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`PCT/NZ2007/000087
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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 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%.
`
`‘
`
`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 CO2 is recycled for further use.
`
`The co—solvent may be removed by evaporation under vacuum.
`
`Preferably the feed material is contacted with a continuous flow of solvent.
`
`Preferably the feed material is contacted with one or more batches of solvent.
`
`The lipid and solvent streams may be fed continuously.
`
`20
`
`25
`
`Optionally, the feed material and co-solvent streams may be mixed prior to contacting with
`
`C02.
`
`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
`
`the feed material (also referred to herein as the “extract”). Where the feed material is
`
`contacted with more than one batch of solvent, or the solvent is cooled in a number of steps,
`
`there will be multiple “extract” products.
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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 comprises greater
`
`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% phosphatidyl serine.
`
`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 more preferably 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
`
`15
`
`20
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`25
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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 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
`
`Figure 4 is a scheme drawing illustrating a fourth preferred process of the current invention
`
`10
`
`ABBREVIATIONS AND ACRONYMS
`
`In this specification the following are the meanings of the abbreviations or acronyms used.
`
`“CL” means cardiolipin
`
`“PC” means phosphatidyl choline
`
`“PI” means phosphatidyl inositol
`
`15
`
`“PS” means phosphatidyl serine
`
`“PE” means phosphatidyl ethanolamine
`
`“PA” means phosphatidic acid
`
`“PL” means plasmalogen
`
`“PP” means phosphonolipid
`
`2O
`
`“ALP” means alkylacylphospholipid
`
`“SM” means sphingomyelin
`
`“CAEP” means ceramide aminoethylphosphonate
`
`“GS” means ganglioside
`
`“N/D” means not detected
`
`25
`
`“C02” means carbon dioxide
`
`10
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`GENERAL DESCRIPTION OF THE INVENTION
`
`As discussed in the Background, it is known that supercritical C02 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 found that the phospholipids PS, CAEP and CL; and
`gangliosides are Virtually insoluble in C02 and a C1-C3 monohydricalcohol co-solvent, and
`
`that SM, ALP, PL and PP are 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 number of 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
`
`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 C02 before the co-solvent is introduced to
`
`remove much or all of neutral lipids. This reduces the neutral lipid content in the C02+co-
`
`solvent extract leading to an extract enriched in soluble phospholipids and/or gangliosides.
`
`The form of the feed material depends on the source of the lipids and its lipid composition.
`
`For example dairy lipid extracts high in phospholipids may be substantially solid even at
`
`30
`
`elevated temperatures. Egg yolk and marine lipids in comparison have a lower melting point.
`The presence of neutral 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 more fluid by the
`
`inclusion of some ethanol or water.
`
`11
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`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 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
`fiom the residual 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-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 fractions is to carry out extractions under progressively more
`
`favourable extraction conditions, such as increasing the temperature.
`
`We have found that co—solvent concentrations below about 10% produce very little extract of
`
`phospholipids and/or gangliosides. 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, ALP, PL, PP and PI, while
`the lipids PS, CL and GS remain virtually insoluble.
`
`Every substance has its own “critical” point at which the liquid and vapour state of the
`
`substance become identical. Above but close to the critical 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 above the critical
`
`point of a substance. The term “subcritica ” as used herein refers to the pressure-temperature
`
`region equal to or above the vapour pressure for the liquid, but below the critical
`
`temperature. The term “near-critical” as used herein encompasses both “supercritical” and
`
`“subcritical” regions, and refers to pressures and temperatures near the critical 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”.
`
`When interpreting each statement in this specification that includes the term “comprising”,
`
`features other than that 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
`
`3O
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`35
`
`12
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`The invention consists in the foregoing and also envisages constructions of which the
`
`following gives examples only.
`
`EXAMPLES
`
`The experimental process is described, with reference to figure 1, as follows.
`
`A measured mass of feed material containing lipids to be fractionated was placed in basket
`
`BKl with a porous sintered steel plate on the bottom. Basket BKI was placed in a 300 mL
`
`extraction vessel EXl. The apparatus was suspended in heated water bath WBl and
`
`10
`
`maintained at a constant temperature through use of a thermostat and electric heater.
`
`In the continuous extraction mode of operation, liquid C02 from supply bottle B1 was
`
`pumped using pump P1 into extraction vessel EX] until the pressure reached the desired
`operating pressure, after which valve VI was operated to maintain a constant pressure in the
`extraction vessel. After passing through valve V1, the pressure was reduced to the supply
`
`15
`
`cylinder pressure of 40 to 60 bar, which caused the CO2 to be converted to a lower density
`
`fluid and lose its solvent strength. Precipitated material was captured in separation vessel
`
`SEPl, and the C02 exited from the top of separator SEPl and was recycled back to the feed
`
`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
`
`20
`
`extraction was optionally carried out using C02 only until all of the compounds soluble in
`C02 only, such as neutral lipids, were extracted. When no further extract was produced by
`C02 extraction, ethanol co-solvent with or without added water was added to the CO2 at the
`
`desired flow ratio from supply bottle B2 using pump P2. Ethanol and further extracted
`
`25
`
`material were separated from the CO2 in separator SEPl and periodically removed through
`
`valve V2. After the desired amount of ethanol had been added the ethanol flow was stopped
`
`and the C02 flow continued alone until all the ethanol had been recovered from the system.
`The remaining CO2 was vented and the residual material in basket BKI was removed and
`dried under vacuum. The extract fraction was evaporated to dryness by rotary evaporation.
`
`30
`
`In the batch extraction mode of operation CO2 alone was optionally 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 140g of ethanol was pumped from supply bottle B2
`
`13
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`through pump P2 into extraction vessel EXl. The system was left for 15 minutes to allow the
`
`system to equilibrate, after which time the C02 flow was started and valve V1 opened to
`
`maintain a constant pressure and allow ethanol and dissolved compounds to flow through to
`
`separator SEPl. This process was repeated twice more, after which the CO; was vented and
`
`the residual material in basket BKl was removed and dried under vacuum.
`
`Extract and residue fractions were analysed for phospholipid content and profile by 31P-
`
`NMR. The phospholipid mass fractions reported here are for phosphatidylcho line (PC),
`
`phosphatidylinositol (PI), phosphatidylethanolamine (PE), plasmalogens (PL),
`
`10
`
`phosphonolipids (PP), alkylacylphospholipids (ALP), sphingomyelin (SM), ceramide
`
`aminoethylphosphonate (CAEP), phosphatidylserine (PS), and cardiolipin (CL).
`
`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/NZZOOS/000262 (published as W0 2006/041316).
`
`20
`
`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 C02
`
`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 ethanol at a concentration
`
`25
`
`in C02 of 25%. The total ethanol and water added was 880g. The composition of the fraction
`
`extracted with C02 and 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 C02 and ethanol, while the residual fraction is substantially
`
`enriched in phosphatidylserine (PS). Phosphatidylserine levels are virtually undetectable in
`
`30
`
`the extract phase indicating very low solubility in C02 and ethanol, and almost complete
`
`recovery of phosphatidylserine in the residue phase.
`
`14
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`
`
`Phos-oholi ids
`
`2.2
`58.3
`4.9
`37.0
`
`om osition, %
`
`Example 2: Fractionation of dairy lipid extract A, 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 firstly
`
`C02 alone to extract 50 % of the feed material (extract 1), which is neutral lipids only, and
`
`then using 95% aqueous ethanol at a concentration in C0; 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
`
`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
`
` 22
`
`Comosition, %
`
`Other
`PhosthLipidS
`.
`
`Other compounds
`
`58.3
`
`25 5
`
`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
`
`continuous extraction mode of operation at 60°C and 300 bar as for example 1, using firstly
`
`C02 alone to extract 41 % of the feed material (extract 1), which is neutral lipids only, and
`
`then using 95% aqueous ethanol at a concentration in C02 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
`
`residue fraction is higher than found in example 1 and example 2 at 20.7 %. The
`
`15
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`RIMFROST EXHIBIT 1055 page 0808
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`3O
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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 though it also contains a high level of neutral lipids.
`
`Table 3
`
`
`
`Yield
`PE
`PS
`PI
`PC
`% 0f feed
`to
`um -
`b.)
`—---m -
`
`O\ \l
`.8
`7
`
`
`
`9—\qu
`
`
`Com-osition, %
`Other
`Other compounds
`Phosholi nids
`
`
`
`
`Example 4: Fractionation of dairy lipid extract A, 40°C
`
`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 C02 alone to extract 54 % of
`
`10
`
`the feed material (extract 1), which is neutral lipids only, and then using 95% aqueous
`
`ethanol at a concentration in C02 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 975 g. 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
`
`
`
`
`Com osition, %
`.
`Other
`Other compounds
`SM Phospholi nids
`
`PE
`
`Yleld
`PS
`% of feed
`PC
`PI
`
`-
`_- 2-8
`- --
`
`
`.3
`
`'
`
`
`.9
`34
`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 mode of operation at 300 bar and 60°C without the
`
`prior C02 only extraction step. The ethanol (95% aqueous ethanol) mass fraction 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
`
`16
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`physical properties of the 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.
`
`5
`
`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
`
`
`
`
`
`Com osition, %
`Other
`Yield
`SM Phos-holi nids
`PE
`PS
`PI
`PC
`% of feed
`
`-—---———m
`
`—_----_—
`
`
`_Im-——
`
`
`
`
`
`
`
`
`
`Other compounds
`
`10 Example 6: Fractionation of dairy phospholipid concentrate using
`the batch extraction process
`
`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%
`
`15
`
`of the feed mass was extracted in three sequential extractions each consisting of 140g of
`
`ethanol (95% aqueous ethanol) in 300mL of C02. The composition of the extracted and final
`
`residual fractions are shown in Table 6. In this example 22% of the feed lipid was extracted,
`
`significantly higher than that obtained in the continuous extraction example (example 5) and
`
`using a lower total quantity of ethanol co—solvent. The phosphatidylserine concentration in
`
`20
`
`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 shows the increase in total extracted
`
`material by allowing a greater contacting time to more completely dissolve the soluble
`fraction.
`
`25
`
`Table 6
`
`
`
`
`I
`,-
`Other
`Other compounds
`
`% of feed
`PC
`PI
`PS
`PE
`SM Phos-nholiids
`
`
`15-4 ”III——
`
`
`
`'
`
`-
`
`-
`
`-
`
`- -133
`
`17
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`Example 7: Fractionation of dairy lipid extract B, ethanol mass
`fraction 10%
`
`This example relates to extraction of dairy lipid extract B, a total lipid extract obtained from
`
`high fat whey protein concentrate processes disclosed in PCT intemational applications
`
`PCT/NZZOO4/000014 (published as W0 W02004/066744).
`
`with composition shown in Table 7 (feed). The 5other 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
`
`10
`
`300 bar and 60°C. 52% of the feed mass was extracted using C02 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
`
`however extract some additional neutral lipid that is not extracted using C02 alone. In this
`
`15
`
`case, both the PS and SM are enriched in the residue.
`
`
`Table 7
`
`
`
`
`Com . osition, %
`Other
`Other compounds
`
`
`SM Phospholipids
`
`
`
`5 7.
`69.0
`1.3
`
`
`
`--—__mm
`8.7
`21.8
`12.0
`5.9
`
`
`PE
`
`PS
`
`20
`
`25
`
`Example 8: Fractionation of dairy lipid extract B, ethanol mass
`fraction 30%
`
`In this example 40g of dairy 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 C02 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 C02 (extract 2). Phospholipid profiles for the
`
`extract and residual fractions are shown in Table 8. Both PS and SM are enriched in the
`
`residue
`
`1 8
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`2.4
`
`Com-osition, %
`Other
`Phos holi - ids
`
`Other compounds
`
`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
`
`15
`
`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 mass ratio with C02, without the C02 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
`
`material was substantially faster using the ethanol co—solvent than using C02 only. The
`
`extract material was substantially extracted using less than the total of 150g of ethanol in
`
`4850g of C02 used, while typically 10 kg of C02 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
`
`extracted using the continuous extraction mode of operation at 300 bar and 60°C, and 95%
`
`aqueous ethanol at a concentration of 25%. 45% of the feed mass was extracted as neutral
`
`lipids using C02 alone. A further 49% of the feed material was extracted using ethanol and
`
`CO2 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
`
`material are substantially enriched compared with non-detectable levels in the feed material.
`
`25
`
`30
`
`19
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`RIMFROST EXHIBIT 1055 page 0812
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`
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`Com-osition, %
`
`hos - holi - ids
`
`Other
`
`Other compounds
`
`Example 11: Fractionation of egg yolk phospholipid extract
`
`This example relates 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
`
`continuous extraction mode of operation at 300 bar and 60°C, and 95% aqueous ethanol at a
`
`concentration of 28%. 50% of the feed mass was extracted as neutral lipids using C02 alone.
`
`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.
`
`10
`
`15
`
`
`Table 10
`
`Com nosition, %
`
`Yield
`
`
`% of feed
`
`
`
`(1)
`_
`
`
`
`
`LIN-ul! O\
`:9reto“H
`
`
`
`Other compounds
`Other
`SM Phos holiids
`
`- -.E-_
`-
`
`Z iw
`
`00 N
`27-6
`
`
`
`“re 'O
`Ulr—A QN
`HmP. \o
`
`76mm
`
`xtract
`
`g,aQ.S('0
`
`Example 12: Fractionation of Hoki head lipid extract
`
`This example relates to fractionation of a Hoki head lipid extract with phospholipid profile
`
`20
`
`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% aqueous ethanol at a
`concentration of 31%. 1% of the feed mass was extracted as neutral lipids using C02 alone.
`
`A further 72% of the feed material was extracted using ethanol and CO2 with a total ethanol
`
`25
`
`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
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`Table 11
`
`
`
`Other
`- hos h
`
`Other compounds
`
`m—
`6-2
`
`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 CO2. No lipid was extracted using C02 alone.
`
`10
`
`79% of the feed material was extracted using ethanol and CO2 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.
`
`Table 12
`
`
`Comosition, wt%
`Other
`
`Phospholipid Other compounds
`s
`
`
`
`
`
`
`
`PI
`
`-
`
`PS
`1
`
`-
`
`PE
`
`SM
`3
`
`
`
`
`
`2 8
`23 4
`0.0
`
`
`15
`
`20
`
`Example 14: Fractionation of dairy lipid extract A with propan-Z-ol
`co-solvent
`
`In this example 39g 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 C02. 48% of the feed material was extracted
`
`as neutral lipids using C02 alone. 23% of the feed material was further extracted using the
`
`25
`
`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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`comparable concentration of ethanol. The levels of PS observed in the extracted fraction is
`
`also higher suggesting the propan-Z-ol is not as selective as ethanol. On this basis alone
`
`ethanol would be the preferred co-solvent.
`
`Table 13
`
`_ Com - osition, %
`
`PE
`
`Other
`SM Phos-1101i nids
`
`Other compounds
`
`3.4
`
`4.2
`
`10
`
`15
`
`20
`
`25
`
`Example 15: Fractionation of soy lecithin
`
`This example relates to fractionation of a soy lecithin (Healtheries Lecithin natural dietary
`
`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 002.
`
`No lipid was extracted using C02 alone. 91% of the feed material was extracted using
`
`ethanol and C02 with a total ethanol flow of 520g. Phospholipid profiles for the extract and
`
`residual fractions are shown in Table 14. PC and PE are preferentially extracted and are
`
`significantly enriched in the extract. There are no detectable levels of PS or SM in this
`
`example.
`
`Table 14
`
`
`
` Com osition, %
`Yield
`Other
`Other compounds
`% of feed
`PC
`PI
`PS
`PE
`SM Phosholi ids
`
`N
`_-_ 12-3 III-__-
`\DN
`
`Cl'l
`NU.)o»—- \1
`
`
`
`
`.
`18.4
`0.0
`0.0
`12.4
`35.2
`
`
`Example 16: Continuous fractionation of egg yolk lipids
`
`This example relates to fractionation of an egg yolk lipid extract containing 15%
`
`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 pumped into the top of
`
`a 10L pressure vessel, and contacted with C02 containing 8.7 % of 98% aqueous ethanol
`flowing upwards through the 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 raffmate phase
`
`22
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`was periodically withdrawn from the bottom of the vessel. The lipid feed rate was 1.5 kg/hr.
`The C02+ co—solvent flow rate was 27 kg/hr.
`
`The extract phase was predominantly neutral lipids but contained 20% of the 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 pumped into the top of the pressure vessel
`
`and contacted with C02 in upflow. The overall concentration of ethanol in C02 under steady
`
`state processing conditions was 5.9%. In this case 50% of the mass ofphospholipids in the
`
`feed were extracted. The composition of the extract phase consisted of between 60% and
`
`70% PC, with the balance mostly PB. 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 of the feed material was extracted using a
`batch stirred tank method at 250 bar and 60°C using C02 and ethanol (containing 5 % water)
`
`at a concentration of 30.5 %. The lipid was placed in the stirred tank, C02 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 C02 and ethanol after stirring for 1 hour by
`sampling the extract phase at constant pressure. P