TW200808817A - Product and process - Google Patents

Abstract

The present application relates to processes for fractionating a feed material into soluble and insoluble components, by contacting a feed material and a solvent and subsequently separating the solvent containing the soluble components from the insoluble components, wherein the feed material comprises one or more of: at least 1% w/w phosphatidyl serine, at least 1% w/w sphingomyelin, at least 1% w/w cardiolipin, or at least 0.3% w/w gangliosides, and wherein the solvent comprises one or more C1-C3 monohydric alcohols and water (wherein the water content of the one or more alcohols is 0 to 40% v/v).

Description

200808817 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to a fractionation method. More particularly, the present invention relates to a method of fractionating a feed material comprising a linal (e.g., a fat-filled glutamate, a heart-filled or sphingolipid, a phospholipid), a nerveolipid, or a combination thereof. [Prior Art] Filling is the main component of all biofilms, and includes glycerol phosphate (for example, phospholipid choline (PC), phospholipid oxime ethanol (PE), phospholipid creatinine (PI), cardiolipin (CL) And phospholipids serine (PS) and sphingolipids (such as neurolemin (SM)). Ganolysin (GS) is a component of the cytoplasmic membrane that regulates cellular signaling events. These components are particularly important in immune, brain function and neurodegenerative disorders. Phospholipids and lyophiles are involved in cellular signal transduction events leading to, for example, cell growth, cell differentiation, cell proliferation, and progressive cell death (apoptosis). A reasonable amount of such ingredients can be found in milk, soy products, eggs, animal tissues, marine animals, plants, microorganisms and other sources. A well-known source is a bovine milk globule membrane (mfgm) known to contain a useful amount of sphingomyelin, brain glycol, ribose, linsenoic acid and other structures (Jensen 2002). Phospholipids and sphingolipids have been involved in many health benefits including brain health, sports nutrition, skin health, eczema treatment, anti-infection, wound healing, intestinal microflora improvement, anti-cancer activity, arthritis reduction, improvement 120457 .doc 200808817 Cardiovascular Health and Treatment of Metabolic Syndrome. The alcohol-water mixture has previously been used to separate or enrich phospholipid PC, sputum and PI and/or for neuroglycolipids. Wu & Wang (2004) describes the use of aqueous ethanol solutions and different temperature treatments to fractionate soy lecithin. The conditions are optimized to produce high PC and high PI fractions. The higher oil (neutral lipid) content was found to significantly increase the yield of the PC fraction. However, the yield, purity and recovery of the PI fraction are significantly reduced. The lecithin-ethanol mixture was stirred for 1 hour and then centrifuged to form a PC-rich fraction (supernatant) and a PI-rich fraction (precipitate). After removing the solvent, the PI fraction was treated with acetone to remove neutral lipids. The ethanol aqueous solution was used at a concentration of 84%, 89%, 95%, and 99% at a solvent to egg phospholipid ratio of 5:1. The lecithin-ethanol mixture was found to have an optimum moisture content of 24%, which was achieved by using a 5:1 95% ethanol-lecithin ratio. The yield, purity and recovery of the PC fraction increased with increasing temperature and maximum purity was obtained at 4 (TC). The optimum solvent-to-sample ratio was 6:1 (in terms of PC yield and recovery) or 10: 1 (for PI). The maximum purity of PC is achieved when fractionated twice, but the purity of the PI fraction decreases as the number of fractionation increases. The optimal fractionation requires 40% of the feed sample oil content. Higher temperature fractionation is used. Applying a lower temperature after fractionation increases the purity of the PC fraction, but the yield and recovery of the PC fraction is low. No PS-rich fraction, SM-rich fraction, CL-rich fraction or GS-rich Part of the method. The traditional method for obtaining high PS fractions is not to enrich the mixture containing PS, but to use phospholipase D, serine and PC or PC-containing phospholipid mixtures (US 5,700,668 A, KR2003086128 A and JP 2079990 A). The phospholipase D enzyme is used herein to convert PC to PS. 120457.doc 200808817 In JP 2002241385 A, a phospholipid mixture is dissolved in an alcohol, followed by a metal salt to precipitate PS, and JP 3047192 A and JP 2805522 B2 disclose Application of saturated hydrocarbons, alcohols and Mixing the aqueous solvent centrifugation partitioned chromatography separation of PE, PC and SM.

JP 8322472 A discloses a method for concentrating acidic lipids from plant (soybean) egg fat. Treating plant-derived phospholipids (suitably at least 40%) at a temperature above 30 ° C and preferably at 35-8 ° C with a lower alcohol containing less than or equal to 15% water and preferably less than or equal to 10% water Soy lecithin of phospholipids). The lower alcohol fraction is then removed. The ratio of phospholipid to lower alcohol by weight is preferably greater than 15. Further, it is preferred to repeat the treatment 3-5 times, wherein each treatment results in a ratio of phospholipid to lower alcohol of more than 1 by weight. The resulting acidic phospholipid concentrate preferably contains 60% or more of PI and phosphatidic acid (PA). A more detailed method is described in JP 95 15 1260 A and US Pat. No. 5,833,858 A, wherein the first step comprises treating the plant-derived lecithin with a solvent comprising 85% to 100% lower alcohol, followed by removing the solvent portion containing the lower alcohol to obtain A phospholipid mixture, and the second step comprises treating the phospholipid mixture with an aqueous solution containing 75% or less ethanol. The water-soluble component contained in the phospholipid mixture obtained in the first step is effectively removed in the second step. Therefore, a lipid mixture containing a high concentration of acidic phospholipids PI and PA can be relatively easily obtained from lecithin of plant origin. However, methods for concentrating PS, SM, CL or GS are not disclosed. JP 1026548 5 A describes the preparation of a highly concentrated mixture of PA and PI and a mixture of PE and PC from lecithin using ammonia-containing ethanol. Another more elaborate method is disclosed in US 5,2 14,171 A, which is used to control the dissolution of PC, PE and PA by the use of an alcohol solvent (by appropriately adjusting the acidity of the solvent) I20457.doc 200808817 , adjusting the pH to above 8 to dissolve PC and PE and adjusting to below 5 to dissolve PA) to carry out the extraction step to fractionate the phospholipid mixture into one or two or more of PC, PE, PI and PA or Part of more. The PI is substantially insoluble in the solvent used in the process and is thus primarily recovered in the extraction residue. Other parameters that affect the solubility of the fraction of the phospholipid mixture to be fractionated are the water content of the alcohol solvent, the temperature, and the choice of base and acid to adjust the pH. To further separate the PC and PE rich fractions, a divalent metal 'salt or trivalent metal salt solution is used. A method of concentrating PS, SM, CL or GS is not disclosed. To produce an SM-rich fraction, a mixture containing 5-15% SM is dissolved in an alcohol (optionally containing water) and the solution is concentrated, followed by extraction with an aliphatic hydrocarbon and precipitation with an aliphatic ketone (WO 2000/45828 A1) . A slightly similar method is disclosed in WO 94/18289 A1, in which a phospholipid is mixed with a lower aliphatic alcohol (methanol, ethanol, propanol, butanol) and a substantially non-polar solvent (hydrocarbon or chloroform) to form a non-polar The SM was precipitated by adding a ketone or decyl acetate or ethyl acetate. These methods have the disadvantage of using a variety of solvents to achieve the desired separation. In small-scale laboratory practice, the butanol-water-diisopropyl ether fractionation method is an effective way to prepare GS-rich fractions (Ladish & Gillard 1985). This method can be applied to total lipid extracts of protoplasts, cells or animal tissues. The dried total lipid extract was partitioned in a 3-component solvent system consisting of diisopropyl ether, 1-butanol and 50 M aqueous NaCl (6/4/5, body/volume/volume). The neuroglycolipid is nearly quantitatively soluble into the lower aqueous phase and the other lipids are solubilized to a mixture of the three solvents to produce the upper organic phase. The salt in the aqueous phase containing the nerve glycolipid and other low molecular weight impurities were then removed by gel filtration. God, two, and. Commercial scale manufacturing of sugar sorghum usually involves heating under alkaline conditions with nerve-containing sugars such as white milk or whey protein concentrate (eg

For example, US 5,831,079, US 5,795:980, JP 93035 155 B, JP 3615798 B2).庳 Phonetic, too, ^ Under this special condition, the dried glycolipid GD3 hydrolyzes and forms a nerve-glycolipid. Other common methods include ultrafiltration (eg jp 3 176698 B2, WO 91/074 1 π,

1/07417), column chromatography (eg DE422119G

Al, CA 2002155 a, tt 252310 B) or both (eg jp 2207090 A - CN 1379034 A) 产生 produces a composition having at least 4% nerolipid as follows: by adding ethanol to milk or dairy products To achieve a final ethanol concentration of 3g_7g vol% and recover / ▲ ' and then add ethanol to the sink to obtain an ethanol concentration of 7〇_9〇 volume%, Jiang Ha 1 A, private protein precipitation and membrane filtration of ethanol Part (US 5,844,104). The preparation of a composition rich in nerve glycolipids is also described in US 6,265,555. This patent claims a method for making an antioxidant containing a neuroglycolipid from a milk or milk-derived material (white whey, buttermilk, whey, and whey protein concentrate) which comprises the following steps: The glycolipid material is dispersed in the ethanol solution to make the ethanol concentration 95%; the dispersion is heated to a temperature of 50-thief to dissolve the nerve glycolipid and simultaneously produce a first precipitate containing the protein; Removing; removing the supernatant from the first precipitate to a temperature lower than 〇t to produce a second sink containing the nerve sugar wax; and recovering the second sink. In the present specification, reference is made to the patents of the patents, other external documents, or the like. It is generally used for the purpose of providing a description of the features of the present invention. Unless otherwise specified, no reference to the _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ General knowledge section. It is an object of the present invention to provide a method of producing a product containing a desired amount of a specific squamous cell and/or a neuroglycolipid or at least providing a sputum to the public.

Use the choice. ', 夯 [Summary of the Invention] Accordingly, the present invention provides a method for separating (four) soluble components and insoluble components from a feeding material, comprising: (4) providing a feeding material comprising: _ or more of the following: (! And at least 1% by weight of the phospholipid lysine; (η) at least 1% by weight/weight of sphingomyelin; (ηι) at least 1% by weight/weight of cardiolipin; or (iv) at least 〇·3% Weight/weight 祌 through the glycolipid; (b) ^ for the solvent containing <_ ρ η - ^ η and the genus ClrC3-sterol and water, wherein the water content of the one or more alcohols is 0 to 40% Volume/volume; (C) contacting the feed material with a solvent and then separating the solvent containing the soluble component from the insoluble component; (sequentially separating the soluble component and the solvent. The feed material preferably comprises less than 10% protein and Less than 10% lactose, more preferably less than 1%, of the feed material comprises protein and lactose. 'Second or the instrument material contains more than i% of the fat-filled tyrosine acid. Town material 120457.doc -12- 200808817 preferably contains more than 2% phospholipid lysine. The feed material preferably contains more than 5% phospholipid Alternatively, the feed material comprises greater than 1% sphingomyelin. The feed material preferably comprises greater than 5% sphingomyelin. The feed material preferably comprises greater than 15% sphingolipid. The feed material comprises greater than 1% cardiolipin. The feed material preferably comprises greater than 2% cardiolipin. The feed material preferably comprises greater than 5% cardiolipin. Alternatively, the feed material comprises 祌·3% 祌The glycolipid. The feed material preferably comprises more than 1% of the nerve-glycolipid. The feed material preferably comprises more than 2% of the nerve-glycolipid. The feed material of the present invention can be derived from terrestrial animals, marine animals, land. Plants, marine plants or microorganisms (such as microalgae, yeast and bacteria). Feeding materials are preferably derived from sheep, goats, pigs, mice, buffalo, camels, yaks, horses, donkeys, llamas, cows or humans. The incoming material is selected from the group consisting of: tissue, tissue part, organ, organ file, milk, milk part, colostrum, colostrum part, blood and blood part. The feed material is preferably derived from dairy material, soybean material, egg. Animal organization Animal organ or animal blood. The feed material is more preferably selected from the group consisting of a composition comprising nipple mussels, a composition comprising egg lipids, and a composition comprising marine biological lipids. The feed material for use in the method of the invention. The best part is the milk. The feeding material is selected from: white milk, white milk, beta whey, beta whey, white whey, white whey, whey, whey. Part, colostrum and colostrum 120457.doc -13· 200808817. The feed material may comprise a milk fat globule. The feed material may have been genetically modified. When it is a solid, it is preferably a solid form in contact with the solvent. Previously, the material can be milled at a low temperature in the rain of the contact agent, preferably at a low temperature for the average particle size of the material to be achieved. The solvent of the present invention preferably comprises: ethanol, n-propanol, iso (alcohol selected from the group consisting of: methanol and mixtures thereof; and (b) 0-40% by volume/volume of water.

More preferably, the solvent contains between 1 and 10% by volume of water between _2 G% of the body volume. The y most L alcohol is preferably ethanol. The solvent of ::! = = = preferably comprises a 95% aqueous solution of ethanol. The ratio of the two-part tt agent to the feed material is preferably between the four (4) and the better. : between 1 and 60. Loose agent and feed material main 60 The party of the invention Du Fu than the sheep is between 25··1 to 40··. ‘Li Yugong is at 3〇. His Majesty is better than 5 〇 °c into the wide Qiao Ming method j line. It is. Preferably, the method of the present invention is carried out at a higher than 55 tn. The method of the present invention can be carried out in a plurality of steps of the method of the present invention. The soil is less than or equal to 4. Preferably, the method is performed at less than or equal to (TC. Ordering. 120456.doc of the present invention, 14-200808817 Steps of Jiajin _ between 1 training. Step (10) using =::: method two materials (four) η The material can be disk-batch I, the feed material can be contacted multiple times; or the feed material is contacted with the solvent above the batch. It is better to use the solvent to flow through the material contact. The stomach material (4) (4) the agent and feed 3, the source is mixed with the solvent and the feed &#.» cut mixing for stirring. ^ and the feed material is better hunted by high shear also provides the product produced by the method of the invention, which is in the solvent The insoluble component (also referred to herein as "residue)) and the soluble core dissolved in the solvent after contact with the shelling material are "extracts"). When the material is fed, the article is also referred to as the batch > When the granule is contacted or in a plurality of quenching solvents, a plurality of "extract" products will be present. The product preferably contains more sphingolipid than the feed material. The product preferably contains more than 3% of sphingomyelin. More preferably contains more than 10 sense sphingomyelin. The product preferably contains more than 15% of the gods Preferably, the sheath phosphorus product contains more phospholipids than the feed material. The product preferably comprises greater than 5% phospholipids. The product preferably comprises greater than 30% gamma-serine: the best product Contains more than Congzhisheng lipid lanolinic acid. The product preferably contains more neuroglycolipid than the feed material. The product preferably contains more than 2% of neuroglycolipids. The product preferably contains large 220457.doc 200808817 at 4 % of neuroglycolipids. The product preferably comprises more than 6% of neuroglycolipids. The product preferably contains more heart fat than the feed material. The product preferably contains more than 5% cardiolipin. The product preferably contains greater than 10% of the heart wax. The product preferably contains more than 25% of the heart fat. Abbreviations and acronyms in this manual, the following is the meaning of abbreviations or acronyms used. nPL" means phospholipids; nCL&quot It means cardiolipin (diphospholipid glycerol); "PC" means phospholipid choline; ΠΡΙ" means phospholipid 醯 inositol; ΠΡΑΠ means phosphatidic acid; "PS means phospholipid 醯 lysine; ΠΡΕ" means phospholipids; amines; nSM" means nerves Sphingomyelin; ''DHSM' means dihydrosphingosine; phospholipid; nGSn means neuroglycolipid; "MFGM" means creamy globule membrane; "N/D" means not detected. [Embodiment] The present invention is based on the confirmation that the solubility of rouge in a short-chain monohydric alcohol solvent is different. Using this solubility difference, the inventors have discovered that insoluble phospholipids (including PS and CL) can be fractionated from soluble phospholipids (including SM) and 120457.doc -16 - 200808817 GS. Many of the variables in this method can be manipulated as follows to optimize the fractionation: (1) The type of material being fed. Various feed materials can be used in the present invention.

For example, the feed material can be derived from sheep, goats, pigs, mice, K cattle road I yak, horses, donkeys, American riding, cattle or human tissues, milk first gift or blood. Alternatively, the feed material may be derived from soy or other plant material, egg, animal tissue or organ, marine animal, marine plant or microbe. The feed material can have been genetically modified. (2) The particle size of the feed material (if a solid feed stream is used). The small particle size (iv) increases the total surface area to dissolve the soluble fat. (3) Solvent type. When the scorpion scorpion, ethanol, n-propanol and isopropanol and mixtures thereof have up to 4% by volume/volume of water, these alcohols are suitable solvents. However, a 95% by volume/volume aqueous solution of ethanol is a preferred solvent due to the lowest solubility of PS, CL and GS and the highest solubility of SM. The inventors have found that (4) the semen and/or nerve knot hearing lipids of the single-dissolved (four) unified branch can simplify the subtlety. Avoid the use of multiple _ systems and / or acid and test capabilities to simplify the removal of the solution from the residue and extracts and the subsequent use of recovered ethanol. In addition, the use of a mono-solution (such as ethanol) means that the solvent is not used in combination with a solvent or a solvent such as acetone, and does not produce a solvent-lipid reaction 120457.doc -17-200808817. This condition means maintaining the natural state of the residue and the extract product. (4) The ratio of solvent to feed stream. This ratio affects three main aspects: purity, pS, CL, and/or GS yield and throughput: Purity: The higher the solvent ratio, the higher the purity of the ps, cla/ or GS obtained in the residue.

• Yield: The higher the solvent ratio, the lower the yield of PS, CL and/or GS. All of these compounds have very low but limited solubility in the solvent, and thus an increase in the ratio increases the loss of the extract. • The higher the production volume and solvent ratio, the lower the production volume. Those skilled in the art will appreciate that there is a competitive constraint in obtaining high purity products, but there are also ways to maximize yield and throughput. (5) Extraction temperature. The higher the 2 degree, the higher the purity of the PS, coffee, or still in the residue. The back temperature can increase the solubility of some of the phospholipids in the dissolved (four) order. ^ = 2 and: or the solubility still increases with temperature, decreasing the yield of (5). Temperature, the degree is actually limited by the solvent stagnation point]. As above, a method of achieving high yield in obtaining a high-purity product. There is a bunch of 'but' but also fine (6) extraction time. The time allowed for the stroke to proceed. Obviously, the process costs: less. However, to be long enough, the batch extraction that can be carried out is to dissolve the soluble scales in suspension. In this case, the extraction of the feed stream introduced by the pull form is extremely important. 120457.doc 200808817 Ibid. In the process of obtaining high-purity products, there is a method of increasing the production volume. There are ... bundles, but there is also the most (7) agitation. The poly is sufficient to keep the particles suspended and to facilitate extraction by breaking any formation. Those skilled in the art will understand that it is most beneficial to have a mix of men and women under the squat. However, although the disturbance is generally _ version will be collectively broken, but the two shear mixing can also

Shihari's granules aggregate to form aggregates. When suboptimal agitation is used as the kneading speed, the impeller type and the size factor begin to function, it is a real risk of proportional increase. When the feed material of the Tianbei mussels in March is solid, it may also be separated by a packed bed under the unstirred debt. The solvent can be passed through a packed bed of grounded feed material at a low temperature to extract the highest solubility (and lowest solubility) and then rise to the south temperature (staged or continuous) until the maximum is reached in the residue ten. The desired final temperature for purity PS, CL and/or GS. This method uses /JHL degrees instead of the ratio of solvent to feed stream. The PC was selectively dissolved at a low temperature (10-2 QC). The subsequent temperature step can then be followed to remove the more soluble phospholipids (pE, SM & ρι) until a final step between 5 〇 and 60 C. The resulting packed bed is rich in turmeric and/or ^TM and neurolipids and is a powdered sink. (8) The ratio of water to solvent at a low water content (<6%), relative to other phospholipids and/or a good recovery of purity South 'PS and/or CL and the resulting Ps, CL and/ Or 120457.doc -19- 200808817 The part of GS that is insoluble in solvents is a powder that is easy to handle. SM is also highly soluble in solvents at low water contents. As the water content increases, PS becomes more soluble in the solvent, while the solubility of PC and SM decreases, resulting in poor separation and lower yield. (9) pH Although the acidic condition makes the PS slightly enriched, the pH does not affect the fractionation in the range of 2-9. However, the use of acid complicates the process and results in extravagant costs. As will be appreciated by those skilled in the art, the above variables can be manipulated as necessary to optimize the composition of the soluble product (e.g., SM content). The term "source" as used herein means originating from. The term "comprising" as used in this specification means ''consisting at least in part." In the statements of ", there may be features other than those beginning with the term. Terms such as "include" will be interpreted in the same manner. Unless otherwise stated, the percentages are based on weight/weight solids. The present invention consists of the above and is also contemplated to be constructed by the following examples only. The method of the invention disclosed in PCT/NZ2004/000014 (published as WO W02004/066744) A PCT/NZ2005/000262 (published in WO 04/04 1316). These feed streams are obtained from the MFGM-rich portion of the bovine white de-milk, which have different phospholipids and are composed of glycosidic fat. Raw material stream A: high lipid content (modified Rose) - Modified Roese-Gottlieb (up to 94% lipid), approximately 32% sarcophagus and 2.3% nerolipid. Extracted with near-critical dimethyl ether with > The milk is rich in MFGM to produce this feed stream. Feed stream B: contains 65% by weight of high-concentration phospholipids. Extraction of bovine white milk with low lactose content by supercritical C〇2 prior to near-critical dimethyl ether extraction Rich in MFGM Min (to remove neutral lipids) to generate this feed stream.

Feed stream C: Feed stream C has a very high lipid content and a higher proportion of scale than feed stream A, but is almost free of gangliolipids. This feed stream is produced by extracting the MFGM-rich portion of the bovine white milk which has a low lactose content by near-critical dimethyl ether. The composition of the phospholipids and neuroglycosmic lipids of the three feed streams are listed in Table 1 below. Table 1 · Filling of raw material stream A - C and nerve knots. Composition of raw materials ABC % PL 31.0% 65.6% 41.7% Pk% PC 23.7% 23.4% 26.9% PI 8.2% 8.1% 6.8% PS 12.7% 12.2% 10.4 % PE 33.1% 32.9% 31.8% SM 18.3% 18.8% 18.8% DHSM 3.7% 4.2% % PS 3.9% 8.0% 4.3% % SM 6.8% 15.1% 7.8% GS 2.3% 3.6% 0.6% Above 50t: Temperature Each feed stream was introduced into an excess of solvent (95% ethanol) and stirred for a predetermined period of time. In this process, 120457.doc -21 - 200808817 fat-soluble in ethanol is dissolved in ethanol to produce an extract stream, leaving no material (four) residue "), the material results in an enriched content Ps&GS. The details of the extraction are provided in the following Examples 1 a-1 d. The choice to introduce this specific feed stream into the solvent. Feed stream B is a solid gel at all actual processing temperatures, so in B Before the extraction, it requires low temperature milling. Therefore, for this method, the raw material stream A and the raw material stream c are the easiest to handle. The raw material streams are liquid under the alkaloid and thus can be pumped. The bottom layer is also solid and can therefore be fed via a pulverizer. Since the 甩& alcohol is in any form at the high temperature (liquid or cold phantom such material stream is uniformly dissolved, it provides two

The feed stream B needs to be milled into a fine powder (which requires low temperature milling) before it is introduced into the solvent. This additional processing step adds cost and makes the feed stream B significantly more expensive to process than the feed stream A or feed stream C. There is also a risk of insufficient milling of the raw stream B, which would jeopardize the process and result in lower purity. However, this problem can be overcome by first dispersing the feed stream in a small amount of solvent. Due to the high to medium lipid content, feed stream A and feed stream c are liquid at elevated temperatures, and due to the low neutral lipid content, the feed stream is solid at all practical temperatures. Example la 1· 100 mL of 95% ethanol was placed at 6 Torr. In the water bath, 1 〇 () m] L Schott bottle. 2. Stir the ethanol from the overhead mixing state (janke & Kunkel IKA-WERK RW20 overhead stirrer) until the temperature reaches 6 〇〇c. 120457.doc -22- 200808817 3 • Weigh approximately 2.5 gram of feed stream A into a 1 〇 ml syringe. 4 • The receiver heats the syringe to c to liquefy the feed stream. 5. The liquefied feed stream was then added to the ethanol over a period of 5 minutes by depressurizing the syringe. 6. The feed stream/ethanol mixture was then stirred at 6 Torr for 2 hours. 7. The receiver passes the solution through Whatman 4 filter paper and collects 235 g of insoluble material (residual material). The ethanol mixture passing through the filter paper contained a residual 2.265 gram of phosphorous gambling material (extract). 8. Analysis of the phospholipid content of the residue and extract fraction by P-NMR. As shown in Table 2, the residue was found to be highly enriched in ps (unmeasured (3 § content is also found in the extract composition as shown in Table 2). Example lb 1 · 100 mL 95% Ethanol was placed in a 1 〇〇 mL Schott bottle in a 6 (rc bath heated jacket) 2. Stirred ethanol by a magnetic stirrer (Janke & Kunkel IKA Lab〇rtechnik r (: i^ book magnetic stirrer) Until the temperature reaches 6 ° C. 3 · Add 2 · 1744 g of raw material stream a to the 1 〇mi syringe. 4. Then heat the syringe to 6 〇〇q to liquefy the feed stream. 5 · Then by pressing The syringe was used to add the liquefied feed stream to the ethanol over a period of 5 minutes. 6. The mixture of the feed stream/ethanol was then stirred under 6 Torr for 2 hours. 7. The solution was then passed through a trade "cracking 4 filter paper and collecting 〇i45 g. The insoluble material π (the object) passed through the ethanol mixture of the filter paper containing the residual 2. 〇 294 g of the disc grease material (extract). 120457.doc -23 - 200808817 8. The phospholipid content of the residue fraction was analyzed by 31P-NMR. As shown in Table 2, the residue was found to be rich in p S (unmeasured g § content). Example lc A method for treating a solid lipid-rich substrate is shown. 1. Low temperature milling 24.6 grams of raw material stream B. 2. Place the milled material in a 1 l Schott bottle and at a temperature of < 4 ° C 2 (10) mL·95% ethanol was poured into the bottle. 3. Place the bottle in a temperature-controlled water bath set at 65. Stir as in the case.

Next, 800 mL of 95% ethanol was added to the mixture in the bottle at a temperature of 65 ° C to obtain a 1:50 raw material to solvent ratio. The feed stream/ethanol mixture was continuously stirred and the temperature was maintained at 65. After 3 hours, the ethanol mixture was poured through a trade to add 4 filter paper and 6.8 grams of insoluble material (residue) was collected. The ethanol mixture (extract) passing through the filter paper contained 17.3 grams of residual phospholipid material. The phospholipid content of the residue and the extract fraction was analyzed by 31 P-NMR. The residue was found to be highly enriched as shown in Table 2 and GS. The extract composition was found to have an enriched content as shown in Table 2. Example Id This example shows that when the feed stream contains low levels of lactose, The ps is substantially enriched. 1. Place 100 grams of 95% ethanol in a 100 mL beaker on a hot plate with a magnetic stirrer and stir as in Example lb while raising the temperature to 65. 120457.doc -24- 200808817 2. 2.0 g of feed stream C was added to the ethanol and the mixture was continuously stirred for 2 hours under starvation. The mixture was then poured through Whatman 4 filter paper and 〇12邑 insoluble material (residue) was collected. The ethanol mixture (extract) passing through the filter paper contained 1.87 grams of residual phospholipid material. 4. The phospholipid content of the residue and the extract fraction was analyzed by 31P-NMR. As shown in Table 2, the residue was found to be highly enriched in ps. The extract composition was also found to have a high content of SM as shown in Table 2. Table 2 shows the results obtained by this method. Table 2 - Comparison of the results obtained by the method of Example 1.

3

laA b Miscellaneous %|2.2 6 7 .8-.7.06 0 2.1852221.0.

Jrl31.04.5324222:25.1 5

A

1CB residue [1.916.1117.53] Extract % 3 9 Residue or 5 % 6 27. .71 6.1.5.57 9 3.1844272.0.

Idc Ϊ172 63 .63 6.3.2 295.2.3422 6%9.3 8 8 0.7. 5 2· ο ο ο 6.0.0. 50 9441 29 6.132220. %n.64 2.8.80

%PS % SM+DHSM 32.7 1.0 0.8 10.2 38.9 1.0 23.0 1.9 1.7 17.4 73.7 0.0 0.5 9.3

GS 6.0 2.7 Example le-Cryogenic Extraction This example demonstrates the low enrichment temperature resulting in a low processing temperature of >8. 1. Low temperature obstructs 0.7857 grams of feed stream B and is placed in a 50 mL centrifuge tube. 2. Then add 47·5 mL of 95% ethanol to the tube 120457.doc -25- 200808817 at 37T: then add the tube to the mixing in a mechanical shaker water bath set at 37t: In the rack. 3. The stirring was then carried out at 3 rc for 2 hours and then the mixture was passed through Whatman 4 filter paper. 0·28 g of insoluble material (residue) was collected and found to have a p s content of 19% by weight (31 Ρ-NMR analysis). Example 2: Multi-step mixing tank for dairy feed stream Ethanol extraction of ginseng examples shows that a multi-step stirred tank extraction method using a solvent to raw material ratio lower than the solvent raw material ratio used in Example i can also achieve a significant set of ps&Gs . The example feed stream B is also used in this example. Feed stream 3 was cryogenically milled to a fine powder prior to introduction into the solvent as in Example 。. Do this as follows:

500 grams of the cryogenically milled feed stream B was placed in a 5 l vessel and 5 L of 95% ethanol was added at 65 °C. The vessel was periodically shaken and the temperature was maintained at 65 ° C by placing the vessel in a hot water bath. After J, the V stops and causes the insoluble material to sink to the bottom of the container. 4. Decanse the solvent phase and retain. 5. Recycle the solid material from this first extraction, cold and low temperature. 6. Will. The remilled material was placed in a 5 L vessel and 5 l, 5% ethanol was added at 6 cavities. The vessel was then periodically shaken and the temperature was maintained at 65 by placing the vessel in a hot water bath. I20457.doc -26- 200808817 7·1 small day shoud' The solution was filtered through Whatman 4 filter paper and the powder residue was collected. The solvent is recovered and combined with the solvent from the first extraction. 8. Place the powder residue in a 5 L vessel and add 5 L 95% ethanol at 65. The capacity was periodically shocked and the temperature was maintained at 65 ° C by placing the vessel in a hot water bath. 9. After 2 hours and 30 minutes, the mixture was filtered through Whatman 4 crepe paper. The solid recovered from the cold drying 'received 125.3 g of powder ("residue)). The filtrate was collected and evaporated ("extract 4"). 1 〇· The combined solvent phase from step 7 was maintained at 2 〇 The phospholipids were further precipitated during the 4 days. The solution was then filtered through Whatman 4 filter paper. The powder residue was collected and lyophilized (78 8 g powder, extract 1 ′). The filtrate was collected and at <0 in a freezer. Storage under the arm for 24 hours resulted in further precipitation, after which it was passed on Whatman 4 filter paper. The filtrate was then collected, evaporated and lyophilized to yield 11 〇 6 gram of powder ("extract 2"). The solid was filtered by lyophilization to yield 85. g g powder ("Extract 3"). 11. Analysis of the phospholipid content of the residue and extracts 1-4 from 31 p-nmr. The residue was found to be highly rich in PS and GS. The composition is shown in Table 3. 'Table 3 shows the results obtained by this method. 120457.doc -27- 200808817 Table 3 · Residues and extracts from multi-step extraction Partial composition B _ Output residue extract 1 Extract 2 Extract 3 Extract 4 Yield (solid, 25% 16% 22% 17% 6% % PL 52 47 70 73 L 63 PL PC 1 11 53 24 to % PI 23 10 0 1 1 ία PS 46 6 0 1 I \J PE 28 36 28 31 D SM 0 37 18 43 JO 17 %PS 24 3 0 1 % SM 0 17 12 31 L 11 GS 6.0 4.2 0.0 0.4 Example 3: Packed bed ethanol extraction of solid feed stream This example shows the separation of phospholipids using a packed bed method without agitation Solid mixture. Use dairy feed stream B, but the general method can be applied to soy-printed phospholipids and phospholipid concentrates from animal organs and tissues. The raw material stream is cryogenically milled into a fine powder. This method uses temperature instead of solvent and feed stream. It is developed to overcome the use of feed stream B (or other feed stream containing very low levels of neutral lipids) in processes such as those described in the Examples and Example 2. Observed the "glue, and aggregation problems. It is believed that the "glue" system is caused by the phospholipid phosphine test (pc) which is not fully resolved and forms a gel. This filler is too ° 一 一 一 一 耩 耩 耩 耩 耩 耩 耩 使用 使用 使用 使用〇-20.〇 Alleviate this problem. In the degree II, the PC selectively dissolves and does not form a coagulation due to the absence of dissolved mm' removed from the packed bed. J20457.doc -28- 200808817 The temperature rising step is the final step between (PE^SM^PI)i^L. The resulting packed bed is rich and is in the form of 6f>〇r.,, =, and sucrose as a powdery precipitate. c above the temperature mom ~

This method is also advantageous in that Tps, delivery is soluble and can experience significant losses. Another advantage of donating this technique is the need to reduce the need for shear mixing. Although the mixture is generally broken, the aggregates are broken.

The particles aggregate to form aggregates. When the 佬尔Αί里 TV is given to m and ~ the field is used for optimal mixing, this situation is - ask. For example, when the factors such as the straightness and the degree of the storage tank, the mixing speed, the type of the enthalpy, and the size of the sump are used to open the privilege, the risk is proportional to the increase. , the possibility of slow fluid dynamics to form aggregates of raw materials. In addition, this method uses non-mixed (layered) flats in contact with ethanol, so there is minimal collision and smaller. The process was carried out in the following manner: 1. A known amount of milled feed stream B (5 1 〇 62 g) was dispersed evenly inside the jacketed sintered vessel. 2. Connect the sintered jacket to the overflow water bath with pump. 3. Set the water bath to l〇 °C. 4. Pour 100 mL of 95% ethanol (2 times the volume of the feed stream B, for example 1 g of the parent feed B to 20 mL of ethanol) into a container and place in a water bath. 5. After the ethanol and the bed reached i〇°c, the ethanol was poured into the top of the sinter and passed through the bed into the flask. 6. Recycle ethanol and continuously pour through the bed for 24 minutes. Put the bed in the raft. 〇 120457.doc -29- 200808817 The soluble material is soluble in ethanol. 7. The ethanol and the first phospholipid extract were then collected and the ethanol was evaporated to give 磷脂6133 g of the phospholipid mixture described in Table 4 as a decanted extract. 8. Increase the temperature of the water bath to T2 (20 ° C). 9·Summer 1〇〇 mL fresh 95% ethanol and placed in a water bath. 1〇 After the ethanol and the bed reached D2 (2〇\:), the acetamidine was poured into the top of the sinter and passed through the bed into the flask. _ π· Recovery of ethanol and continuous pouring through the bed for 28 minutes. Put the bed in the plus. ^ The soluble material is soluble in ethanol. 12. Next, ethanol and a second phospholipid extract were collected to obtain 1 〇 21 g of a phospholipid mixture described in Table 4 as an extract at 20 °C. 13 · Increase the temperature of the water bath to 3 ° ° C. 14. Measure 1 mL of fresh 95% ethanol and place in a water bath. 15. After the ethanol and the bed reached 30, the ethanol was poured into the top of the sinter and passed through the bed into the flask. Φ 16· Recovery of ethanol and continuous pouring through the bed for 30 minutes. In the bed, 3 (Γ(: soluble material) is dissolved in ethanol. 17. Then ethanol and the third phospholipid extract are collected to obtain a phospholipid mixture of 30 ° C extract described in Table 4 of 〇·97〇g. 18. Increase the temperature of the water bath to 50 ° C. 〇 19 · Measure 1 〇〇 mL of fresh 95% ethanol and place in a water bath. 20. After the ethanol and the bed reach 50t:, pour the ethanol into the sinter And flow through the bed into the flask. 21 • Recover ethanol and pour through the bed for 5 3 minutes. Dissolve the soluble material in the bed under 120 .. 120457.doc -30- 200808817. Ethanol and the fourth phospholipid extract were obtained as a mixture of phospholipids described in Table 4 as an extract of 50 ° C. 23. The temperature of the water bath was raised to 6 ° C. 24. One mL of fresh 95% ethanol was measured and placed. In a water bath. 2 5 · After ethanol and bed (4) c, pour ethanol into the top of the sinter and flow through the bed into the flask. 26. Recover ethanol and pour through the bed for 22 minutes. 〇. The soluble material in the underarm is dissolved in ethanol. 27. The ethanol and the fifth phospholipid extract are then collected to obtain 〇117 The fat-filled mixture of 6 0 C % is described in Table 4. 28. The residue of the collection bed was 1 277 g, which was found to contain a high content of 1 >8 and a neuroglycolipid. It is described as residual in Table 4. Table 4 shows the results obtained by this method. Table 4: Results obtained by the method of Example 3

Feed stream yield (solid) % of pores _%PL

PC PI PS PE SM 1>HSM 4.4.8.520 1· 2 8 3 L o· 12 4 2 0 .818.5.53 441.0.35142. 2-751-7-8 390.0.26265. .95 5.2.31 f2.28308 .

% PS % SM

5 .2

27 50°C 60°C Extract Extract 10.5% 2.3% 73.0 64.4 12.3 4.6 11.1 24.1 4.7 17.1 48.2 43.4 17.7 8.1 4.5 1.9 3.5 11.0 16.2 6.4 120457.doc -31 - 200808817 The extracts from each step can be combined or kept Independent phospholipid fraction. For example, 2CTC and machine extract are at least 25% (10) product, while other extracts can be used as a PC/PE phospholipid mixture. Discussion of Examples 1-3 The method used in the example [3] is the laboratory (4). It is estimated that these methods can be used to designate the materials in the poem 4, and the inventors consider the following factors. Feed Stream (Feed Material) Two different feed streams were used in Example 1. And the feed stream C proves to be the easiest to use because it is liquid at temperatures above 4 Gt and is therefore easily placed in a solvent. 〇: On the one hand, the raw material is at 0. . The above temperature is a gelatinous solid. The following is hard and can be used to reduce the feed stream to a fine operation using a low temperature barrier to allow sufficient dispersion in the extraction solvent. The low temperature impediments require the feed stream B to be the three raw material stream streams tested' although this problem can be overcome by first dissolving the feed stream in less t solvent as described. :: === Currently believes that "glue, is caused by the incompletely dissolved (four)^ stone choline (pc). The stream read between the feed streams c, because the feed stream A contains a much higher: dinuclear fat ratio (meaning residue (using 3 〇:1 solvent and raw material flow θ 3 with a reasonable amount of PS (32.7% PS) And about 6% of the nerve knots, so the feed stream A is better. Therefore, although the use of raw residues contains a higher content of disc fat, many health applications need to produce 120457.doc -32- 200808817 Rich in PS and ganglioside products, making feed stream a the preferred feed stream for both. Also 'by-product from feed stream A ("extract" ") (using 3〇: 溶剂 solvent and The feed stream ratio yields 10.2% hydrazine. However, this product is also rich in neutral mussels and only contains 37.5% phospholipids. The product can be further treated with supercritical c〇2, degummed or acetone to remove neutral lipids. .

As discussed, the residue produced by the extraction of feed stream c produces the highest enrichment (73.7%) 'but the saccharide content of this product is lower than the residue produced by feed stream A and feed stream B. & nerve knot rouge content. Raw stream C contains less impurities f than feed stream A. In this case, it is possible to reduce the lactose and ash content of the above raw materials. 8 The purity of the extract produces a very positive effect. Finally, although raw material stream B is the most undesirable raw material stream, its residue is similar to the residue obtained by extraction with feed stream A, with a similar content of PS (23%) and nerve glycolipids. A (four). By-products ("extracts") are more noticeable because they contain very low levels of neutral lipids, high levels of phospholipids (63.7%) and 14.1% SM. As a by-product ("Extract II"), although the raw material flowing is the preferred raw material stream of the method described in the above, since the total raw material stream solid is converted into a product rich in ps, the yield has a problem. The production of this method is low, and 91% of the feedstock is solid. However, the extraction yield of feed stream C is even lower: 6% of the feed stream solids are converted to PS. However, the purity of the Ps product is extremely high. Product (Example 2) 120457.doc -33- 200808817 The second example, which can be independently preserved, differs from Example 1 in that it produces a product Γ extract "). As summarized in Table 3, the extraction produced up to four by-products, the most noticeable being the 31% SM product (Extract 3). Packed bed extraction (Example 3) The most intriguing aspect of the technology is likely to be during the extraction process.

Gu ° 顚然' can also produce SM-containing products by specifically manipulating the ethanol temperature in the step. The field/method completely divides the raw material stream 3 into a part containing high-stem sphingosine and squama. However, one of the disadvantages of this option is that it is difficult to process the feed stream B as described. Solvent: raw material flow ratio, and solvent to raw material ratio was also found to directly affect the PS concentration in the insoluble portion. = The rule is that the higher the ratio of the granules, the more the % purity in the insoluble fraction is. For example, in Example 1, it was found that the ratio of solvent to raw mash was 30:1, which gave 32.7%, PS fraction. Apricot you _ , I knife The field uses 5 〇: 1 solvent to raw material ratio, the part is 38.9% PS. # 、和3〇·1 〉Valley ratio also has advantages. This lower solvent ratio allows for smaller extraction vessels under full conditions, thus minimizing costs and improving production. Temperature and time: Temperature also affects Ps purity in the residue. The equivalent number has obvious practical limits on the operation of the king's rules. It is recommended to make the temperature as high as economically and practically as possible for the duration required to obtain a reasonable amount of ps in the final product. The inventors here focus on products containing at least 3% ps. In this study, both the feed stream A and the feed stream c in the method of Example 1 gave a product of more than 3% by weight. Example 4: Effect of water content on dairy lipid extract (ethanol fraetiojjadojj)

This example demonstrates the effect of water content on PS enrichment at 65 °C. 8 g of dairy lipid extract (feedstream B) was mixed in 4 ml of 95% ethanol (0% to 4% water). The mixture was then quickly heated to 65. It is also strong and turbulent. The sample was held at 65 ° C for 3 hours under periodic shaking. The sample was then filtered and the solid was air dried and then dried under vacuum. The sample is branched by up nmr. The results are shown in the table $. At lower water contents, the relative purity of PS is higher than that of other phospholipids, "the recovery rate is good and some are easy to process into powder. As the water content increases, the recovery of hydrazine decreases" and the PCTM content in the product increases. Produces a fraction containing more water and hydrogenated scales. Table 5

120457.doc -35 - 200808817 实例例5: Sample and solvent ratios of dairy lipid extracts (the effect of ethanol fractionation on the feed stream... The actual example is not the effect of sample-to-solvent ratio on PS enrichment at 65t. 2 g of the gifted lipid extract (feedstream B) was added to 2 〇 (5) 丨 % % ethanol and mixed at 65 C. Cold ethanol was added to obtain the desired sample to solvent ratio 'and then mixed. In the refrigerator for 1 After an hour, the samples were again mixed, then heated to 65 ° C and re-mixed. The sample was allowed to stand underneath 65. Then mixed and filtered. All mixing was carried out using ... Gabitin. Drying under vacuum The collected solids were analyzed by 3 spectroscopy. The results are shown in the following Table 6. The pS enrichment was improved by increasing the amount of solvent. 74.3 in the insoluble material at a sample to solvent ratio of 1:1 〇〇 The % phospholipid is PS. When phospholipids account for 56.6% of this fraction, the amount of Ps in this sample is 42%. Table 6 Sample: Solvent yield (% solids) % PL 4 1 Lip composition (%) PC PI PS PE CM Material 23.9 7.9 11.3 Jl MZj 33 1 O丄Vx 99 ? 1:10 35 56.9 9.8 15.3 24.2 mJ . X 35.9 11.6 1:20 21 55.7 3.6 20.9 35.9 32.0 4.7 1:50 17 56.1 1.8 12.7 67.0 13.5 1 5 1:10» 15.4 56.6 1.2 10.2 74.3 8.7 1.0 Example 6 ·· PH value for dairy lipid extract (raw material Effect of Ethanol Fractionation of Stream B) τ This example shows the effect of pH on PS enrichment at 65 ° C. Add dairy lipid extract (0.8 g) (feed stream B) to 4 〇 ml at 65 C In ethanol, ethanol contains 5% water, 5% glacial acetic acid or 5% ammonia water 120457.doc -36-200808817 (2.5%) and the pH values of the three mixtures are 7.7, 3.2 and Η, respectively. The mixture was mixed by a periodic shaking for 2 hours, followed by filtration. Dry (4) in a straight space and analyzed by "Ρ NMR. The results are shown in Table 7 below. The acidic conditions gave a good 1 > 8 enrichment, but the results were similar in the broad Yang range. Table 7 --------- Raw material 'yield (solid %) % PL Phospholipid composition (%) ^ PC 23.9 PI 丨·~一圆 _ 7.9 PS 11.3 PE 33.1 SM —--- 29 ? pH value =3.2 solid 24 59.7 3.5 23.3 44.5 23.8 X Λ pH = 7.7 solid 31 46.8 5.5 16.3 42.1 28.9 Ο Λ 3 5 0 pH = 10.0 solid 33 52.0 4.8 17.7 393 31.4 Mountain U 5.0 Example 7: Solvent on dairy lipid extract ( Effect of fractionation of feed stream c); This example demonstrates the effect of alcohol chain length on PS enrichment. 100 g of a solvent with 5% water was heated to 65 ° C on a stirred hot plate (since the lower boiling point of sterol, 5 〇 t for methanol). After reaching the temperature, 2 g of the dairy lipid extract (feed stream c) was added and mixed for 2 hours. The sample was removed from the heat and filtered under vacuum. The insoluble fraction was then dried on Whatman 4 filter paper and the soluble fraction was rotary evaporated. Record all weights for mass S balance. The sample was analyzed by 3 NMR. The results are shown in Table 8. 120457.doc 37- 200808817 Table 8 Yield (% solids) % PL ~~ Lipid composition PC PI PS CM Material 41.7 26.9 6.8 10.4 X Kj OitX 99 Q Sterol insoluble soluble 42 • 53 13·1 56.6 0.9 31.3 11.2 6.4 64.7 14 *^1.0 21.7 1 1.3 07 c Ethanol insoluble soluble 7 93 89.3 41.2 0.8 29.6 7.8 6.4 82.5 1? 6.0 5? Z l .Z> N/D 0〇A n-propanol insoluble soluble 3 96 75.2 36.8 0.5 31.5 6.6 4.3 8g.〇3.6 3Z.O 2.6 33 6 N/D 96 ? Isopropanol insoluble soluble 6 93 86.6 0.7 11.0 80.5 m\J 4.6 0.6 Example 8: Ethanol fractionation of phospholipids from various sources (sample ratio solvent ι: ι〇) This example demonstrates that the method of the present invention is widely applicable to a variety of lipid matrices using a sample of 1:1 Torr with a > trough ratio and a temperature of 50 °C. This example also shows that the phospholipid is substantially concentrated by the method of the invention when present in the feed material. Extracts from the following sources of animal extracts (Q. 5 g): • Bovine liver, • Bull heart, • Shark spinal cord, • Lemma JTish, and • New Zealand hoki head Weigh in a test tube and add 5 g of 95% ethanol. The tube was stoppered and placed in a 5 〇χ water bath with periodic shaking and 2 hours of ultrasonic treatment. The $ ί was removed and centrifuged (3000 rpm, 2 min) and the supernatant was decanted from the solid. The solvent was partially removed under a stream of argon 120457.doc -38 - 200808817 and then under vacuum. The phospholipid profile was determined by Mp NMR. The results are shown in Table 9. In all cases, the insoluble fraction is enriched with the desired phospholipids PS and CL. Scale fats (especially phospholipids from lemonfish) have a higher content of polyunsaturated fatty acids than dairy lipids, which increase the solubility of phospholipids in ethanol. The soluble fraction is rich in SM. Table 9 Yield (% solids) % PL Phospholipid composition (%) PC PI PS PE SM CL Bovine liver raw material 56.7 44.9 10.4 2.2 23.0 3.6 8.3 Insoluble 12 73.0 9.1 29.1 6.2 33.8 N/D 16.0 Solubility __ 88 43.2 56.9 3.8 0.9 20.7 4.3 6.1 Beef heart material 61.1 21.7 4.4 2.0 20.8 5.4 26.4 Insoluble 27 72.9 4.4 14.0 5.1 21.6 6.6 41.1 Solubility 73 55.4 34.9 1.0 0.5 25.5 6.6 15.8 Shark spinal cord material 22.9 35.7 1.4 12.6 47.5 2.8 N/D insoluble 22 43.5 21.3 2.7 23.4 46.8 3.9 N/D Lemonfish raw material 63.7 46.2 7.9 3.5 25.0 5.2 7.9 Insoluble 26 76.1 8.3 27.6 10.7 29.4 N/D 19.6 Solubility 74 50.5 58.7 2.0 1.3 24.6 5.8 4.2 New Zealand sorghum without raw material 33.2 49,6 5.3 6.3 22.8 3.1 Insoluble 23 26.1 25.6 10.2 13.4 22.1 10.6 Soluble 77 36.2 52.2 4.5 5.4 24.5 2.9

New Zealand's unneeded PE and SM concentrations have been brought together due to the difference in resolution during the analysis. Example 9: Ethanol fractionation of phospholipids from various sources, (sample ratio, reagent enthalpy) 120457.doc -39- 200808817 This example demonstrates the enrichment of PS from feedstock materials with low PS content. 100 g of 95% ethanol was heated to 65 ° C on a stir plate. After reaching the temperature, 2 g of sample was added and mixed for 2 hours. The sample was removed from the heat and filtered under vacuum. The insoluble fraction was intercepted on Whatman 4 filter paper and then the insoluble fraction was dried and the soluble fraction was rotary evaporated to reduce the volume. Record all weights for mass balance. The sample was analyzed by 31P NMR. The results are shown in Table 10. Table 10

Yield (% solids) % PL Phospholipid composition (%) PC PI PS PE SM CL Soy lecithin material 66.3 33.5 19.8 N/D 29.1 N/DN/D Insoluble 24 69.0 6.4 39.4 Li 29.1 N/DN/D Soluble 76 73.2 53.0 7.7 N/D 21.8 N/DN/D Egg material 27.7 70.2 1.8 1.6 18.8 4.7 N/D insoluble 4 29.1 8.4 14.7 48.4 18.0 N/DN/D Solubility 96 30.3 71.0 1.8 N/D 18.0 4.4 N/D Industrial application The invention can be used to provide products having high levels of specific phospholipids/sphingolipids, including phospholipids, riboskinates, and sphingomyelins. These products can be used in many applications, including infant formula, brain health, sports nutrition, drug delivery, dermatological applications, and arthritis. references

Jensen, R.G. 2002. The Composition of Bovine Milk Lipids: January 1996 to December 2000. J. Dairy Sci. 85: 120457.doc -40- 200808817 295-350

Ladisch, S.; Gillard, B. 1985. A solvent partition method for microscale ganglioside purification. Anal. Biochem. 146: 220-231

Padley F.B.; Gunstone, F.D.; Harwood^ J.L. 1986. Occurrence and characteristics of oils and fats, at "Tfie

Lipid Handbook (Gunstone, F.D.; Harwood, J.L.; Padley, F.B.), pp. 49 -1 70. Chapman and Hall, London-NY o

Wu? Y" Wang, T. 2004. Fractionation of crude soybean lecithin with aqueous ethanol. JAOCS 81(7): 697-704 〇 120457.doc -41 -

Claims (1)

200808817 X. Patent Application Range: 1. A method for fractionating a feed material into a soluble component and an insoluble component, which comprises: (a) providing a feed material comprising one or more of the following: (0 at least 1 % weight/weight of phospholipid lysine; (ii) at least 1% w/w of sphingolipid; (iii) at least 1% w/w of cardiolipin; or (π) at least 3%·3% by weight/ Weight gangliosides; () seven, a solvent containing one or more C1-C3 monohydric alcohols and water, wherein the water content of the one or more alcohols is from 〇 to 4% by volume/volume; c) = the feed material is contacted with the solvent and then the solvent containing the soluble components is separated from the insoluble components; (4) separating the soluble components and the solvent as appropriate. 2. If the claim 1 is too, Soil, 'eight in the feed material contains less than 10% protein and less than 10% lactose. Greed /, medium >, at 1%% of the feed material contains the egg 'which makes the feed material The feed material containing more than 1% of the phosphonium contains more than 2% of the sarcophagus, wherein the feed material Containing more than 5% of phospholipids 3. The method of claim 1 and lactose. 4. The method of claim & serine. 5. The method of claim 醯 胺 胺. Method 醯 胺 胺 120 120 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 Sphingomyelin. 9. The method of claim 1, sphingomyelin 10. The method of claim 1, lipid 11. The method of claim 1, lipid 12. The method of claim 1, lipid. If the method of claim 1 is followed by the method of lipolysis, 14. If the method of j stomach 1 is requested, the glycolipid is obtained. 15. If the method of claim 1 is used, the conclusion is as follows: 16. In claims 1 to 15, Any material containing more than 15% of the gods 1% of the heart stone method 'where the feed material contains more than 5%, the method wherein the feed material contains more than 3% · 3% of the nerve from the land gas (8) ^ 〗 In the method, the material in the source is source, S/ animal, land plant, marine plant or Microorganisms such as micro, yeast and bacteria. 17. The method of claim 16, 苴,, ,,甲甲, into the material is derived from sheep, I hands, pigs, mice, buffalo, slightly squat, alley φ , 午H, yak, horse, donkey, llama, cow or human. ^ 120457.doc 200808817 18. The method of claim 6 or 丨7, wherein the feed material is selected from: tissue, tissue part , organs, organs, milk, milk, colostrum, colostrum, blood and blood. The method of claim 16, wherein the feed material is derived from dairy material, macroscopic material, egg, animal tissue, animal organ or animal blood. 20. The method of claim 6, wherein the feed material is selected from the group consisting of a composition comprising a dairy lipid, a composition comprising an egg lipid, and a composition comprising a marine biolipid. The method of any one of claims 1 to 20, wherein the feed material is a milk portion. The method of claim 21, wherein the feeding material is selected from the group consisting of: buttermilk, buttermilk fraction, beta whey, whey fraction, whey whey, whey whey fraction, Whey, whey, colostrum and colostrum. 23. The method of any one of the preceding claims, wherein the feed material comprises a milk fat globule film. The method of any one of claims 1 to 2, wherein the feed material has been genetically modified. 25. The method of claim 2 to 2, wherein the feed material is in solid form. 26. The method of claim 25, wherein the feed material is cryogenically milled prior to contact with the solvent. 27. The method of claim 26, wherein the feed material is milled at a low temperature prior to contact with the solvent to obtain an average particle size of 4571_5 mm 120457.doc 200808817 degrees. 28·If requested! To any of the 27 廿山斗贝 I, where the solvent comprises: (a) an alcohol selected from the group consisting of: T, ethanol, n-propanol, isopropanol, and mixtures thereof; and (b) 0 - 40% by volume/volume of water σ 29. The method of claim 28, wherein the direct agent comprises between 0 and (10)^ volume/volume. 3 0 ·If the method of the item 2 8 is made, the Lizhong 兮汾 / / Μ Μ, 丨 between 1 and 10% of the volume / limb product, water. The method of any one of claims 28 to 30, wherein the alcohol is ethanol. 32. The method according to any of the preceding claims, wherein the solvent comprises a 95% aqueous solution of ethanol. 3 3 · As requested! The method of any of the preceding claims, wherein the ratio of the solvent enthalpy feeding material is between (4) and (10)". I. Feeding material 枓 Monthly method 3 3, wherein the "(4) ^ ^ ^ ^ 20:1 and 60:1. I. The ratio of the feed material is between 35. According to the method of claim 33, it is between 25:1 and 4〇α. The ratio of bamboo rafts + is between 36 如 ° C as required in the request ^ to ^. , z, wherein the method is above 37. The method of claim 36, which is considered to be the method of claim 36, the two of which are performed above 5 (rc). The team is as requested! The method of 项 糸 is carried out under two hunger. The steps of lowering or raising the temperature are carried out. 'The method is based on a number of 120457.doc 200808817 40. The system is below or equal to 4. (: proceeding. 41 · as requested. Item 4 〇> π, the method, where the method is below or equal to 0. (: proceeding. 42 · as requested The method of any one of the above, wherein the step (c) is performed between 00 and 00, wherein the step (c) is performed for 2 hours. 44. If the method material of any one of the requirements is contacted once. 45. If the method of any of the claims is in contact with the same batch of solvent, multiple times. 46. The method of any one of the above-mentioned methods is in contact with a plurality of solvents. 47. The method according to any one of claims 1 to 46, wherein the feeding material is The packed bed is a method of contacting the solvent with the feed of 48. The contact page of the tenth of claims 1 to 46, wherein the mixture is mixed with the grain and the feed material by stirring. 49 = the method of claim 48, Wherein the scramble is made by high (four) mixed. Port 50 - a product produced by a method such as a request to an office - 5) - as requested in (4) of the product 'where the product is a soluble material. The money of claim 50, wherein the silk is an insoluble material. 53. The product of claim 5 or 51, the middle, and the T忒 product contain more sphingomyelin than the feed. With the feed force of the solvent with the feed ^ where the feed material is hunted by the use of the solution I20457.doc 200808817 54. The product of claim 50 or 51, wherein the product contains greater than 3% lysine. 5 5 The product of claim 50 or 51 wherein the product comprises greater than 1% sphingomyelin. 56. The product of claim 50 or 51, wherein the product comprises greater than 15% neurolephospholipid. The product of item 50 or 52, wherein the product contains more than the feed material The product of claim 50 or 52, wherein the product comprises greater than 5% phospholipid quinic acid. 5 9 · The product of 50 or 52, wherein the product comprises greater than 3的脂脂丝酸酸。 6. The product of claim 50 or 52, wherein the product comprises greater than 7% by weight of the fat-loading serine. 61. The product of claim 50 or 52, wherein The product contains more neuroglycolipid than the feed material. 62. The product of claim 50 or 52, wherein the product comprises greater than 2% of a nerve knot. 63. The product of claim 5 or 52, wherein the product comprises greater than 4% nerve knot fat. 64. The product of claim 5, or 52, wherein the product comprises greater than 6% of a neurolipid. 65. The product of claim 5, or 52, wherein the product contains more cardiolipin than the feed material. 120457.doc 200808817 66. The product of claim 50 or 52, wherein the product comprises greater than 5% cardiolipin. 67. The product of claim 50 or 52, wherein the product comprises greater than 10% heart stone rouge. 68. The product of claim 50 or 52, wherein the product comprises greater than 25% cardiolipin. 120457.doc 200808817 VII. Designated representative map: (1) The representative representative of the case is: (none) (2) The symbolic symbol of the representative of the prince is simple: 8. If there is a chemical formula in this case, please reveal the best indication of the characteristics of the invention. Chemical formula: • (none) 120457.doc