(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
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`
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`(19) World Intellectual Property Organization
`International Bureau
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`|l||||l|||||l|llllllllllllllllll||||||||||I|||||ll||l||||||||l|l||||llllllllllll
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`(10) International Publication Number
`(43) International Publication Date
`WO 01/76385 A1
`18 October 2001 (18.10.2001)
`
`
`(51)
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`International Patent Classification7: A23J 1/08, C1 13
`1/00, 7/00, A23D 9/013, A23] 7/00
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`an
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`International Application Number:
`
`PCT/lBOl/00963
`
`[DE/DE]; Pappelweg 9, D-59302 Oelde (DE). WITT,
`Willi [DE/DE]; Kleeweg 34, D—49545 Tecklenburg (DE).
`BEST, Bernd [DE/DE]; Annastrasse 13, D-64546 Mar-
`felden (DE).
`
`an
`
`International Filing Date:
`
`12 April 2001 (12.04.2001)
`
`(74)
`
`Agents: SPECHT, Peter et a1.; Jollenbecker Strasse 164,
`D-33613 Bielefeld (DE).
`
`an
`
`Filing Language:
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`an
`
`Publication Language:
`
`English
`
`(81)
`
`English
`
`(30)
`
`Priority Data:
`100 18 213.5
`60/271,209
`
`12 April 2000 (12.04.2000)
`23 February 2001 (23.02.2001)
`
`DE
`US
`
`Designated States (national): AE, G, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ,
`DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR.
`HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR,
`LS, LT, LU, LV, MA. MD, MG, MK, MN, MW, MX, M2,
`NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI. SK, SL, TJ, TM,
`TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW.
`
`Designated States (regional): ARlPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
`patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
`patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE,
`IT, LU, MC, NL, PT, SE, TR), OAPI patent (BF, BJ, CF,
`CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).
`
`Applicant (for all designated States except US): WEST-
`FALIA SEPARATOR INDUSTRY GMBH [DE/DE];
`Wemer-Habig-Strasse 1, D—59302 Oelde (DE).
`
`(84)
`
`Inventors; and
`Inventors/Applicants (for US only): HRUSCHKA,
`Steffen, M. [DFJDE]; Aenne—Brauksiepe-Str. 7, D-59302
`Oelde (DE). KIRCHNER, Stefan [DE/DE]; Elsenkermstr.
`61, D-33334 Giitersloh (DE). RASSENHOVEL, Jiirgen
`
`Published:
`with international search report
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`(71)
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`(72)
`(75)
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`01/76385A1
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`[Continued on next page]
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`
`(54) Title: METHOD FOR THE FRACTIONA'I'ION OF OIL AND POLAR LIPID-CONTAINING NATIVE RAW MATERIALS
`USING ALCOHOL AND CENTRIFUGATION
`
`Solubility of phospholipids as a function of
`alcohol concentration
`
`
`
`PhospholipldSolubility
`
`
`
`(gl1009solutlon)
`
`
`
`0%
`
`I01
`
`20%
`
`30%
`
`(0%
`
`50%
`
`601
`
`70%
`
`301'.
`
`Alcohol Concentration % WIw
`
`O (57) Abstract: A process for the production of polar lipid-rich materials and preferably phospholipids. Preferably the polar lipid—rich
`materials are separated and recovered from native raw materials by extraction with water-soluble organic solvent and use of density
`W
`separation to separate the resulting mixture.
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`BNSDOCID: <WO
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`76385A1_|_>
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`WO 01/76385 A1 ||||||||||||I||||||||l||||lllll|llllllllll|||||l||||||l|l|l|l||||||l||||llllllll
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`— before the expiration of the time limit for amending the
`claims and to be republished in the event of receipt of
`amendments
`
`For two—letter codes and other abbreviations, refer to the "Guid—
`ance Notes on Codes andAbbreviations " appearing at the begin-
`ning ofeach regular issue of the PCT Gazette.
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`0176385A17|7>
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`WO 01/76385
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`PCT/I ROI/00963
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`METHOD FOR THE FRACTIONATION OF OIL AND POLAR
`LIPID-CONTAINWG NATIVE RAW MATERIALS USING
`ALCOHOL AND CENTRIFUGATION
`
`FIELD OF THE INVENTION
`
`The present invention relates to a process for the separation and recovery of polar
`lipid—rich fractions from mixtures such as native raw materials. Other fractions in. the raw
`materials can also be recovered.
`
`IO
`
`BACKGROUND OF THE INVENTION
`
`Examples of polar
`
`lipids
`
`include phospholipids
`
`(eg. phosphatidyl choline,
`
`phosphatidyl
`
`ethanolamine,
`
`phosphatidyl
`
`inositol,
`
`phosphatidyl
`
`serine,
`
`phosphatidylglycerol, diphosphatidylglycerols), ccphalins, sphingolipids (sphingomyelins
`
`15
`
`and glycosphingolipids), and glycoglycerolipids.
`Phospholipids are composed of the
`following major structural units: fatty acids, glycerol, phosphoric acid, amino alcohols,
`
`lipids, playing
`They are generally considered to be structural
`and carbohydrates.
`important roles in the structure of the membranes of plants, microbes and animals.
`
`Because of their chemical structure, polar lipids exhibit a bipolar nature, exhibiting
`solubility or partial solubility in both polar and non-polar solvents. The term polar lipid
`within the present description is not
`limited to natural polar lipids but also includes
`
`20
`
`chemically modified polar lipids. Although the term oil has various meanings, as used
`
`herein, it will refer to the triacylglycerol fraction.
`
`25
`
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`
`One ofthe important characteristics of polar lipids, and especially phospholipids,
`
`is that they commonly contain polyunsaturated fatty acids (PUFAs:
`
`fatty acids with 2 or
`
`more unsaturated bonds).
`
`In many plant, microbial and animal systems,
`
`they are
`
`especially enriched in the highly unsaturated fatty acids (HUFAs: fatty acids with 4 or
`
`more unsaturated bonds) of the omega—3 and omega-6 series. Although these highly
`
`unsaturated fatty acids are considered unstable in triacylglycerol
`
`form,
`
`they exhibit
`
`enhanced stability when incorporated in phospholipids.
`
`The primary sources of commercial PUFA-rich phospholipids are soybeans and
`
`canola seeds. These biomaterials do not contain any appreciable amounts of HUFAs
`
`unless they have been genetically modified.
`
`The pliOSpholipids (commonly called
`
`lecithins) are routinely recovered from these oilseeds as a by—product of the vegetable oil
`
`extraction process. For example, in the production of soybean or canola oil, the beans
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`(seeds) are first heat—treated and then crushed, ground, and/or flaked,
`
`followed by
`
`extraction with a non—polar solvent such as hexane. Hexane removes the triacylglycerol—
`
`rich fraction from the seeds together with a varying amount of polar lipids (lecithins).
`
`The extracted oil is then de-gummed (lecithin removal) either physically or chemically as
`
`a part of the normal oil refining process and the precipitated lecithins recovered. One
`
`disadvantage of this process is the use of the non-polar solvents such as hexane presents
`
`toxicity and flammability problems that must be dealt with.
`
`The crude lecithin extracted in the “de-gumming” process can contain up to about
`
`33% oil (triacylglycerols). One preferred method for separating this oil from the crude
`
`lecithin is by extraction with acetone. The oil (triacylglycerols) is soluble in acetone and
`
`the lecithin is not. The acetone solution is separated from the precipitate (lecithin) by
`
`centrifugation and the precipitate dried under first a fluidized bed drier and then a vacuum
`
`drying oven to recover the residual acetone as the product is dried. Drying temperatures
`
`of 50-70°C are commonly used. The resulting dried lecithins contain approximately 2-
`
`4% by weight of oil (triacylglycerols). Process temperatures above 70°C can lead to
`
`thermal decomposition of the phospholipids. However, even at temperatures below 70°C
`
`the presence of acetone leads to the formation of products that can impair the organoleptic
`
`quality of the phospholipids. These by—products can impart musty odors to the product
`
`and also a pungent aftertaste.
`
`To avoid use of non—polar solvents such as hexane and avoid the negative side
`
`effects of an acetone—based process, numerous processes have also been proposed
`
`involving the use of supercritical fluids, especially supercritical C02. For example, U.S.
`
`Patent No. 4,367,178 discloses the use of supercritical C02 to partially purify crude soy
`
`lecithin preparation by removing the oil from the preparation. German Patent Nos. DE-A
`
`30 11 185 and DE-A 32 29 041 disclose methods for de-oiling crude lecithin with
`
`supercritical C02 and ethane respectively. Other supercritical processes have been
`
`proposed which include adding small amounts of hydrocarbons such as propane to the
`
`supercritical C02 to act as entraining agents. However, supercritical fluid extraction
`
`systems are very capital expensive and cannot be operated continuously.
`
`Further,
`
`extraction times are long and the biomaterials must be dried before extraction, and this
`
`increases the difficulties of stabilizing the resulting dry product with antioxidants. All of
`
`these factors make the supercritical process one of the most expensive options for
`
`extracting and recovering polar-lipid material or mixtures of these materials. As a result,
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`alternative processes using extraction with liquid hydrocarbons at lower pressures have
`
`been described. For example U.S. Patent No. 2,548,434 describes a method for de-oiling
`
`oilseed materials and recovering crude lecithin using a liquid hydrocarbon at
`
`lower
`
`pressures (35-45 bars) but elevated temperatures (79° to 93°C). U.S. Patent No.
`
`5,597,602 describes a similar process that operates at even lower pressures and
`
`temperatures. However, even with these improvements supercritical fluid extraction
`
`remains very expensive and is not currently used to produce phospholipids for food use
`
`on a large commercial scale.
`
`The primary commercial source of HUFA-rich polar lipids is egg yolk. Two
`
`primary methods are used for the recovery of egg phospholipids on an industrial scale.
`Both require the drying of the egg yolk before extraction.
`In the first process the dried
`
`egg yolk powder is extracted first with acetone to remove the triacylglycerols. This is
`
`then followed by an extraction with pure alcohol to recover the phospholipids.
`
`In the
`
`second process, pure alcohol is used to extract an oil/lecithin fraction from the dried egg
`
`yolk. The oil/lecithin phase is then extracted with acetone to remove the triacylglycerols,
`
`leaving behind a lecithin fraction. Both of these methods require the use of acetone,
`
`which has the disadvantages discussed above.
`
`Canadian Patent No. 1,335,054 describes a process for extracting fresh liquid egg
`
`yolk into protein, oil and lecithin fractions by the use of ethanol, elevated temperatures,
`
`filtration and low temperature crystallization with further filtration.
`
`The purity of the
`
`lecithin product is not disclosed. However one skilled in the art would expect that the
`
`lecithin fraction produced by this process would not be very pure. There would still be
`
`very significant amounts of oil associated with the lecithin because the chilling process
`
`would primarily remove the triglycerides containing saturated fatty acids.
`
`Those
`
`containing some unsaturated fatty acids would remain more soluble at low temperatures.
`
`Additionally, the filtration and the chilling/filtration processes employed in this method
`
`would be labor intensive and difficult to turn into a continuous process.
`
`In light of the
`
`current state of the art, there remains a need for an improved extraction technology for
`
`food-grade polar lipid products that is less expensive to operate and which protects the
`
`overall quality of the HUFAs in the polar lipid products.
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`SUMMARY OF THE INVENTION
`
`In accordance with the present invention, an improved process is provided for
`
`recovering polar
`
`lipids
`
`from native biomaterials, which does not
`
`involve the
`
`disadvantages of the prior art. One embodiment of the invention resides in a process for
`
`recovering polar lipids and/or polar lipid-containing mixtures from biomaterials using
`
`both high and low water—soluble organic solvent concentrations and centrifugation.
`
`In accordance with an embodiment of the present
`
`invention, a process for
`
`fractionation of an oil—, polar lipid-, and protein-containing mixture is provided. The
`
`process includes the steps of adding a high concentration of water-soluble organic solvent
`
`10
`
`to the mixture, separating protein from the mixture by subjecting the mixture to density
`
`separation, e.g., using gravity or centrifugal force, to form a protein-rich fraction and a
`
`polar lipid/oil—rich fraction,
`
`reducing of the concentration of water-soluble organic
`
`solvent
`
`in the polar
`
`lipid/oil-rich fraction, and subjecting this fraction to density
`
`separation, e.g., using gravity or centrifugal force, to form a polar lipid—rich fraction and
`
`15
`
`an oil-rich fraction.
`
`In accordance with another embodiment of the present invention, a process for
`
`recovering polar lipid from a polar lipid-containing mixture employing the use of a water-
`
`soluble organic solvent, wherein the relatively high solubility of polar lipid in a water-
`
`soluble organic solvent, in which the water-soluble organic solvent comprises greater than
`
`68 percent by weight of the aqueous solution, followed by process steps which utilize
`
`water—soluble organic solvent concentrations of from about 5 to about 35% by weight, are
`
`employed to assist in the recovery.
`
`In accordance with another embodiment of the present invention, a process for
`
`fractionation of an oil-, polar lipid—, and protein-containing mixture is provided. The
`
`process includes the steps of adding a high concentration water-soluble organic solvent to
`
`the oil-, polar lipid—, and protein—containing mixture, and separating protein from the
`
`mixture to form a protein-rich fraction and a polar lipid/oil—rich fraction. As used herein,
`
`the term “high concentration water-soluble organic solvent” will mean greater than 68
`
`percent organic solvent, preferably greater than 80 percent organic solvent, more
`
`preferably greater than 90 percent, more preferably from about 80 percent to about 95
`
`20
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`25
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`percent organic solvent.
`
`Preferably,
`
`the process steps are conducted under oxygen-reduced atmospheres
`
`that can include use of inert or non—reactive gases (e.g. nitrogen, carbon dioxide, argon,
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`etc), use of solvent vapors, use of a partial or full vacuum, or any combination of the
`
`above.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`The present
`
`invention may be more readily understood by reference to the
`
`following figures, wherein
`
`FIG.
`
`1
`
`is a graphical representation of the solubility of phospholipids, a form of
`
`polar lipids, as a function of alcohol concentration.
`
`FIG. 2 is a graphical representation of a phospholipid extraction process (as an
`
`example of a polar lipid extraction process) based on a high concentration of alcohol.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`Because of their bipolar nature, polar lipids (including phospholipids) are of
`
`significant commercial interest as wetting and emulsifying agents. These properties may
`
`also help make HUFAs in the phospholipids more bioavailable, in addition to enhancing
`
`their stability. These properties make phospholipids ideal forms of ingredients for use in
`
`nutritional supplements, food, infant formula and pharmaceutical applications.
`
`We have unexpectedly found that polar lipids are soluble not only in high water-
`
`soluble
`
`organic
`
`solvent
`
`concentrations
`
`(e.g.,
`
`at water-soluble
`
`organic
`
`solvent
`
`concentrations greater than about 68% w/w) but also in low water-soluble organic solvent
`
`concentrations (less than about 35% water-soluble organic solvent w/w) (FIG. 1). As
`
`used herein, water-soluble organic solvent concentration means the weight percentage of
`
`water—soluble organic solvent in an aqueous solution. The aqueous solution includes
`
`added water and water present in the materials.
`
`For the purpose of this invention,
`
`phospholipids are described as “soluble” if they do not settle or separate from the
`
`continuous phase (sometimes also called supernatant or light phase) when subjected to
`
`centrifugation by equipment described in this invention.
`
`In the water-soluble organic
`
`solvent concentration range from about 35% w/w to about 68% w/w water-soluble
`
`organic solvent, polar lipids exhibit significantly lower solubility. The present invention
`
`exploits this property of polar lipids (enhanced solubility/dispersibility at low water-
`
`soluble organic solvent concentrations), which can then be exploited in several ways
`
`(along with the high solubility of phospholipids in high water-soluble organic solvent
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`concentrations) to develop processes for inexpensively extracting and recovering polar
`
`lipids, and especially phospholipids, from native biomaterials.
`
`Native biomaterials that are rich in HUFA-containing polar lipids include fish,
`
`crustaceans, microbes, eggs, brain tissue, milk, meat and plant material
`
`including
`
`oilseeds. As used herein, the terms fish, crustaceans, microbes, eggs, brain tissue, milk,
`
`meat and plant material
`
`including oilseeds will
`
`include genetically modified versions
`
`thereof. The content of phospholipids in these materials is generally low, usually ranging
`
`from 0.1% to about 4% by wet weight. As a result large amounts of raw materials need to
`
`be processed to recover these phospholipids. Because of the high costs of prior extraction
`
`techniques, phospholipids and especially HUFA-enriched phospholipids were very
`
`expensive and therefore restricted to use in the infant formula, pharmaceutical and
`
`cosmetic industries. One of the advantages of the present invention is that it provides for
`
`the extraction of polar lipids, and in particular phospholipids, in a cost-effective manner.
`
`A polar lipid recovery process utilizing high concentrations of water—soluble
`
`organic solvent in a polar lipid/oil concentration step followed by the use of low water-
`
`soluble organic solvent concentrations in a step recovering the polar lipids from the oil
`
`phase is outlined in FIG. 2. Liquid egg yolk is used as the polar—lipid rich biomaterial in
`
`this example.
`
`It is understood, however, that other polar lipid-containing biomaterials
`
`(e.g. fish, crustaceans, microbes, brain tissue, milk, meat and plant material including
`
`oilseeds) could also be processed in a similar manner with minor modification to the
`
`process.
`
`In the first step of the process 12, the material is dried, if necessary. For a more
`
`efficient recovery of the protein, the material is optionally subjected to size reduction 14.
`
`A water-soluble organic solvent (e.g., alcohol) is added 14. The concentration of water—
`
`soluble organic solvent
`
`in the water-soluble organic solvent/water solution is at least
`
`about 68% w/w, preferably at least about 80% w/w, preferably at least about 90% w/w,
`
`more preferably from about 80 to about 95% w/w, more preferably from about 85 to
`
`about 95% w/w, and more preferably from about 90 to about 95% w/w. The more
`
`moisture that is present
`
`in the material, the greater the amount and/or the higher the
`
`concentration of the water-soluble organic solvent that will be needed to achieve the
`
`desired concentration when mixed with the material.
`
`In other words, if the material is
`
`relatively dry,
`
`less water—soluble organic solvent and/or lower concentration water-
`
`soluble organic solvent can be employed. On the other hand, if the material is relatively
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`wet, more water—soluble organic solvent and/or higher concentration water-soluble
`
`organic solvent must be employed.
`
`The denatured protein 20 is then separated by density separation 18.
`
`Since
`
`proteins are not soluble in high concentrations of water-soluble organic solvent, they
`
`precipitate (while the polar lipids and oil dissolves in the high concentration water-soluble
`
`organic solvent) and the precipitated proteins 20 are separated from the polar lipid/oil-
`
`enriched fraction 22 by density separation 18, e.g., using gravity or centrifugal force.
`
`Using egg yolk as an example, this results in two fractions being recovered: (1) a fraction
`
`with approximately 60-95% oil (as % dry weight) and about 5-40% dry weight as polar
`
`lipids; and (2) a protein fraction, preferably with more than 90% of the protein of the egg
`
`yolk.
`
`If it is desired to separate the polar lipid from the oil, the oil/polar lipid fraction 22
`
`is mixed 26 with water 24 to a final concentration of water-soluble organic solvent in
`
`water of from about 5 to about 35% w/w, preferably from about 20 to about 35% w/w,
`
`. more preferably from about 25 to about 30% w/w. A less desirable alternative would be
`
`to dry the oil/polar lipid fraction 22 and then add a water-soluble organic solvent, and
`
`water as necessary, to achieve the desired concentration of water-soluble organic solvent.
`
`The polar lipid is then separated from the oil by means of density separation 28. A polar
`
`lipid-enriched fraction 30 and an oil—enriched fraction 32 are formed. Further processing
`
`can be performed on the polar lipid-enriched fraction 30 and/or oil—enriched fraction 32 as
`
`desired or necessary.
`
`For example, counter-current washing/centrifugation or cross-
`
`current washing/separation of the oil and polar lipid products can be employed to improve
`
`the purity of the products and economics of the overall process.
`
`In an alternative embodiment, the drying step can be eliminated. For example,
`
`instead of drying a material such as eggs, wet eggs can be used. The process is similar to
`
`that described above, however, the drying step is eliminated. As a result, a larger amount
`
`and/or higher concentration of water-soluble organic solvent is employed to precipitate
`
`the protein.
`
`Because of the simplicity of the equipment required in the process, the entire
`
`process can very easily be conducted under a reduced—oxygen atmosphere (e.g., nitrogen,
`
`a preferred embodiment of the process), further protecting any HUFAs in the polar lipids
`
`from oxidation. For example, a gas tight decanter can be used to separate protein from
`
`the mixture. A suitable decanter is model CA 226—28 Gas Tight available from Westfalia
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`Separator Industry GmbH of Oeldc Germany, which is capable of continuous separation
`
`of protein from suspensions with high protein solids content in a centrifugal field. A gas
`
`tight separator useful for separating polar lipids from oil is model SC 6-06—576 Gas Tight
`
`available from Westfalia Separator Industry GmbH of Oelde Germany, which is capable
`
`5
`
`of continuous separation of polar lipids from oil in a centrifugal field.
`
`The concentration of water-soluble organic solvent in the protein removal step is
`
`preferably greater than about 68% w/w, more preferably greater than about 70% w/w,
`
`more preferably greater than about 80% w/w, more preferably greater than about 90%
`
`w/w.
`
`In principle,
`
`it
`
`is believed that
`
`the higher the water-soluble organic solvent
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`concentration,
`
`the
`
`stronger
`
`the protein contraction, but
`
`the more nonpolar
`
`the
`
`aqueous/water-soluble organic solvent phase, more polar lipids may be dissolved in the
`
`oil phase. The appropriate concentration and temperature must therefore be found, for
`
`example, by conducting a few preliminary experiments (centrifuge tests), for each raw
`
`material.
`
`15
`
`The present invention,
`
`in various embodiments, includes components, methods,
`
`processes, systems and/or apparatus substantially as depicted and described herein,
`
`including various embodiments, subcombinations, and subsets thereof. Those of skill in
`
`the art will understand how to make and use the present invention after understanding the
`
`present disclosure.
`
`The present invention, in various embodiments, includes providing
`
`20
`
`devices and processes in the absence of items not depicted and/or described herein or in
`
`various embodiments hereof, including in the absence of such items as may have been
`
`used in previous devices or processes, e.g., for improving performance, achieving ease
`
`and/or reducing cost ofimplementation.
`
`The foregoing discussion of the invention has been presented for purposes of
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`25
`
`illustration and description. The foregoing is not intended to limit the invention to the
`
`form or forms disclosed herein. Although the description of the invention has included
`
`description of one or more embodiments and certain variations and modifications, other
`
`variations and modifications are within the scope of the invention, e. g., as may be within
`
`the skill and knowledge of those in the art, after understanding the present disclosure.
`
`It
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`30
`
`is
`
`intended to obtain rights which include alternative embodiments to the extent
`
`permitted,
`
`including alternate,
`
`interchangeable and/or equivalent structures, functions,
`
`ranges or steps to those claimed, whether or not such alternate, interchangeable and/or
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`BNSDOCID: <WOi
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`0176385A17l7>
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`

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`WO 01/76385
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`9
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`PCT/lBOl/00963
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`equivalent structures,
`
`functions,
`
`ranges or steps are disclosed herein, and without
`
`intending to publicly dedicate any patentable subject matter.
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`BNSDOCID: <WO
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`0176385A17L>
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`

`

`W0 01 /76385
`
`We claim:
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`10
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`PCT/l ROI/00963
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`1.
`
`A process for fractionation of an oil-, polar lipid-, and protein-containing
`
`mixture, comprising the steps:
`
`(a)
`
`adding a water-soluble organic solvent
`
`to said mixture and
`
`separating protein from said mixture to form a protein-rich fraction and a polar
`
`lipid/oil—rich fraction;
`
`(b)
`
`reducing the concentration of water—soluble organic solvent in said
`
`polar lipid/oil-rich fraction; and
`
`(c)
`
`subjecting the water/water-soluble organic solvent and polar
`
`lipid/oil-rich fraction to density separation to form a polar lipid-rich fraction and
`
`an oil—rich fraction.
`
`2.
`
`The process of Claim 1, wherein the separation of protein of step (a)
`
`comprises the steps:
`
`(3)
`
`adding water-soluble organic solvent to said oil-, polar lipid-, and
`
`protein-containing mixture to obtain a water-soluble organic solvent concentration
`
`ofa least about 68% w/w; and
`
`(b)
`
`separating by density separation the resulting mixture into a
`
`protein—rich fraction and a polar lipid/oil-rich fraction
`
`3.
`
`The process of Claim 1, wherein said oil-, polar lipid—, and protein-
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`10
`
`15
`
`20
`
`containing mixture is derived from eggs.
`
`4.
`
`The process of Claim 1, wherein water—soluble organic solvent
`
`is
`
`recovered from the protein-rich fraction and the polar lipid/oil—rich fraction after the
`
`density separation.
`
`25
`
`30
`
`5.
`
`The process of Claim 1, wherein said polar lipid/oil—rich fraction formed in
`
`step (a) comprises from about 5% to about 40% by weight polar lipid and from about
`
`60% to about 95% by weight oil.
`
`6.
`
`The process of Claim 1, wherein said protein-rich fraction formed in step
`
`(a) comprises from about 80% to about 95% by weight protein on a dry basis.
`
`7.
`
`The process of Claim 1, wherein said oil-, polar lipid-, and protein-
`
`containing mixture further comprises cholesterol and a substantial amount of said
`
`cholesterol reports to said oil-rich fraction pursuant to the separation of step (c).
`
`8.
`
`The process of Claim 1, wherein said water—soluble organic solvent in step
`
`(a) is present in a water—soluble organic solvent/water mixture in which said water-soluble
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`BNSDOCID: <WO
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`0176385A17L>
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`

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`WO 01/76385
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`1 l
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`PCT/lBOl/00963
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`organic solvent comprises from about 80% to about 95% by weight of said water-soluble
`
`organic solvent/water mixture.
`
`9.
`
`The process of Claim 1, wherein said water-soluble organic solvent in step
`
`(c) is present in a water-soluble organic solvent/water mixture in which said water—soluble
`
`organic solvent comprises from about 5% to about 35% by weight of said water-soluble
`
`organic solvent/water mixture.
`
`10.
`
`The process of Claim 1, wherein said water-soluble organic solvent is
`
`recovered by countercurrent washing, evaporation or drying.
`
`11.
`
`The process of Claim 1, wherein said polar lipid—rich fraction is dried to
`
`recover water-soluble
`
`organic
`
`solvent, washed with an water—soluble
`
`organic
`
`solvent/water mixture comprising greater than about 80% by weight water-soluble
`
`organic solvent in order to precipitate residual protein and further dried to recover the
`
`water-soluble organic solvent.
`
`12.
`
`The process of Claim 11, wherein the addition of said water-soluble
`
`organic solvent results in the precipitation of at least some of said protein, which is
`
`recovered by density separation.
`
`13.
`
`The process as claimed in Claims 1-12, wherein said water-soluble organic
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`10
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`15
`
`solvent comprises a polar solvent.
`
`14.
`
`The process as claimed in Claims 1-13, wherein said water-soluble organic
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`20
`
`solvent comprises an alcohol.
`
`15.
`
`The process as claimed in Claims l-l4, wherein said water—soluble organic
`
`solvent comprises a C1-C3 alcohol.
`
`16.
`
`The process as claimed in Claims 1-15, wherein said water-soluble organic
`
`solvent comprises iSOpropanol, ethanol or mixtures thereof.
`
`17.
`
`The process as claimed in Claims 1-16, wherein the pH during processing
`
`is from pH 4 to about pH 10.
`
`18.
`
`The process as claimed in Claims 1—17, wherein said mixture is obtained
`
`from at least one of eggs, fish, crustaceans, microbes, brain tissue, milk, meat and plant
`
`material including oilseeds.
`
`19.
`
`The process as claimed in Claims 1-18, wherein at least 60% of the polar
`
`lipids originally present in the mixture are recovered in a polar lipid-rich fraction.
`
`20.
`
`The process as Claimed in Claims 1-19, wherein at least 80% of the polar
`
`lipids originally present in the mixture are recovered in a polar lipid-rich fraction.
`
`25
`
`30
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`BNSDOCID: <WO
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`01 76385A1_l_>
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`

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`WO 01/76385
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`12
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`PCT/I BO] /00963
`
`21.
`
`A process for recovering polar lipid from a polar lipid-containing mixture
`
`employing the use of a water-soluble organic solvent, wherein the relatively high
`
`solubility of polar lipid in an aqueous solution of the water-soluble organic solvent, in
`
`which the water-soluble organic solvent comprises more than 68 percent by weight of the
`
`aqueous solution, followed by employing the use of a water—soluble organic solvent,
`
`wherein the relatively high solubility of polar lipid in an aqueous solution of the water—
`
`soluble organic solvent, in which the water-soluble organic solvent comprises less than 35
`
`percent by weight of the aqueous solution, are employed to assist in said recovery.
`
`22.
`
`The process as claimed in Claim 21, wherein said mixture is obtained from
`
`at
`
`least one of eggs,
`
`fish, crustaceans, microbes, brain tissue, milk, meat and plant
`
`material including oilseeds.
`
`23.
`
`The process of any of Claims 1-22, wherein said polar lipid comprises a
`
`phospholipid.
`
`24.
`
`The process of any of Claims 1-23, wherein at
`
`least a portion of said
`
`process is performed in an oxygen—reduced atmosphere.
`
`25.
`
`The process as claimed in Claims 1-24, wherein said mixture is obtained
`
`from at least one of eggs, fish, crustaceans, microbes, brain tissue, milk, meat and plant
`
`material including oilseeds.
`
`26.
`
`The process as claimed in Claim 1, wherein said steps of adding and
`
`10
`
`15
`
`20
`
`subjecting are repeated at least once.
`
`27.
`
`A process for fractionation of an undried oil—, polar lipid-, and protein-
`
`containing mixture, comprising the steps:
`
`(a)
`
`adding water-soluble organic solvent to said oil-, polar lipid-, and
`
`protein—containing mixture to obtain a water—soluble organic solvent concentration
`
`ofa least about 68% w/w; and
`
`(b)
`
`separating by density separation the resulting mixture into a
`
`protein-rich fraction and a polar lipid/oil-rich fraction.
`
`28.
`
`An oil—containing, polar lipid—containing or protein-containing product
`
`produced by any ofthe processes of Claims 1-27.
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`25
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`30
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`BNSDOCID. <WO
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`0 1 76385A17l7>
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`WO 01/76385
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`PCT/IBOl/00963
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`201630‘A40'A50'A60'lo70%BO'1.90'L
`10'.‘ Figure1.Solubilityofphosphoiipidsasafunctionofalcohol
`
`concentration
`
`N
`
`\n
`
`v—
`
`0.5-
`
`0%
`
`
`
`Alcoho!Concentration%w/w
`
`(uounios SOUL/5)
`
`Imuqnios pidnoudsoud
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`/
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`SUBSTITUTE SHEET (RULE 26)
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`WO 01/76385
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`PCT/IBOl/00963
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`
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`Dry Polar Lipid-and Oil-
`Containing Material
`10
`
`
`
`Size Reduction, e.g., Crush,
`Grind and/or Flake
`12
`
`
`
`
`
`Water-Soluble
`
`Organic Solvent
`
`14
`
`
`
`
`
`
`Density
`Polar Lipid
`Separation
`and Oil
`
`
`22
`18
`
`
`Protein
`20
`
`Mix
`26
`
`
`
` FIG. 2
`
`ensity
`Separation
`28
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`O176385A17L>
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`SUBSTITUTE SHEET (RULE 26)
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`HVTFHUNATTCNNAHJSEMURCIIIIEPCHWT
`
`tnterna.
`
`it Application No
`
`PCT/IB 01/00963
`
`CLASSIFICATION OF SUBJECT MATTER
`TPC 7
`A23J1/08
`CllBl/OO
`
`CllB7/00
`
`A23D9/013
`
`A23J7/00
`
`According to international Patent Classification (IPC) orto both national classification and IPC
`B. FIELDS SEARCHED
`Minimum documentation searched (classification system followed by classification symbols)
`IPC 7
`A23J CllB A23D
`
`Documentation searched