the product contains more gangliosides than the feed material. More preferably
`the product comprises greater than 2% gangliosides. Even more preferably the product
`comprises greater than 4% gangliosides. Most preferably the product comprises greater than
`6% gangliosides.
`
`Preferably the product contains more cardiolipin than the feed material. More preferably the
`product comprises greater than 5% cardiolipin. Even more preferably the product comprises
`greater than 10% cardiolipin. Most preferably the product comprises greater than 25%
`cardiolipin.
`
`Preferably the product contains more acylalkyphospholipids and/or plasmalogens than the
`feed material. More preferably the product comprises greater than 5%
`acylalkyphospholipids and/or plasmalogens. Even morepreferably the product comprises
`greater than 10% acylalkyphospholipids and/or plasmalogens. Most preferably the product
`comprises greater than 25% acylalkyphospholipids and/or plasmalogens.
`
`Preferably the product contains more aminoethylphosphonate and/or other phosphonolipids
`than the feed material. More preferably the product comprises greater than 5%
`aminoethylphosphonate and/or other phosphonolipids. Even more preferably the product
`comprises greater than 10% aminoethylphosphonate and/or other phosphonolipids. Most
`preferably the product comprises greater than 25% aminoethylphosphonate and/or other
`phosphonolipids.
`
`10
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`15
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`20
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`BRIEF DESCRIPTION OF THE DRAWINGS
`The invention may be more fully understood by having reference to the accompanying
`
`drawings wherein:
`
`Figure 1 is scheme drawingillustrating a preferred 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 drawingillustrating a third preferred process ofthe current invention
`Figure 4 is a scheme drawingillustrating a fourth preferred process of the current invention
`
`10
`
`ABBREVIATIONS AND ACRONYMS
`In this specification the following are the meaningsof the abbreviations or acronyms used.
`
`“CL” meanscardiolipin
`
`“PC” means phosphatidyl! choline
`
`“PT” means phosphatidyl inositol
`
`15
`
`“PS” means phosphatidyl serine
`
`“PE” means phosphatidyl ethanolamine
`
`“PA” means phosphatidic acid
`
`“PL” means plasmalogen
`
`“PP” means phosphonolipid
`
`20
`
`“ALP” means alkylacylphospholipid
`
`“SM” means sphingomyelin
`
`“CAEP” means ceramide aminoethylphosphonate
`
`“GS” means ganglioside
`
`“N/D” meansnot detected
`
`25
`
`“CO” means carbon dioxide
`
`10
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`GENERAL DESCRIPTION OF THE INVENTION
`
`As discussed in the Background,it is known that supercritical CO. with up to 12.5% ethanol
`as a co-solvent can extract the phospholipids PC, and to a much lesser extent, PE and PI
`from soy or egg. Surprisingly, we have foundthat the phospholipids PS, CAEP and CL; and
`gangliosides are virtually insoluble in CO. and a C)-C3 monohydricalcohol co-solvent, and
`that SM, ALP, PL and PPare soluble. Therefore it is possible to separate the soluble
`phospholipids from the insoluble phospholipids and gangliosides to achieve fractions
`enriched in one or other of the desired components.
`
`10
`
`There are a numberof factors affecting the operation of the process:
`
`=
`
`Feed material and feed preparation
`
`= Extraction temperature and pressure
`
`15
`
`=" Co-solvent concentration
`
`= Total solvent throughput
`
`«
`
`Solvent flow rate and contacting conditions
`
`20
`
`25
`
`30
`
`It is advantageous to start with a feed material containing at least 5 % by mass of lipids, and
`ideally at least 2 % by mass of phospholipids, particularly PS, SM, CL, ALP, PL, PP, CAEP
`and/or gangliosides.
`
`The feed material can be processed using pure CObefore the co-solventis introduced to
`remove muchorall of neutral lipids. This reduces the neutral lipid content in the CO)+co-
`solventextract leading to an extract enriched in soluble phospholipids and/or gangliosides.
`
`The form of the feed material depends on the sourceofthe lipids andits lipid composition.
`For example dairylipid extracts high in phospholipids may be substantially solid even at
`elevated temperatures. Egg yolk and marine lipids in comparison have a lower melting point.
`The presence ofneutral lipids also tends to produce a more fluid feed material. To promote
`good contacting it may be beneficial to prepare the feed material. Solid materials containing
`lipids may be able to be cryomilled. Lipid feed materials can also be made morefluid by the
`inclusion of some ethanol or water.
`
`11
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`Changing the processing conditions oftemperature, pressure, co-solvent concentration, and
`total solvent usage, influences the amount of material extracted, the purity of the final
`product, and the recovery (orefficiency) of the process. For example, the virtually insoluble
`lipids such as PS, GS, CAEP and CL, have very slight solubilities so that excessive use of
`solvent, or very favourable extraction conditions, can result in small losses of PS, GS and CL
`from the residual fraction. A high purity product may be achieved, but with a reduced yield.
`Conversely the enrichmentof soluble lipids will be greater if smaller amounts of the other
`lipids are co-extracted, but the total yield will be lower. Processing economics, and the
`relative values of the products, will determine where this balance lies. A further option to -
`obtain multiple enriched fractionsis to carry out extractions under progressively more
`favourable extraction conditions, such as increasing the temperature.
`
`Wehave foundthat co-solvent concentrations below about 10% producevery little extract of
`phospholipids and/or gangliosides. At higher concentrations the rate of material extracted
`increases rapidly. We have found the co-solvent concentrationsofat least 20%, and more
`preferably 30% achieve high levels of extraction ofPC, PE, SM, ALP, PL, PP and PI, while
`the lipids PS, CL and GS remain virtually insoluble.
`
`Every substancehas its own “critical” point at which the liquid and vapour state of the
`substance becomeidentical. Above but close to thecritical point of a substance, the
`substance is in a fluid state that has properties of both liquids and gases. The fluid has a
`density similar to a liquid, and viscosity and diffusivity similar to a gas. The term
`“supercritical” as used herein refers to the pressure-temperature region abovethecritical
`point of a substance. The term “subcritical” as used herein refers to the pressure-temperature
`region equal to or above the vapour pressure for the liquid, but belowthecritical
`temperature. The term “near-critical” as used herein encompasses both “supercritical” and
`“subcritical” regions, and refers to pressures and temperatures near thecritical point.
`
`Percentages unless otherwise indicated are on a w/w solids basis.
`
`The term “comprising” as used in this specification means “consisting at least in part of”.
`Wheninterpreting each statementin this specification that includes the term “comprising”,
`features other thanthat or those prefaced by the term may also be present. Related terms
`such as “comprise” and “comprises”are to be interpreted in the same manner.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
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`The invention consists in the foregoing and also envisages constructions of which the
`
`following gives examples only.
`
`EXAMPLES
`
`10
`
`15
`
`20
`
`25
`
`30
`
`The experimental process is described, with referenceto figure 1, as follows.
`
`A measured mass of feed material containing lipids to be fractionated was placed in basket
`BK1 with a poroussintered steel plate on the bottom. Basket BK1 was placed in a 300 mL
`extraction vessel EX1. The apparatus was suspended in heated water bath WB1 and
`maintained at a constant temperature through use of a thermostat and electric heater.
`
`In the continuous extraction mode of 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 through valve V1, the pressure was reducedto the supply
`cylinder pressure of 40 to 60 bar, which caused the CO> to be converted to a lower density
`fluid and lose its solvent strength. Precipitated material was captured in separation vessel
`SEP1, and the CO, exited from the top of separator SEP1 and was recycled back to the feed
`pump through coriolis mass flow meter FM1 and cold trap CT1 operated at -5°C. Extracted
`material was collected periodically from separator SEP1 by opening valve V2. The
`extraction was optionally carried out using COonly until all of the compounds soluble in
`CO, only, such as neutral lipids, were extracted. When no further extract was produced by
`CO, extraction, ethanol co-solvent with or without added water was added to the CO>at the
`desired flow ratio from supply bottle B2 using pump P2. Ethanol and further extracted
`material were separated from the CO, in separator SEP1 and periodically removed through
`valve V2. After the desired amountofethanol had been added the ethanol flow was stopped
`and the CO. flow continued alone until all the ethanol had been recovered from the system.
`The remaining CO2 was vented and the residual material in basket BK1 was removed and
`dried under vacuum. The extract fraction was evaporated to dryness by rotary evaporation.
`
`In the batch extraction mode of operation COz alone wasoptionally passed continuously
`through the apparatus, as for the continuous flow mode of operation, until all CO2 alone
`extractable material was removed. The CO2 flow was then stopped and valve V1 closed to
`maintain the pressure. Approximately 140gof ethanol was pumped from supply bottle B2
`
`13
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`WO 2007/123424
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`through pump P2 into extraction vessel EX1. The system wasleft for 15 minutes to allow the
`system to equilibrate, after which time the CO, flow wasstarted and valve V1 opened to
`maintain a constant pressure and allow ethanol and dissolved compoundsto flow 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.
`
`Extract and residue fractions were analysed for phospholipid content and profile by a
`NMR.The phospholipid massfractions reported here are for phosphatidylcholine (PC),
`phosphatidylinositol (PI), phosphatidylethanolamine (PE), plasmalogens (PL),
`phosphonolipids (PP), alkylacylphospholipids (ALP), sphingomyelin (SM), ceramide
`aminoethylphosphonate (CAEP), 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.
`
`15
`
`Example 1: Fractionation of dairy lipid extract A, ethanol] mass
`fraction 25%
`
`Lipid extract A is a total lipid extract obtained by a processes disclosed in PCT international
`applications PCT/NZ2005/000262 (published as WO 2006/041316).
`
`20
`
`25
`
`30
`
`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 (extract 1) using CO2
`only. This extract contained no phospholipids, and was entirely neutral lipids. A further 31%
`of the feed material (extract 2) was extracted using 95% aqueous ethanolat a concentration
`in COz of 25%. The total ethanol and water added was 880g. The composition of the fraction
`
`extracted with COand ethanol (extract 2), and the composition of the residual fraction are
`
`shown in Table 1. The extract is enriched in phosphatidylcholine (PC) and sphingomyelin
`(SM)which are more soluble in COand ethanol, while the residual fraction is substantially
`enriched in phosphatidylserine (PS). Phosphatidylserine levels are virtually undetectable in
`the extract phase indicating very low solubility in CO2 and ethanol, and almost complete
`recovery of phosphatidylserine in the residue phase.
`
`14
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`Table 1
`
` 4.9
`
`osition, %
`Other
`Phospholipids
`
`Other compounds
`
`37.0
`
`Example 2: Fractionation of dairy lipid extract A, ethanol mass
`fraction 31%
`
`41g of dairy lipid extract A, with composition as for example 1 was extracted using the
`
`10
`
`continuous extraction mode of operation at 60°C and 300bar as for example 1, using firstly
`
`COzalone to extract 50 % of the feed material (extract 1), which is neutral lipids only, and
`
`then using 95% aqueous ethanol at a concentration in COz2 of 31%. 33% of the feed material
`
`was extracted (extract 2). The total ethanol and water added was 1150g. The composition of
`
`the residual fraction is shown in Table 2. The higher ethanol concentration gives a more
`
`15
`
`complete extraction of lipids and the concentration of phosphatidylserine in the residue
`
`fraction is higher than found in example 1 at 19.3 %.
`
`Table 2
`
`
`
`
`Composition, %
`Other
`
`Phospholipids
`SM_|
`PE
`PS
`PI
`PC
`
`2.8|43|13.2|7.8 2.2 58.3
`
`
`|-[|-|-|-|- f
`|-|
`
`25.5
`
`
`Other compounds
`
`Yield
`
`20
`
`Example 3: Fractionation of dairy lipid extract A, ethanol mass
`fraction 43%
`
`40g of dairy lipid extract A, with composition as for example 1 was extracted using the
`
`25
`
`continuous extraction modeof operation at 60°C and 300 baras for example 1, using firstly
`
`CO, alone to extract 41 % of the feed material (extract 1), which is neutral lipids only, and
`then using 95% aqueousethanol at a concentration in CO2 of 43% to extract 32 % of the feed
`
`(extract 2). The total ethanol and water added was 960g. The composition of extract 2 and
`residual fractions are shown in Table 3. The concentration of phosphatidylserine in the
`
`30
`
`residue fraction is higher than found in example 1 and example 2 at 20.7 %. The
`
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`
`concentration of SM in the extract, at 12.5 % by mass,is enriched relative to the feed, at 7.8
`% by mass, even thoughit also contains a high level of neutral lipids.
`
`Table 3
`
`i
`'% of feed|
`
`Phospholipids
`
`Composition, %
`Other
`
`Other compounds
`
`Example 4: Fractionation of dairy lipid extract A, 40°C
`
`10
`
`39g of dairy lipid extract A, with composition as for example 1 was extracted using the
`continuous extraction mode of operation at 300 bar using firstly CO? alone to extract 54 % of
`the feed material (extract 1), which is neutral lipids only, and then using 95% aqueous
`ethanol at a concentration in CO2 of 30 % to extract 12 % of the feed (extract 2). The
`temperature in this example was 40°C. The total ethanol and water added was 975g. The
`composition of the extracted and residual fractions are shown in Table 5. The degree of
`extraction of SM is lower than for examples 1 to 3 at 60°C, but the concentration in the
`
`15
`
`extract is higher. The concentration of PS in the residue, at 12.4 %, is lower than examples 1
`to 3.
`
`Table 4
`
`
`
`
`Composition, %
`Yield
`;
`Other
`Other compounds
`
`
`
`PC
`% of feed|
`PI
`PS
`PE
`SM_|
`Phospholipids
`
`Feed[|112|28
`Er
`
`Extract2|12|27.9|
`a
`12
`
`
`
`34
`
`
`
`
`
`Residue
`9.9
`
`20
`
`Example 5: Fractionation of dairy phospholipid concentrate
`
`40g of a dairy phospholipid concentrate with composition as shown in Table 5 (feed) was
`extracted using the continuous extraction modeof operation at 300 bar and 60°C withoutthe
`prior COonly extraction step. The ethanol (95% aqueous ethanol) massfraction in CO2 was
`
`25
`
`30%. The total ethanol and water added was 1026g. The composition of the extracted and
`
`residual fractions are shown in Table 5. Only 11% of the feed lipid was extracted, so the
`
`enrichment of phosphatidylserine in the residue is not significant, but the concentration did
`
`increase from 8% to 8.8%. The poor degree of extraction in this example is due to the
`
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`physical properties ofthe solid feed material limiting mass transfer. In comparison, the dairy
`lipid extract in examples 1 through 4,is liquid at the processing temperature and better
`extraction rates are observed.
`
`Different feed preparation methods and/or longer equilibration times and/or greater solvent
`quantities are expected to increase the amount of extractable material.
`
`Table 5
`
`
`
`Yield
`% of
`feed]
`
`PC
`
`PI
`
`PS
`
`PE
`
`Composition, %
`Other
`Phospholipids
`
`SM_|
`
`Other compounds
`
`
`
`
`Nuvopomam &an
`
`
`Pp 1 a
`
`
`
`
`10
`
`Example 6: Fractionation of dairy phospholipid concentrate using
`the batch extraction process
`
`15
`
`20
`
`25
`
`19g of a dairy phospholipid concentrate with composition as described in example 5 was
`extracted using the batch extraction mode of operation at 300 bar and 60°C.A total of 22%
`of the feed mass wasextracted in three sequential extractions each consisting of 140g of
`ethanol (95% aqueous ethanol) in 300mL of CO. The composition of the extracted andfinal
`residual fractions are shown in Table 6. In this example 22% ofthe feed lipid was extracted,
`significantly higher than that obtained in the continuous extraction example (example 5) and
`using a lowertotal quantity of ethanol co-solvent. The phosphatidylserine concentration in
`the residue has increased from 8% to 11.2%; and the sphingomyelin concentration in the
`extract has increased from 15.1 to 16.7 %. This example showsthe increase in total extracted
`material by allowing a greater contacting time to more completely dissolve the soluble
`fraction.
`
`Table 6
`
`
`
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`Example 7: Fractionation of dairy lipid extract B, ethanol mass
`fraction 10%
`
`This examplerelates to extraction of dairy lipid extract B,a total lipid extract obtained from
`high fat whey protein concentrate processes disclosed in PCT international applications
`PCT/NZ2004/000014 (published as WO WO2004/066744).
`
`10
`
`with composition shown in Table 7 (feed). The ‘other compounds’ listed include 2-3%
`gangliosides and about 3% lactose, both absent in dairy lipid extract A. In this example 42g
`of dairy lipid extract B was extracted using the continuous extraction mode of operation at
`300 bar and 60°C. 52% of the feed mass was extracted using CO. alone (extract 1). Only 3%
`of the feed lipid was further extracted using 460g of 95% aqueous ethanol (extract 2), and
`the extract contained less than 10% phospholipids. The extraction of phospholipids does not
`occur to any significant extent for ethanol mass fractions of 10% or lower. The ethanol does
`howeverextract some additional neutrallipid that is not extracted using CO2 alone.In this
`
`15
`
`case, both the PS and SM are enriched in the residue.
`
`
`
`
`|veeFaae|% of feed|
`
`
`
`PC
`PI
`PS
`PE
`SM Phospholipids
`
`
`7,
`2.
`.
`.
`5.7
`1.3
`69.0
`
`0.3
`
`
`
`Composition, %
`
`20
`
`25
`
`Example 8: Fractionationofdairy lipid extract B, ethanol mass
`fraction 30%
`
`In this example 40g ofdairy lipid extract B was extracted using the continuous extraction
`mode of operation at 300 bar and 60°C. 51% of the feed mass was extracted using CO2 alone
`(extract 1). A further 7% of the feed material was extracted using 760g of 95% aqueous
`ethanol at a mass concentration of 30% in CO: (extract 2). Phospholipid profiles for the
`
`extract and residual fractions are shown in Table 8. Both PS and SM are enriched in the
`
`residue
`
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`
`Table 8
`
`Other compounds
`
`mposition, %
`Other
`
`Example 9: Fractionation of dairy lipid extract A, ethanol mass
`fraction 3%
`
`This example shows that when the co-solvent concentration is below 10% by mass, no
`
`phospholipids are extracted.
`
`10
`
`In this example 27g of dairy lipid extract A, as described in example 1, was extracted using
`the continuous extraction mode of operation at 300 bar and 60°C,using 98% ethanol at 3 %
`
`by massratio with CO2, without the CO. only extraction step. 62% of the feed mass was
`
`extracted. No detectable phospholipids were extracted. This extract represents 90% of the
`
`neutral lipid present in the feed material. The rate of extraction of neutral lipid from the feed
`
`15
`
`material was substantially faster using the ethanol co-solvent than using CO2 only. The
`
`extract material was substantially extracted using less than the total of 150g of ethanol in
`
`4850g of CO2 used, while typically 10 kg of CO2 alone is required for extraction of neutral
`
`lipids, as in example 1.
`
`20
`
`Example 10: Fractionation of egg yolk lecithin
`
`This example relates to fractionation of a commercially available egg yolk lecithin, with
`
`phospholipid profile shown in Table 9. No phosphatidylserine was detected in the feed lipid,
`indicating concentration levels <0.5%. In this example 34g of the feed material was
`
`25
`
`extracted using the continuous extraction mode of operation at 300 bar and 60°C, and 95%
`
`aqueousethanol at a concentration of 25%. 45% of the feed mass wasextracted as neutral
`
`lipids using CO, alone. A further 49% of the feed material was extracted using ethanol and
`
`CQ,with a total ethanol flow of 640g. Phospholipid profiles for the extract and residual
`
`fractions are shown in Table 9. In this example, the phosphatidylserine levels in the residual
`
`30
`
`material are substantially enriched compared with non-detectable levels in the feed material.
`
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`
`:
`
`Residue|6|174[80|5.9|19.1
`
`
`
`
` Composition, %
`ad
`a
`
`Niga Nl”s
`PC
`Phospholipids
`% of feed|
`wfZjZ sfNya Bealo
`[56.4
`
`
`
`~”n
`
`Example 11: Fractionation of egg yolk phospholipid extract
`
`This examplerelates to fractionation of an egg yolk phospholipid fraction with phospholipid
`profile shown in Table 9. In this example 40g of the feed material was extracted using the
`continuousextraction mode of operation at 300 bar and 60°C, and 95% aqueousethanol at a
`concentration of 28%. 50% of the feed mass was extracted as neutral lipids using COalone.
`A further 46% of the feed material was extracted using ethanol and CO2 with a total ethanol
`flow of 800g. Phospholipid profiles for the extract and residual fractions are shown in Table
`10. In this example, the phosphatidylserine levels in the residual material are substantially
`enriched compared with levels in the feed material, while sphingomyelin is enriched in the
`extract relative to the feed.
`
`Table 10
`
`Example 12: Fractionation of Hoki head lipid extract
`
`This example relates to fractionation of a Hoki head lipid extract with phospholipid profile
`shown in Table 11. In this example 25g of the feed material was extracted using the
`continuous extraction mode of operation at 300 bar and 60°C, and 95% aqueousethanolat a
`concentration of 31%. 1% of the feed mass was extracted as neutrallipids using CO, alone.
`A further 72% of the feed material was extracted using ethanol and CO, with a total ethanol
`flow of 940g. Phospholipid profiles for the extract and residual fractions are shown in Table
`11. In this example, the phosphatidylserine levels in the residual material are substantially
`enriched compared with levels in the feed material. Some PS is also observed in the extract
`‘phase. The alkylacylphosphatidylcholine (AAPC), a type of alkylacylphospholipid, is
`completely extracted.
`
`20
`
`10
`
`15
`
`20
`
`25
`
`RIMFROST EXHIBIT 1055
`
`RIMFROST EXHIBIT 1055 page 0813
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`
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`

`WO 2007/123424
`
`PCT/NZ2007/000087
`
`Table 11
`
`Example 13: Fractionation of bovine heart lipid extract
`
`This example relates to fractionation of a bovine heart phospholipid lipid extract with
`phospholipid profile shown in Table 9. In this example 40g of the feed material was
`extracted using the continuous extraction mode of operation at 300 bar and 60°C, and 95%
`aqueous ethanol at a concentration of 33% in CO. Nolipid was extracted using CO2zalone.
`79% of the feed material was extracted using ethanol and CO) with a total ethanol flow of
`960g. Phospholipid profiles for the extract and residual fractions are shown in Table 12. The
`phosphatidylserine levels in the residual material are substantially enriched compared with
`levels in the feed material. Cardiolipin is also significantly enriched in the residue.
`
`10
`
`15
`
`Table 12
`
`
`
`Composition, wt%
`ste|NO|=
`
` ©|o0]9)lanl
`
`Other
`Phospholipid| Other compounds
`
`PS_| PE|SM s
`
`:
`—
`8
`2.3
`
`8.2 | 18.6|0.8|0.4|8.6
`
`2
`14.1]
`4.7
`]23.4] |
`0.0
`
`
`
`
`
`20
`
`25
`
`Example 14: Fractionation of dairy lipid extract A with propan-2-ol
`co-solvent
`
`In this example 39¢ of the dairy lipid extract A, as described in example 1, was extracted
`using the continuous extraction mode of operation at 300 bar and 60°C, and 95% aqueous
`propan-2-ol at a mass concentration of 35% in CO>. 48% ofthe feed material was extracted
`as neutral lipids using CO2 alone. 23% ofthe feed material was further extracted using the
`propan-2-ol co-solvent and CO2 with a total propanol mass of 810g. Phospholipid profiles
`for the extract and residual fractions are shown in Table 13. The phosphatidylserine levels in
`
`the residual material are substantially enriched, and the result is comparable to results for
`examples 1 and 2. A slightly lower total PS level is achieved than for example 2 using a
`
`21
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`

`WO 2007/123424
`
`PCT/NZ2007/000087
`
`comparable concentration of ethanol. The levels of PS observedin the extracted fraction is
`
`also higher suggesting the propan-2-ol is not as selective as ethanol. On this basis alone
`
`ethanol would be the preferred co-solvent.
`
`Table 13
`
`. Composition, %
`Other
`Phospholipids
`
`SM_|
`
`PE
`
`Other compounds
`
`4.2
`
`;
`
`;
`
`Example 15: Fractionation of soy lecithin
`
`This example relates to fractionation of a soy lecithin (Healtheries Lecithin natural dietary
`
`10
`
`supplement, Healtheries of New Zealand Limited) with composition shown in Table 9 . In
`
`this example 42g of feed material was extracted using the continuous extraction mode of
`
`operation at 300 bar and 60°C, and 95% aqueous ethanol at a concentration of 33% in COz.
`
`Nolipid was extracted using COz2alone. 91% of the feed material was extracted using
`
`ethanol and CO2 with a total ethanol flow of 520g. Phospholipid profiles for the extract and
`
`15
`
`residual fractions are shown in Table 14. PC and PEare preferentially extracted and are
`
`significantly enriched in the extract. There are no detectable levels of PS or SM in this
`
`example.
`
`Table 14
`
`mposition, %
`Other
`Yield
`Phospholipids
`SM_|
`PE
`PS
`PI
`PC
`% of feed)
`Feed||22.2|12.3|
`22.2
`
`Extract|9|:; : 51.4
`
`:
`;
`
`Other compounds
`
`35.2
`
`20
`
`Example 16: Continuous fractionation of egg yolk lipids
`
`This example relates to fractionation of an egg yolk lipid extract containing 15%
`
`25
`
`phospholipids and the balance mostly neutral lipids by HPLC analysis. The phospholipid
`
`fraction contains 55% PC, 29% PE, and 14% PI . The feed lipid was pumpedinto the top of
`
`a 10L pressure vessel, and contacted with CO, containing 8.7 % of 98% aqueous ethanol
`flowing upwards throughthe vessel at 300 bar pressure and temperature of 60°C. An extract
`phase was continuously taken from the top of the contacting vessel, and a raffinate phase
`
`22
`
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`WO 2007/123424
`PCT/NZ2007/000087
`was periodically withdrawn from the bottom ofthe vessel. The lipid feed rate was 1.5 kg/hr.
`The CO2+ co-solvent flow rate was 27 kg/hr.
`
`The extract phase was predominantly neutral lipids but contained 20% ofthe phospholipids
`present in the feed stream. The phospholipids in the extract fraction consisted of between
`70% and 100% PC, with the balance mostly PE. This represents a preferential extraction of
`PC over other phospholipids.
`
`In a second experiment, feed lipid was premixed with 98% ethanol (with 2 % water) at a
`concentration of 10.2% lipid. This mixture was pumpedinto the top of the pressure vessel
`and contacted with CO2 in upflow. The overall concentration of ethanolin COz2under steady
`state processing conditions was 5.9%. In this case 50% of the mass ofphospholipids in the
`feed were extracted. The composition ofthe extract phase consisted of between 60% and
`70% PC, with the balance mostly PE. The presence of PI and other phospholipids in the
`extract was not detectable by the HPLC method used.
`
`Example 17: Fractionation of green-lipped mussel lipid extract
`
`This example relates to fractionation of a green-lipped mussel lipid extract with phospholipid
`profile shown in Table 11. In this example 12.2g ofthe feed material was extracted using a
`batch stirred tank method at 250 bar and 60°C using CO, and ethanol (containing 5 % water)
`at a concentration of 30.5 %. The lipid was placedin the stirred tank, CO. was added to give
`the desired pressure and then the 95 % ethanol was added in during constant stirring. 65 % of
`the feed material was then extracted using CO, and ethanolafter stirring for 1 hour by
`sampling the extract phase at constant pressure. Phospholipid profiles for the extract and
`residual fractions are shown in Table 15. In this example, the CAEPlevels in the residual
`material are substantially enriched compared with levels in the feed material. The
`alkylacylphosphatidylcholine (AAPC), a type of alkylacylphospholipid, is partially
`extracted.
`
`10
`
`15
`
`20
`
`25
`
`
`
`
`Table 15
`
`Ty
`[es
`
`
`
`Composition, %
`
`Yield
`SM_|AAPC]| CAEP
`PE
`% ofeos PC 0 PS
`ras
`\o
`
`3.77
`
`
`Other compounds
`
`23
`
`RIMFROST EXHIBIT 1055
`
`RIMFROST EXHIBIT 1055 page 0816
`
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`

`

`WO2007/123424
`Example 18: Fractionation o