Lysophosphatidylcholine Composition

Abstract

The present invention provides marine lysophosphatidylcholine compositions for use in pharmaceuticals, dietary supplements and functional foods, as well as methods for making the marine lysophosphatidylcholine compositions.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention provides marine lysophosphatidylcholine compositions for use in pharmaceuticals, dietary supplements and functional foods, as well as methods for making the marine lysophosphatidylcholine compositions.

[0002] 2. Background of the Invention Lysophosphatidylcholine (LPC) is a compound produced by partial hydrolysis of a phosphatidylcholine (PC) molecule, resulting in the removal of one of the fatty acid groups attached to the PC molecule.

[0003] Lysophosphatidylcholine, which contains one mole of fatty acid per mole of lipid in the sn-1 position, is found in trace amounts in most animal tissues (higher concentrations are membrane-disrupting). It is generated by hydrolysis of dietary and biliary phosphatidylcholine and is absorbed intact in the intestine, but is re-esterified before being excreted in the lymph. In addition, lysophosphatidylcholine is formed in most tissues by hydrolysis of phosphatidylcholine by the superfamily of phospholipase A2 enzymes, as part of the deacylation / reacylation cycle that controls the overall molecular species composition of the latter, as described above. In the plasma of animal species, significant amounts of lysophosphatidylcholine are formed by the action of the enzyme lecithin:cholesterol acyltransferase (LCAT), which is secreted from the liver. Lysophosphatidylcholine catalyzes the transfer of fatty acids from the sn-2 position of phosphatidylcholine in plasma to free cholesterol, forming cholesterol esters, and of course, lysophosphatidylcholine is composed of a mixture of mainly saturated fatty acids and molecular species with mono- and dienoic fatty acid components. In plasma, lysophosphatidylcholine is bound to albumin and lipoproteins, reducing its effective concentration to safe levels. The identification of a highly specific phospholipase A2γ in peroxisomes specific for the generation of sn-2-arachidonoyl lysophosphatidylcholine suggests that lysophosphatidylcholine may be related to eicosanoid generation and signaling. Elevated levels of 26:0-lysophosphatidylcholine in blood have been reported to be characteristic of Zellweger spectrum disorders (a consequence of defects in peroxisome biogenesis).

[0004] Lysophosphatidylcholine has proinflammatory properties and is known to be a pathological component of oxidized lipoproteins (LDL) in plasma and atherosclerotic lesions; for example, a specific enrichment of 2-arachidonoyl-lysophosphatidylcholine has been reported in carotid atherosclerotic plaques of type 2 diabetic patients. Recently, lysophosphatidylcholine has been found to have several functions in cell signaling, and specific receptors (coupled to G proteins) have been identified. Lysophosphatidylcholine activates specific phospholipase C, which releases diacylglycerol and inositol triphosphate, resulting in increased intracellular Ca2+ and protein kinase C activation. Lysophosphatidylcholine also activates mitogen-activated protein kinases in certain cell types, promoting demyelination in the nervous system. In vascular endothelial cells, lysophosphatidylcholine induces the important proinflammatory mediator cyclooxygenase-2 (COX-2), a key enzyme in prostaglandin synthesis. Elevated levels of lysophosphatidylcholine have been identified in cervical cancer and may be useful in diagnosing this disease. Some biological effects of lysophosphatidylcholine may simply be due to its ability to diffuse readily into membranes, altering their curvature and indirectly affecting the properties of membrane proteins.

[0005] WO2015048554 discloses methods for identifying compounds that modulate trafficking via the Mfsd2a protein and other potential uses of LPC compositions. Summary of the Invention

[0006] The present invention provides marine lysophosphatidylcholine compositions for use in pharmaceuticals, dietary supplements and functional foods, as well as methods for making the marine lysophosphatidylcholine compositions.

[0007] Thus, in some preferred embodiments, the present invention provides marine lysophosphatidylcholine (LPC) compositions or concentrates characterized in that they comprise LPC in an amount of about 10% to about 100% (w / w) of the composition and an omega-3 fatty acid content of 5% to 50% (w / w) of the composition, and optionally have one or more of the following features or properties: a) a 2-LPC:1-LPC ratio of 1:8 to 18:1; b) a phosphatidylcholine (PC) content of less than 10% (w / w) of the composition; c) a phosphatidylethanolamine (PE) content of less than 1.2% (w / w) of the composition; d) a neutral lipid content of 5% to 65% w / w of the composition; e) a 2-LPC ether content of less than 1.0% (w / w); and f) an EPA:DHA ratio of 1:1 to 3:1, or a DHA:EPA ratio of 1:1 to 5:1.

[0008] In some preferred embodiments, the composition comprises 60%-100% LPC (w / w) of the composition. In some preferred embodiments, the composition comprises 70%-90% LPC (w / w) of the composition. In some preferred embodiments, the composition comprises 20%-50% LPC (w / w) of the composition. In some preferred embodiments, the composition comprises 20%-30% LPC (w / w) of the composition. In some preferred embodiments, the composition comprises 10%-20% LPC (w / w) of the composition.

[0009] In some preferred embodiments, the composition has an omega-3 fatty acid content of 30%-50% (w / w) of the composition. In some preferred embodiments, the composition has an omega-3 fatty acid content of 35%-45% (w / w) of the composition. In some preferred embodiments, the composition has an omega-3 fatty acid content of 5%-20% (w / w) of the composition.

[0010] In some preferred embodiments, the composition has characteristic (a). In some preferred embodiments, the composition has characteristic (b). In some preferred embodiments, the composition has characteristic (c). In some preferred embodiments, the composition has characteristic (d). In some preferred embodiments, the composition has characteristic (e). In some preferred embodiments, the composition has characteristic (f). In some preferred embodiments, the composition has two or more of characteristics (a), (b), and (c). In some preferred embodiments, the composition has two or more of characteristics (a), (b), (c), (d), (e), and (f). In some preferred embodiments, the composition has three or more of characteristics (a), (b), (c), (d), (e), and (f). In some preferred embodiments, the composition has four or more of characteristics (a), (b), (c), (d), (e), and (f). In some preferred embodiments, the composition has five or more of characteristics (a), (b), (c), (d), (e), and (f). In some preferred embodiments, the composition has properties (a), (b), (c), (d), (e) and (f).

[0011] In some preferred embodiments, the composition is selected from the group consisting of a krill LPC composition, a herring LPC composition, a herring roe LPC composition, an algae LPC composition, and a Calanus LPC composition.

[0012] In some preferred embodiments, the present invention provides a pharmaceutical or nutraceutical composition comprising the above-described composition and a physiologically acceptable carrier, hi some preferred embodiments, the physiologically acceptable carrier is a lipid carrier.

[0013] In some preferred embodiments, the present invention provides an oral delivery vehicle containing a marine LPC composition, a pharmaceutical composition, or a nutraceutical composition as described above.

[0014] In some preferred embodiments, the present invention provides a lipid composition comprising a first lipid fraction and a second lipid fraction, the first lipid fraction being a marine LPC composition as described above, the second lipid fraction being obtained from a different source than the first lipid fraction and / or comprising less than 20% LPC. In some preferred embodiments, the second lipid fraction is selected from the group consisting of a triglyceride fraction, a diglyceride fraction, a fatty acid ethyl ester fraction, a free fatty acid fraction, and combinations thereof. In some preferred embodiments, the second lipid fraction is a marine lipid fraction comprising EPA and / or DHA. In some preferred embodiments, the present invention provides a pharmaceutical or nutraceutical composition comprising the lipid composition as described above and a physiologically acceptable carrier. In some preferred embodiments, the present invention provides an oral delivery vehicle comprising the lipid composition as just described.

[0015] In some preferred embodiments, the present invention provides a method for producing a lysophosphatidylcholine (LPC) composition with high EPA and DHA content from a marine feedstock containing phospholipids, comprising treating the marine feedstock with a phospholipase not native to the marine feedstock to provide a phospholipase-treated feedstock, and fractionating the phospholipase-treated feedstock to provide a lipid composition with a higher lysophosphatidylcholine content than the starting feedstock. In some preferred embodiments, the feedstock is selected from the group consisting of a krill lipid preparation, a herring lipid preparation, a herring egg lipid preparation, an algae lipid preparation, and a Calanus lipid preparation. In some preferred embodiments, the krill lipid preparation is a Euphausia Superba lipid preparation.

[0016] In some preferred embodiments, the feedstock is contacted with the phospholipase in a solvent. In some preferred embodiments, the solvent is a mixture of water and an alcohol. In some preferred embodiments, the alcohol is ethanol. In some preferred embodiments, the feedstock is contacted with the phospholipase in a mixture of about 85% water and 15% ethanol.

[0017] In some preferred embodiments, the enzyme is phospholipase A1 (PLA1). In some preferred embodiments, the enzyme is phospholipase A1 (PLA1), wherein the enzyme concentration is in the range of 0.1-20 (volume / weight)%, preferably 0.1-15 (volume / weight)%, more preferably 0.1-10 (volume / weight)%, even more preferably 0.1-5 (volume / weight)%, and most preferably 0.1-3 (volume / weight)%. In some preferred embodiments, the enzyme is phospholipase A1 (PLA1), and the method is carried out at a pH of 3-12, preferably 4-10, more preferably 4-9, and most preferably 5-9. In some preferred embodiments, the enzyme is phospholipase A1 (PLA1), and the method is carried out at a temperature of 4-95°C, preferably 4-85°C, more preferably 10-80°C, even more preferably 15-70°C, even more preferably 15-65°C, and most preferably 15-60°C.

[0018] In some preferred embodiments, the raw material has an EPA and DHA content in the following ranges: EPA: 1-70% by weight and DHA: 1-70% by weight, more preferably EPA: 5-70% by weight and DHA: 5-70% by weight, and most preferably EPA: 10-60% by weight and DHA: 10-60% by weight. In some preferred embodiments, the LPC composition has an EPA and DHA content in the following ranges: EPA: 1-100% by weight and / or DHA: 1-100% by weight, more preferably EPA: 5-100% by weight and / or DHA: 5-100% by weight, and most preferably EPA: 10-90% by weight and / or DHA: 10-90% by weight.

[0019] In some preferred embodiments, the LPC composition has an LPC content in the range of 10-100% by weight, preferably 20-100% by weight, more preferably 30-100% by weight, even more preferably 40-100% by weight, and most preferably 50-100% by weight. In some preferred embodiments, the LPC composition obtained by this process comprises LPC at about 20% to about 95% (w / w) of the composition and an omega-3 fatty acid content of 5% to 50% (w / w) of the composition, and is optionally characterized in having one or more of the following features or characteristics: a) a 2-LPC:1-LPC ratio of 1:8 to 18:1; b) a phosphatidylcholine (PC) content of less than 10% (w / w) of the composition; c) a phosphatidylethanolamine (PE) content of less than 1.2% (w / w) of the composition; d) a neutral lipid content of 5% to 65% (w / w) of the composition; e) a 2-LPC ether content of less than 1.0% (w / w); and f) an EPA:DHA ratio of 1:1 to 3:1 or a DHA:EPA ratio of 1:1 to 5:1.

[0020] In some preferred embodiments, the method further comprises formulating the LPC composition for human consumption.

[0021] In some preferred embodiments, the present invention provides an LPC or lipid composition as described above, or produced by a method as described above, for use to supplement the diet of a human subject, preferably under 10 years of age, more preferably under 1 year of age, even more preferably under 1 month of age, and most preferably a newborn.

[0022] In some preferred embodiments, the present invention provides marine lysophosphatidylcholine (LPC) compositions or concentrates characterized in that they comprise LPC in an amount of about 60% to about 100% (w / w) of the composition and an omega-3 fatty acid content of 30% to 50% (w / w) of the composition, and optionally have one or more of the following features or properties: a) a 2-LPC:1-LPC ratio of 1:8 to 18:1, or more preferably 1:1 to 10:1; b) a phosphatidylcholine (PC) content of less than 0.5 to 5% (w / w) of the composition; c) a phosphatidylethanolamine (PE) content of less than 0.5% (w / w) of the composition; d) a neutral lipid content of 5% to 40% w / w of the composition; and e) a 2-LPC:2-LPC ether ratio of 2.5:1 to 4:1.

[0023] In some preferred embodiments, the present invention provides marine lysophosphatidylcholine (LPC) compositions or concentrates characterized in that they comprise LPC in an amount of about 10% to about 20% (w / w) of the composition and an omega-3 fatty acid content of 5% to 50% (w / w) of the composition, and optionally comprise one or more of the following features or properties: a) a 2-LPC:1-LPC ratio of 1:8 to 18:1, or more preferably 1:1 to 10:1; b) a phosphatidylcholine (PC) content of less than 10% (w / w) of the composition; c) a phosphatidylethanolamine (PE) content of less than 1.2% (w / w) of the composition; d) a neutral lipid content of 5% to 65% (w / w) of the composition; e) a 2-LPC ether content of less than 1.0% (w / w); and f) an EPA:DHA ratio of 1:1 to 3:1 or a DHA:EPA ratio of 1:1 to 5:1.

[0024] In some preferred embodiments, the present invention provides marine lysophosphatidylcholine (LPC) compositions or concentrates characterized in that they comprise LPC in an amount of about 20% to about 40% (w / w) of the composition and an omega-3 fatty acid content of 5% to 50% (w / w) of the composition, and optionally have one or more of the following features or properties: a) a 2-LPC:1-LPC ratio of 1:8 to 18:1, or more preferably 1:1 to 10:1; b) a phosphatidylcholine (PC) content of less than 10% (w / w) of the composition; c) a phosphatidylethanolamine (PE) content of less than 1.2% (w / w) of the composition; d) a neutral lipid content of 5% to 65% w / w of the composition; e) a 2-LPC ether content of less than 1.0% (w / w); and f) an EPA:DHA ratio of 1:1 to 3:1 or a DHA:EPA ratio of 1:1 to 5:1.

[0025] In some preferred embodiments, the present invention provides marine lysophosphatidylcholine (LPC) compositions or concentrates characterized in that they comprise LPC in an amount of about 40% to about 60% (w / w) of the composition and an omega-3 fatty acid content of 5% to 50% (w / w) of the composition, and optionally have one or more of the following features or properties: a) a 2-LPC:1-LPC ratio of 1:8 to 18:1, or more preferably 1:1 to 10:1; b) a phosphatidylcholine (PC) content of less than 10% (w / w) of the composition; c) a phosphatidylethanolamine (PE) content of less than 1.2% (w / w) of the composition; d) a neutral lipid content of 5% to 65% w / w of the composition; e) a 2-LPC ether content of less than 1.0% (w / w); and f) an EPA:DHA ratio of 1:1 to 3:1 or a DHA:EPA ratio of 1:1 to 5:1.

[0026] In some preferred embodiments, the present invention provides marine lysophosphatidylcholine (LPC) compositions or concentrates characterized in that they comprise LPC in an amount of about 60% to about 80% (w / w) of the composition and an omega-3 fatty acid content of 5% to 50% (w / w) of the composition, and optionally have one or more of the following features or properties: a) a 2-LPC:1-LPC ratio of 1:8 to 18:1, or more preferably 1:1 to 10:1; b) a phosphatidylcholine (PC) content of less than 10% (w / w) of the composition; c) a phosphatidylethanolamine (PE) content of less than 1.2% (w / w) of the composition; d) a neutral lipid content of 5% to 65% (w / w) of the composition; e) a 2-LPC ether content of less than 1.0% (w / w); and f) an EPA:DHA ratio of 1:1 to 3:1 or a DHA:EPA ratio of 1:1 to 5:1.

[0027] In some preferred embodiments, the present invention provides marine lysophosphatidylcholine (LPC) compositions or concentrates characterized in that they comprise LPC in an amount of about 60% to about 100% (w / w) of the composition and an omega-3 fatty acid content of 5% to 50% (w / w) of the composition, and optionally have one or more of the following features or properties: a) a 2-LPC:1-LPC ratio of 1:8 to 18:1, or more preferably 1:1 to 10:1; b) a phosphatidylcholine (PC) content of less than 10% (w / w) of the composition; c) a phosphatidylethanolamine (PE) content of less than 1.2% (w / w) of the composition; d) a neutral lipid content of 5% to 65% (w / w) of the composition; e) a 2-LPC ether content of less than 1.0% (w / w); and f) an EPA:DHA ratio of 1:1 to 3:1 or a DHA:EPA ratio of 1:1 to 5:1.

[0028] In some embodiments, the above lysophospholipid compositions are provided for use in increasing the amount of EPA and / or DHA in target tissues or organs by oral administration of the lysophospholipid compositions.Preferred target tissues and organs according to the present invention are adrenal gland, blood, bone, bone marrow, brain, adipose (white), kidney (whole), large intestine mucosa, liver, lung, muscle, cardiac muscle, pancreas, pituitary gland, prostate, skin, small intestine mucosa, spleen, gastric mucosa, testis, thymus, and / or thyroid.In some preferred embodiments, the increase in the amount of EPA and / or DHA is compared to an equivalent dose of EPA and / or DHA provided as a phospholipid (i.e., a phospholipid molecule that contains fatty acyl groups at both the SN-1 and SN-2 positions of the phospholipid). [Brief description of the drawings]

[0029] [Figure 1] 1H-NMR spectrum of sample AKB69444-1. [Diagram 2] HPLC chromatogram of the LPC product, sample AKB69444-1. [Diagram 3] 1H-NMR spectrum of sample AKB70005-2. [Figure 4] HPLC chromatogram of the LPC product, sample AKB70005-2. [Diagram 5] 1H-NMR spectrum of sample AKB70005-3. [Figure 6] LPC and HPLC chromatograms of sample AKB70005-3. [Figure 7] 1H-NMR spectrum of sample AKB70005-4. [Figure 8] LPC and HPLC chromatograms of sample AKB70005-4. [Figure 9] 1H-NMR spectrum of sample AKB70005-5. [Figure 10] HPLC chromatogram of the LPC product, sample AKB70005-5. [Figure 11] 1 is a flow chart of the process of the present invention. [Figure 12] 1 is a flow chart of the process of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] definition As used herein, "phospholipid" refers to an organic compound having two fatty acid moieties attached to the sn-1 and sn-2 positions of glycerol and a head group linked by a phosphate residue at the sn-3 position of glycerol. Exemplary head group moieties include choline, ethanolamine, serine, and inositol. Phospholipids include phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid. The fatty acid moiety is a portion of a fatty acid molecule that is linked at the sn-1 or sn-2 positions, for example, by an ester or ether bond. When the fatty acid moiety is a fatty acyl, the fatty chain of the fatty acyl is linked via an ester bond, and when the fatty acid moiety is a fatty acid chain, the fatty chain is linked via an ether bond. Thus, when a particular fatty acid is referred to in connection with the phospholipids of the present invention (e.g., EPA or DHA), it should be interpreted as a reference to the relevant fatty acyl group or its fatty chain.

[0031] As used herein, the term "ether phospholipid" refers to a phospholipid in which the fatty acid moiety at one of the sn-1 or sn-2 positions is an aliphatic chain of fatty acids linked via an ether bond. Ether phospholipids include, for example, alkylacylphosphatidylcholine, alkylacylphosphatidylethanolamine, and alkylacylphosphatidylserine.

[0032] As used herein, the term "lysophospholipid" refers to a phospholipid molecule having a fatty acid moiety at one of the sn-1 and sn-2 positions of the molecule and an -OH group at the other position, resulting in one mole of fatty acid moiety per mole of phospholipid molecule. The term lysophosphatidylcholine (LPC) refers to a phosphatidylcholine molecule having a fatty acid moiety at one of the sn-1 and sn-2 positions of the molecule and an -OH group at the other position, resulting in one mole of fatty acid moiety per mole of lipid. Lysophospholipids may be designated as 1- or 2-lysophospholipids to indicate the position of the -OH group in the molecule. Thus, 1-LPC refers to a lysophosphatidylcholine having an -OH group at the sn-1 position of the molecule and a fatty acid moiety at the sn-2 position. 2-LPC refers to a lysophosphatidylcholine having an -OH group at the sn-2 position of the molecule and a fatty acid moiety at the sn-1 position. When the lysophospholipid is an ether phospholipid, the fatty acid moiety is a fatty alcohol linked via an ether bond at the sn-1 or sn-2 position.When the lysophospholipid is an ester phospholipid, the fatty acid moiety is a fatty acid ester linked via an ester bond at the sn-1 or sn-2 position.

[0033] As used herein, the term "long chain polyunsaturated fatty acid" refers to a fatty acid having 20 or more carbons and that is unsaturated at two or more bonds.

[0034] As used herein, the term omega-3 fatty acid refers to a polyunsaturated fatty acid with a final double bond in the hydrocarbon chain between the third and fourth carbon atoms from the methyl end of the molecule. Non-limiting examples of omega-3 fatty acids include 5,8,11,14,17-eicosapentaenoic acid (EPA), 4,7,10,13,16,19-docosahexaenoic acid (DHA) and 7,10,13,16,19-docosapentaenoic acid (DPA).

[0035] As used herein, the term "physiologically acceptable carrier" refers to any carrier or excipient that is commonly used with oil-based pharmaceuticals, including, but not limited to, oils, starches, sucrose, and lactose.

[0036] As used herein, the term "oral delivery vehicle" refers to any means of delivering oral medication, including, but not limited to, capsules, pills, tablets, and syrups.

[0037] As used herein, the term "food" refers to any food or feed suitable for consumption by humans, non-ruminant animals, or ruminant animals. A "food" can be a prepared and packaged food (e.g., mayonnaise, salad dressing, bread, or cheese food) or an animal feed (e.g., extruded pelleted animal feed or coarse mixed feed). A "prepared food" means any packaged food approved for human consumption.

[0038] As used herein, the term "food" refers to any substance suitable for human or animal consumption.

[0039] As used herein, the term "functional food" refers to a food to which a biologically active supplement has been added.

[0040] As used herein, the term "infant food" refers to food formulated for infants such as formula.

[0041] As used herein, the term "elderly food" refers to food formulated for elderly people.

[0042] As used herein, the term "pregnancy food" refers to food formulated for pregnant women.

[0043] As used herein, the term "dietary supplement" refers to a food used as part of the diet or a food formulated as a dietary supplement.

[0044] As used herein, the term w / w (weight / weight) refers to the amount of a given substance in a composition on a weight basis, unless otherwise specified, and is expressed as a percentage of the total composition weight.For example, a composition that contains 50% (w / w) phospholipid means that the mass of phospholipid is 50% of the total mass of the composition (i.e., 50 grams of phospholipid in 100 grams of composition such as oil).Throughout this specification, when a solvent concentration is specified, the concentration refers to the weight percent of the solvent in the specified solvent solution.As a non-limiting example, 96% ethanol contains 96% ethanol and 4% water.As another non-limiting example, if this specification describes a lipid solution as containing 20% ​​(w / w) dry matter, and the solvent is 95% ethanol, this means that 100g of the solution contains a total of 20 grams of dry matter and 80g of 95% ethanol.

[0045] As used herein, the term "krill meal" refers to a dry powder prepared from krill and having a moisture content of about 3% to about 15%.

[0046] The present invention provides marine lysophosphatidylcholine compositions for use in pharmaceuticals, dietary supplements and functional foods, as well as methods for making marine lysophosphatidylcholine (LPC) compositions. In a particularly preferred embodiment, the LPC composition is prepared from krill phospholipids, such as Euphausia superba or Euphausia pacifica phospholipids. In another preferred embodiment, the LPC composition is prepared from Calanus, herring, herring roe, or algae phospholipids. Suitable krill crude phospholipid extracts, desalted krill phospholipid extracts and methods for processing krill are disclosed in PCT / IB2016 / 000208 and PCT / IB2016 / 000326, each of which is incorporated herein by reference in its entirety.

[0047] Intact phospholipids such as phosphatidylcholine are usually converted to lysophospholipids in the small intestine by the action of phospholipase.Then, lysophospholipids are absorbed in the body.The data presented herein demonstrates that when LPC compositions are orally administered in animal models, their target omega-3 fatty acids in the composition are incorporated into multiple tissues at both a faster rate and in a higher total amount than when they are orally administered in PC (phosphatidylcholine) compositions that do not contain a comparable amount of lysophospholipids.This result is surprising in view of the fact that orally administered PC is known to be efficiently converted to lysophospholipids in the intestine, so no difference would be expected.

[0048] 1. Starting materials The present invention is not limited to the use of a particular biological starting material. In preferred embodiments, a phospholipid extract is prepared from the biological starting material, and the LPC composition is made by enzymatic treatment of the phospholipid extract. The biological starting material may preferably be or may be produced from an algal biomass, a plant biomass, or a marine animal biomass. In preferred embodiments, a marine animal biomass is utilized as the starting material. Suitable marine animal biomass includes, but is not limited to, krill, crab, Calanus, plankton, eggs, crayfish, shrimp, fish, especially herring, and mollusks. The biological starting material may be either fresh or frozen, or may be a material produced from algal, plant, or marine animal biomass, such as a meal, powder, hydrolysate, or coagulate (paste). The paste may be a wet paste or a dry paste. In some preferred embodiments, the biological starting material is krill material, such as krill meal, krill hydrolysate, krill coagulate, or fresh or frozen krill. Any species of krill may be utilized. In a preferred embodiment, the krill is Euphausia superba or Euphausia pacifica.

[0049] In some particularly preferred embodiments, the biological starting material is krill meal. Krill meal may preferably be made by any standard marine meal process. Generally, krill meal is produced by cooking freshly caught krill at low temperature (about 80-85°C), drying to a moisture content of about 5-8% and then grinding. In embodiments where the product is intended for human consumption, it is preferred to package and store the meal under nitrogen without the addition of antioxidants.

[0050] Thus, the process of the present invention may be used with a wide variety of starting materials. In the remainder of the process discussion, reference is made generally to the use of krill meal as a starting material. However, it will be understood that any of the starting materials contemplated herein may be substituted for krill meal in the described process.

[0051] 2. Solvent extraction from krill meal In the first step of the extraction process, the krill meal is mixed with a suitable solvent to extract lipids from the meal. In contrast to prior art methods, the present invention utilizes conditions that preferably extract the maximum amount of lipids from the krill meal at the expense of increased amounts of contaminants in the initial solvent extract. In a preferred embodiment, the solvent is an organic protic solvent, although other solvents known for use in the extraction of food-grade lipids may also be used, such as acetone, hexane, etc. Suitable organic protic solvents include, but are not limited to, n-butanol, n-propanol, isopropanol, nitromethane, ethanol, and methanol. In a particularly preferred embodiment, the protic solvent is ethanol.

[0052] In a preferred embodiment, the concentration of the protic solvent used in the initial solvent extraction step is at least 90%, or preferably about 94%-98%, more preferably about 95%-97%, and most preferably about 96% (e.g., 96% ethanol or methanol).

[0053] In some embodiments, the protic solvent is mixed with the biological starting material in a ratio of protic solvent:biological starting material of about 1:1 to 10:1, preferably about 3:1 to 6:1, more preferably about 4:1 to 5:1, and most preferably about 4.4:1.

[0054] In preferred embodiments, the biological starting material is extracted with a protic solvent at a temperature of about 5° C. to about 65° C., about 20° C. to about 60° C., preferably about 30° C. to about 50° C., more preferably about 30° C. to about 50° C., and most preferably about 40° C. In some embodiments, the extraction time (i.e., the length of time the biological starting material is in contact with the solvent) is about 10 minutes to about 2 hours, preferably about 15 minutes to about 60 minutes, more preferably about 20 minutes to about 45 minutes, and most preferably about 30 minutes.

[0055] Following the extraction step, the crude krill lipid solution containing lipids derived from the krill meal is separated from the solvent / krill meal mixture, for example by decantation and / or filtration. The insoluble material, including proteins and other useful materials, is then dried to recover the ethanol. The remaining protein-rich meal product may then be used in food products, protein supplements, animal feed, and the like. In some embodiments, the decanted solution containing the soluble lipids has a dry matter content of about 4%-9% (w / w), preferably about 5.5%-7.5% (w / w), and most preferably about 6%-7% (w / w), where w / w refers to the weight of the dry matter as a percentage of the weight of the total solution. In preferred embodiments, the dry matter consists essentially of crude krill lipids, and preferably has a lipid content of more than 90%, 95%, 96%, 97%, 98% or 99% (w / w), where w / w refers to the weight percent of lipids of the total weight of the dry matter.

[0056] 3. Desalting and concentration In some embodiments, the crude krill lipid solution is desalted to remove hexane-insoluble materials such as insoluble inorganic salts (e.g., NaCl with small or trace amounts of KCl and / or AlCl3), as well as undesirable compounds such as trimethylamine oxide, and metals such as copper and arsenic. In some preferred embodiments, the LPC composition of the present invention is prepared from a desalted krill lipid composition. Although the method is described with reference to desalted krill lipids above, the method is generally applicable to any lipid fraction, including phospholipids.

[0057] In some embodiments, the crude krill lipid solution is desalted by evaporating the solvent from the crude krill lipid solution to provide a crude krill lipid composition, and then repeatedly washing the crude krill lipid composition with an aqueous solvent. Suitable aqueous solvents include, but are not limited to, ethanol mixed with water or deionized water such that the ethanol concentration is about 40%-70%, preferably about 50%-60%. In these embodiments, the crude krill lipid composition is mixed with the solvent, the lipid phase is recovered, and the aqueous phase is decanted. The washing steps may be repeated, for example, 1, 2, 3, 4, 5 or more times as necessary. The ratio of aqueous solvent:crude krill lipid composition is preferably about 1:1-1:5, more preferably about 1:1-2.5:1, and most preferably about 1:1.7 for each washing step.

[0058] In some embodiments, the crude lipid solution is desalted by chromatography. Suitable chromatographic media include silica gel media, including but not limited to spherical silica gel and derivatized silica gel such as C8 (octyl-functionalized silica) and C18 (octadecyl-functionalized silica), and ion exchange resins such as Dowex™ resin. In embodiments where chromatography is utilized, the crude krill lipid is applied to the chromatographic medium, preferably in a protic solvent, preferably the same solvent (e.g., ethanol) used in the initial extraction. Although standard column chromatographic methods may be utilized, it is preferred to utilize a moving bed chromatography or simulated moving bed chromatography apparatus. The present invention is not limited to a particular type of chromatographic purification apparatus. Indeed, in preferred embodiments, the chromatographic purification apparatus may be a column, a fixed bed apparatus, a simulated moving bed apparatus, or a moving bed apparatus. In some particularly preferred embodiments, the apparatus is a simulated moving bed (SMB) apparatus. Suitable SMB systems are disclosed, for example, in U.S. Pat. Nos. 9,556,116; 9,650,590; and 9,560,333, each of which is incorporated herein by reference in its entirety.

[0059] The dry matter basis composition of the desalted krill lipids may preferably be characterized as follows: In some embodiments, the desalted krill lipids preferably comprise between about 30% (w / w) and 50% (w / w) phospholipids, more preferably between about 35% (w / w) and about 45% (w / w) phospholipids, and most preferably about 40% (w / w) phospholipids, where w / w refers to the weight of phospholipids as a percentage of the total weight of the desalted krill lipid. In some embodiments, the desalted krill lipids preferably comprise between about 32% (w / w) and 52% (w / w) triglycerides, more preferably between about 36% (w / w) and about 48% (w / w) triglycerides, and most preferably about 42% (w / w) triglycerides, where w / w refers to the weight of triglycerides as a percentage of the total weight of the desalted krill lipid. In some embodiments, the desalted krill lipids preferably comprise between about 3% (w / w) and 13% (w / w) free fatty acids, more preferably between about 5% (w / w) and about 11% (w / w) free fatty acids, and most preferably between about 8% (w / w) free fatty acids, where w / w refers to the weight of the free fatty acids as a percentage of the total weight of the desalted krill lipid. In some embodiments, the desalted krill lipids preferably comprise between about 0.5% (w / w) and 5% (w / w) lysophospholipids, more preferably between about 0.8% (w / w) and about 3.2% (w / w) lysophospholipids, and most preferably between about 1.2% and 2.8% (w / w) lysophospholipids, where w / w refers to the weight of the lysophospholipids as a percentage of the total weight of the desalted krill lipid. In some embodiments, the desalted krill lipids preferably contain about 1% (w / w) inorganic salts, more preferably about 0.5% (w / w) inorganic salts, even more preferably about 0.2% (w / w) inorganic salts, and most preferably about 0.1% (w / w) inorganic salts, where w / w refers to the weight of inorganic salts as a percentage of the total weight of the desalted krill lipids. In some embodiments, the desalted krill lipids preferably contain less than about 5 mg N / 100 g, more preferably less than about 3 mg N / 100 g, even more preferably less than about 2 mg N / 100 g, and most preferably less than about 1 mg N / 100 g, where N content serves as a convenient proxy for trimethylamine oxide (TMAO) content.In some embodiments, the desalted krill lipids contain less than about 10 ppm copper (Cu. ++ ), more preferably less than about 5 ppm Cu ++ , and even more preferably less than about 2 ppm Cu ++ , and most preferably less than about 1 ppm Cu ++ In some embodiments, the desalted krill lipids contain less than about 10 ppm total arsenic (As 3+, organic and inorganic), more preferably less than about 5 ppm total arsenic, even more preferably less than about 3 ppm total arsenic, and most preferably less than about 1 ppm total arsenic. In some embodiments, the desalted krill lipids preferably contain between about 0.01% and 2% (w / w) ethyl esters, more preferably between about 0.01% and about 1.5% (w / w) ethyl esters, and most preferably between about 0.01% and about 1% (w / w) ethyl esters, where w / w refers to the weight of ethyl esters as a percentage of the total weight of the desalted krill lipids. In some embodiments, the krill phospholipid composition preferably comprises less than about 5%, 4%, 3% or 2% (w / w) ethyl esters, down to a limit of 0.01% ethyl esters (i.e., between 5% and 0.01% (w / w) ethyl esters, between 4% and 0.01% (w / w) ethyl esters, between 3% and 0.01% (w / w) ethyl esters, or between 2% and 0.01% (w / w) ethyl esters), more preferably less than about 1.5% (w / w) ethyl esters, and most preferably less than about 1% (w / w) ethyl esters, where w / w refers to the weight of ethyl esters as a percentage of the total weight of desalted krill lipids. In some embodiments, the desalted krill lipids have a conductivity of less than about 50 μS / cm when measured in 95% ethanol at 5% dry matter, more preferably less than about 30 μS / cm when measured in 95% ethanol at 5% dry matter, and most preferably less than about 20 μS / cm when measured in 95% ethanol at 5% dry matter. In some embodiments, the desalted krill lipids have a viscosity of about 50-800 mPas at 25° C., more preferably about 100-400 mPas at 25° C., and most preferably about 180-340 mPas at 25° C. In some embodiments, the desalted krill lipid composition has a pH of about 6.7-8.3 when measured in 95% ethanol.

[0060] In some preferred embodiments, the LPC compositions of the present invention are prepared from a phospholipid composition made from a desalted krill lipid composition. Although the method is described above with reference to desalted krill lipids, the method is generally applicable to any lipid fraction that contains phospholipids.

[0061] Thus, in some embodiments, the dry matter content of a lipid composition comprising phospholipids, such as the desalted krill lipid composition described above, is adjusted to a predetermined level by adding or removing solvent, and the resulting mixture is fractionated, such that the phospholipids partition primarily into one phase and the neutral lipids partition into another phase. In some embodiments, a lipid composition comprising phospholipids, such as the desalted krill lipids, is mixed with a suitable protic solvent, preferably ethanol, such that the dry matter (i.e., lipid) content of the resulting solution is about 10%-40% (w / w), preferably about 15%-35% (w / w), more preferably about 18%-30% (w / w), and most preferably about 20%-25% (w / w). In embodiments where the desalting step already provides lipids in a suitable protic alcohol solution, such as when ethanol is used as the chromatography solvent, the desalted krill lipid solution may preferably be evaporated to obtain the desired dry matter content, i.e., about 10% to 40% (w / w), preferably about 15% to 35% (w / w), more preferably about 18% to 28% (w / w), and most preferably about 20% to 22% (w / w). Suitable methods for evaporation include, but are not limited to, evaporation under reduced pressure (e.g., vacuum distillation), falling film evaporation, and removal of the solvent through a membrane.

[0062] After adjusting the dry matter content to the desired level by adding or removing solvent, the solution is fractionated into an upper light phase solution rich in phospholipid content and a lower heavy phase solution containing mainly neutral lipids and high levels of astaxanthin. Preferably, the temperature of the solution during the fractionation process is controlled. In some embodiments, the temperature of the fractionation process is about 0°C to about 20°C, preferably about 5°C to about 15°C, more preferably about 8°C to about 12°C, and most preferably about 10°C.

[0063] In some embodiments, the concentration of the protic solvent may be varied to control the phospholipid concentration in the lipid composition of the upper phase. In some embodiments, the protic solvent has a concentration of about 55% to 100%, more preferably about 65% to 98%. In some embodiments, the protic solvent has a concentration of about 90% to 100%, more preferably about 92% to 98%, and most preferably about 95%. In these embodiments, the phospholipid content of the lipids of the upper phase after fractionation on a dry matter basis is about 50% to 70% (w / w), preferably about 55% to 65% (w / w), and most preferably about 60% (w / w). In yet other preferred embodiments, the protic solvent has a concentration of about 80% to 90% (w / w), more preferably about 82% to 88% (w / w), and most preferably about 85% (w / w). In these embodiments, the phospholipid content on a dry matter basis of the lipids in the upper phase after fractionation is about 70%-90% (w / w), preferably about 75%-85% (w / w), and most preferably about 80% (w / w).

[0064] In some embodiments, the upper and lower phases are separated by centrifugation, preferably low-temperature centrifugation using a two- or three-phase separator. In some embodiments, the centrifugation is performed at about 0° C. to about 30° C., more preferably about 0° C. to about 10° C., and most preferably about 3° C. to about 7° C. In general, the gravitational force utilized depends on the delta T between the phases. The lower the temperature, the larger the delta T obtained. In some preferred embodiments, the G-force used for separation is about 8000×G to about 15000×G.

[0065] In some embodiments, the process step of adjusting the dry matter content described above via a centrifugation step is repeated one or more times.

[0066] In some embodiments, the upper light phase is collected and further processed. The solvent is preferably removed from the upper phase by one or more evaporation steps to obtain a krill phospholipid composition. The krill phospholipid composition preferably comprises about 50%-85% (w / w) phospholipids, and more preferably about 55%-80% (w / w) phospholipids, where w / w refers to the weight of phospholipids as a percentage of the total weight of the composition.

[0067] In some embodiments, the lower heavy phase is collected and further processed. In some embodiments, the solvent is removed from the lower phase to obtain a krill neutral lipid composition. In some embodiments, the lower phase may be fractionated with a protic solvent and subjected to a second centrifugation step to recover additional phospholipids not recovered in the first fractionation step. Again, the solvent is removed from the resulting lower phase to obtain a krill neutral lipid composition. The krill neutral lipid composition in both cases is characterized by containing high levels of astaxanthin. In some embodiments, the krill neutral lipid composition may be combined or blended with a krill phospholipid composition to obtain a lipid composition with desired levels of phospholipids, neutral lipids, and astaxanthin. In some embodiments, the krill neutral lipid composition may be further processed (e.g., by chromatography) to obtain an astaxanthin composition. The astaxanthin composition may then be combined or blended with a krill phospholipid composition to obtain a lipid composition with desired levels of phospholipids and astaxanthin.

[0068] In some embodiments, the process further comprises adding a triglyceride oil, such as a medium chain triglyceride oil or a long chain triglyceride oil, at any stage during the process. For example, triglyceride oil may be added to the collected light phase, heavy phase, phospholipid composition, or neutral lipid composition. In some embodiments, the process steps of adjusting the dry matter content described above via centrifugation and / or evaporation steps are repeated one or more times.

[0069] In some embodiments, the krill phospholipid and neutral lipid composition are produced by further chromatography steps. In these embodiments, at least a portion of the desalted lipid-rich stream is introduced into a polar liquid extraction zone comprising a fixed bed adsorber comprising a macroporous styrene polymer bead-type resin effective for adsorbing neutral lipids. In preferred embodiments, the fixed bed chromatography step provides a polar lipid extract stream comprising solvent and at least 50% polar lipid by weight on a dry basis. In some preferred embodiments, the fixed bed adsorber is intermittently regenerated with a hot ethanol stream at an elevated regeneration temperature of about 40° C. to about 60° C. to obtain a neutral lipid raffinate stream comprising solvent, neutral lipids and astaxanthin. In some embodiments, solvent is recovered from the polar lipid extract stream to obtain the krill phospholipid composition and from the neutral lipid raffinate stream to obtain the neutral lipid composition. These processes are described in detail in co-pending U.S. patent application Ser. No. 14 / 619,102, which is incorporated herein by reference in its entirety.

[0070] In some embodiments, the krill phospholipid composition on a dry matter basis preferably comprises about 5% (w / w) to 35% (w / w) triglycerides, more preferably about 10% (w / w) to about 30% (w / w) triglycerides, and most preferably about 15% to 25% (w / w) triglycerides, where w / w refers to the weight of triglycerides as a percentage of the total weight of the krill phospholipid composition. In some embodiments, the krill phospholipid composition preferably comprises about 2% (w / w) to 13% (w / w) free fatty acids, more preferably about 4% (w / w) to about 11% (w / w) free fatty acids, and most preferably about 4% to 10% (w / w) free fatty acids, where w / w refers to the weight of free fatty acids as a percentage of the total weight of the krill phospholipid composition. In some embodiments, the krill phospholipid composition preferably comprises about 0.5% (w / w) to 10% (w / w) lysophospholipids, more preferably about 0.8% (w / w) to about 7.0% (w / w) lysophospholipids, and most preferably about 5.0% or 3.0% (w / w) lysophospholipids, where w / w refers to the weight of the lysophospholipids as a percentage of the total weight of the krill phospholipid composition. In some embodiments, the krill phospholipid composition preferably comprises about 1% (w / w) inorganic salts, more preferably about 0.5% (w / w) inorganic salts, even more preferably about 0.2% (w / w) inorganic salts, and most preferably about 0.1% or 0.05% (w / w) inorganic salts, where w / w refers to the weight of the inorganic salts as a percentage of the total weight of the krill phospholipid composition. In some embodiments, the krill phospholipid composition preferably comprises less than about 5 mg N / 100 g, more preferably less than about 3 mg N / 100 g, even more preferably less than about 2 mg N / 100 g, and most preferably less than about 1 mg N / 100 g, where N content serves as a convenient proxy for trimethylamine oxide (TMAO) content. In some embodiments, the krill phospholipid composition comprises less than about 10 ppm copper (Cu ++ ), more preferably less than about 5 ppm Cu ++ , and even more preferably less than about 2 ppm Cu ++ , and most preferably less than about 1 ppm Cu++ In some embodiments, the krill phospholipid composition contains less than about 10 ppm total arsenic (As 3+ ), more preferably less than about 5 ppm total arsenic, even more preferably less than about 3 ppm total arsenic, and most preferably less than about 1 ppm total arsenic. In some embodiments, the krill phospholipid composition preferably comprises between about 0.01% and 2% (w / w) ethyl esters, more preferably between about 0.01% and about 1.5% (w / w) ethyl esters, and most preferably between about 0.01% and about 1% (w / w) ethyl esters, where w / w refers to the weight of the ethyl esters as a percentage of the total weight of the krill phospholipid composition. In some embodiments, the krill phospholipid composition preferably comprises less than about 5%, 4%, 3% or 2% (w / w) ethyl esters, down to a limit of 0.01% ethyl esters (i.e., between 5% and 0.01% (w / w) ethyl esters, between 4% and 0.01% (w / w) ethyl esters, between 3% and 0.01% (w / w) ethyl esters, or between 2% and 0.01% (w / w) ethyl esters), more preferably less than about 1.5% (w / w) ethyl esters, and most preferably less than about 1% (w / w) ethyl esters, where w / w refers to the weight of ethyl esters as a percentage of the total weight of the krill phospholipid composition. In some embodiments, the krill phospholipid composition has a conductivity of less than about 50 μS / cm when measured in 95% ethanol, 5% dry matter, more preferably less than about 30 μS / cm when measured in 95% ethanol, 5% dry matter, and most preferably less than about 20 μS / cm, about 10 μS / cm, about 5 μS / cm, or about 1 μS / cm when measured in 95% ethanol, 5% dry matter. In some embodiments, the krill phospholipid composition has a viscosity of about 400-2000 mPas at 35° C., more preferably about 500-1800 mPas at 35° C., and most preferably about 600-1600 mPas at 35° C. In some embodiments, the krill phospholipid composition has a pH of about 6.7-8.3 when measured in 95% ethanol.

[0071] 4. Generation of LPC Composition In some preferred embodiments, the LPC composition of the present invention is prepared from the above phospholipid sources. In some preferred embodiments, the present invention provides a method for producing a lysophosphatidylcholine (LPC) composition with high EPA and DHA content from a marine or other raw material containing phospholipids, comprising treating the marine raw material with a phospholipase that is not native to the marine raw material to obtain a phospholipase-treated raw material, and fractionating the phospholipase-treated raw material to obtain a lipid composition with a higher lysophosphatidylcholine content than the starting raw material. In some preferred embodiments, the raw material is selected from the group consisting of a krill lipid preparation, a herring lipid preparation, a herring roe lipid preparation, an algae lipid preparation, and a Calanus lipid preparation, thereby obtaining a krill LPC composition, a herring LPC composition, a herring roe LPC composition, an algae LPC composition, or a Calanus LPC composition. In some particularly preferred embodiments, the krill lipid preparation is a Euphausia superba lipid preparation. In some preferred embodiments, the raw material has an EPA and DHA content in the following ranges; EPA: 1-70% by weight and DHA: 1-70% by weight, more preferably EPA: 5-70% by weight and DHA: 5-70% by weight, and most preferably EPA: 10-60% by weight and DHA: 10-60% by weight.

[0072] In some preferred embodiments, the feedstock is contacted with a phospholipase in a solvent. The present invention is not limited to the use of any particular phospholipase. In some embodiments, the phospholipase is phospholipase A1 (PLA1). In some particularly preferred embodiments, the enzyme is LECITASE™ Ultra, QUARA™ LowP. In some embodiments, the solvent is a mixture of water and alcohol. In some preferred embodiments, the alcohol is ethanol. In further preferred embodiments, the feedstock is contacted with a phospholipase in a mixture of about 85% water and 15% ethanol. In some particularly preferred embodiments, the enzyme is phospholipase A1 (PLA1) and the enzyme concentration is in the range of 0.1-20% (volume / weight), preferably 0.1-15% (volume / weight), more preferably 0.1-10% (volume / weight), even more preferably 0.1-5% (volume / weight), and most preferably 0.1-3% (volume / weight). In some preferred embodiments, the enzyme is phospholipase A1 (PLA1) and the process is carried out at a pH of 3 to 12, preferably 4 to 10, more preferably 4 to 9, and most preferably 5 to 9. In some preferred embodiments, the enzyme is phospholipase A1 (PLA1) and the process is carried out at 4 to 95°C, preferably 4 to 85°C, more preferably 10 to 80°C, even more preferably 15 to 70°C, even more preferably 15 to 65°C, and most preferably 15 to 60°C.

[0073] In some preferred embodiments, the LPC composition of the present invention is prepared by additional solvent concentration and / or chromatographic separation procedures. In some preferred embodiments, the enzyme-treated LPC composition is concentrated by phase separation in a polar / non-polar solvent system. In some preferred embodiments, the enzyme-treated LPC composition is mixed with a solvent system containing equal amounts of a polar solvent (e.g., methanol) and a non-polar solvent (e.g., heptane). The resulting mixture is stirred and the phases are allowed to separate. The polar lipids (e.g., LPC and PC) partition into the polar phase. In some embodiments, the polar phase is removed, washed with heptane, and the polar phase is recovered. Then, in some preferred embodiments, the polar phase is evaporated to dryness to obtain a concentrated LPC composition. In some embodiments, the LPC composition may be further concentrated by a chromatographic procedure, for example, flash chromatography using silica 60 gel, followed by evaporation to dryness.

[0074] The above method produces an LPC composition having preferred characteristics and / or properties. In some preferred embodiments, the LPC composition has an EPA and DHA content in the following ranges: EPA: 1-100% by weight and / or DHA: 1-100% by weight, more preferably EPA: 5-100% by weight and / or DHA: 5-100% by weight, and most preferably EPA: 10-90% by weight and / or DHA: 10-90% by weight. In some further preferred embodiments, the LPC composition has an LPC content in the range of 10-100% by weight, preferably 20-100% by weight, more preferably 30-100% by weight, even more preferably 40-100% by weight, and most preferably 50-100% by weight.

[0075] In yet another preferred embodiment, the LPC composition obtained by the process comprises: The composition is characterized by comprising about 10% to about 100% LPC (w / w), or 60% to 100% LPC, 70% to 90% LPC, 20% to 50% LPC, 20% to 40% LPC, 20% to 30% LPC, or 10% to 30% LPC, and by having an omega-3 fatty acid content of 5% to 60% (w / w) of the composition, 5% to 50% (w / w) of the composition, 5% to 40% of the composition, or 5% to 30% of the composition, or 20% to 60% (w / w) of the composition, 20% to 50% (w / w) of the composition, or 20% to 40% (w / w) of the composition, or 30% to 50% (w / w) of the composition. Those skilled in the art will recognize that the omega-3 fatty acid content includes the content of omega-3 fatty acyl chains that are linked to the phospholipids and glycerol molecules in the composition by ester or ether bonds. The weight percent of omega-s fatty acyl groups in the composition is preferably: 1 H-NMR or 13 The omega-3 fatty acid content may be determined by other suitable NMR techniques, including C-NMR. Alternatively, the omega-3 fatty acid content may be expressed in grams per 100 grams of fatty acid, as analyzed by gas chromatography, as known in the art. In these embodiments, the lysophospholipid composition preferably contains 10-50g / 100g omega-3 fatty acid, most preferably 20-40g / 100g omega-3 fatty acid, where grams per 100 grams is the weight of omega-3 fatty acid per 100 grams of total fatty acid, as measured by gas chromatography.

[0076] In preferred embodiments, the lysophospholipid composition further has one or more of the following characteristics or properties: a) a ratio of 2-LPC:1-LPC of 1:8 to 18:1, and more preferably 1.2:1 to 8:1, 1.2:1 to 4:1, 1.2:1 to 2:1, 4:1 to 10:1; or 5:1 to 12:1; b) a phosphatidylcholine (PC) content of between 0.5% and 10% (w / w) of the composition, more preferably less than 10%, 9%, 8%, 7%, 6% or 5% (w / w) of the composition; c) a phosphatidylethanolamine (PE) content of less than 1.2%, 1.1%, 1.0%, 0.7%, or 0.5% (w / w) of the composition; d) a neutral lipid content of about 5% to 65% (w / w) of the composition, or more preferably about 45% to 65% (w / w) or 15% to 35% (w / w); e) the ratio of 2-LPC:2-LPC ether is between 15:1 and 50:1, more preferably between 50:1 and 25:1, and the composition preferably contains less than 5%, 4%, 3%, 2%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1% 2-LPC ether; and / or f) EPA:DHA ratio is 1:1~3:1, DHA:EPA ratio is 1:1~5:1.

[0077] In some preferred embodiments, the composition comprises 20%-40% or 23%-35% LPC (w / w) of the composition. In some preferred embodiments, the composition comprises 40%-60%, 40%-70%, or 50%-70% LPC (w / w) of the composition. In some preferred embodiments, the composition comprises 70%-90% LPC (w / w) of the composition. With respect to property (f), if the LPC composition is prepared from krill phospholipids, the LPC composition will preferably comprise an EPA:DHA ratio of 1:1-3:1, more preferably 1.5:1-2:5:1. With respect to property (f), if the LPC composition is prepared from a marine source other than krill (e.g., herring, herring roe, or seaweed), the LPC composition will preferably comprise a DHA:EPA ratio of 1:1-5:1, more preferably 2:1-4:1, and most preferably 2.5:1-3.5:1.

[0078] In some preferred embodiments, the composition has an omega-3 fatty acid content of 35%-45% (w / w) of the composition. In some preferred embodiments, the composition has characteristics (a), (b), (c), (d), (e) or (f) or a combination of characteristics, such as characteristics (a) and (b); (a) and (c); (a) and (d); (a) and (e); (a) and (f); (b) and (c); (b) and (d); (b) and (e); (b) and (f); (c) and (d); (c and (e); (c) and (f); (d). ) and (e);(d) and (f);(e) and (f);(a), (b) and (c);(a), (b) and (d);(a), (b) and (e);(a), (b) and (f);(a), (c) and (d);(a), (c) and (e);(a), (c) and (f);(a), (d) and (e);(a), (d) and (f);(b), (c) and (d);(b), (c) and (e);(b)(c) and)f);(b), ( d), and (e);(b), (d), and (f);(c), (d), and (e);(c), (d), and (f);(a), (b), (c), and (d);(a), (b), (c), and (e);(a), (b), (c), and (f);(b), (c), (d), and (e);(b), (c), (d), and (f);(a), (c), (d), and (e);(a), (c), (d), and (e);(a), (c), (d), and (f);(a), (b), (d) and (e);(a), (b), (d) and (f);(a), (b), (c), (d), and (e);(a), (b), (c), (d), and (f);(b), (c), (d), (e), and (f);(a), (b), (d), (e), and (f);(a), (b), (c), (e), and (f);(a), (b), (c), (d), (e), and (f), and (a), (b), (c), (d), (e), and (f), and other possible combinations. In some preferred embodiments, the composition has two or more of the characteristics of (a), (b), and (c). In some preferred embodiments, the composition has two or more of the characteristics of (a), (b), (c), (d), and (e). In some preferred embodiments, the composition has three or more of characteristics (a), (b), (c), (d), (e) and (f).In some preferred embodiments, the composition has four or more of properties (a), (b), (c), (d), and (e). In some preferred embodiments, the composition has five or more of properties (a), (b), (c), (d), (e), and (f).

[0079] 5. Preparation of LPC Compositions In some preferred embodiments, the present invention provides a pharmaceutical or dietary supplement composition comprising the above LPC composition and a physiologically acceptable carrier. In some preferred embodiments, the physiologically acceptable carrier is a lipid carrier. In some preferred embodiments, the present invention provides an oral delivery vehicle containing the marine LPC composition, pharmaceutical or dietary supplement composition as described herein above.

[0080] In some preferred embodiments, the present invention provides a lipid composition comprising a lipid fraction and a second lipid fraction, the first lipid fraction being a marine LPC composition as described herein above, the second lipid fraction being obtained from a different source than the first lipid fraction and / or comprising less than 20% LPC. In some preferred embodiments, the second lipid fraction is selected from the group consisting of a triglyceride fraction, a diglyceride fraction, a fatty acid ethyl ester fraction, a free fatty acid fraction and combinations thereof. In some preferred embodiments, the second lipid fraction is a marine lipid fraction comprising EPA and / or DHA. In some preferred embodiments, the present invention provides a pharmaceutical or nutraceutical composition comprising the lipid composition as described and a physiologically acceptable carrier. In some preferred embodiments, the present invention provides an oral delivery vehicle comprising the lipid composition just described.

[0081] The LPC composition of the present invention is preferably administered orally. Thus, in some embodiments, the composition of the present invention (such as those described in the previous section) is included in an acceptable excipient and / or carrier for oral ingestion. The actual form of the carrier, and therefore the composition itself, is not important. The carrier may be a liquid, gel, gelcap, capsule, powder, solid tablet (coated or uncoated), tea, etc. The composition is preferably in the form of a tablet or capsule, and most preferably in the form of a softgel capsule. Suitable excipients and / or carriers include vegetable oils, fish oils, krill oils, maltodextrin, calcium carbonate, dicalcium phosphate, tricalcium phosphate, microcrystalline cellulose, glucose, rice flour, magnesium stearate, stearic acid, croscarmellose sodium, sodium starch glycolate, crospovidone, sucrose, vegetable gums, lactose, methylcellulose, povidone, carboxymethylcellulose, corn starch, and the like (including mixtures thereof). Preferred carriers include calcium carbonate, magnesium stearate, maltodextrin, and mixtures thereof. The various ingredients and excipients and / or carriers are mixed and formed into the desired form using conventional techniques. The tablet or capsule of the present invention may be coated with an enteric coating that dissolves at a pH of about 6.0 to 7.0. A suitable enteric coating that dissolves in the small intestine but not in the stomach is cellulose acetate phthalate. Further details of techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).

[0082] In some embodiments, the LPC composition is formulated for oral administration with flavoring or sweetening agents. Examples of useful flavoring agents include, but are not limited to, pure anise extract, imitation banana extract, imitation cherry extract, chocolate extract, pure lemon extract, pure orange extract, pure peppermint extract, imitation pineapple extract, imitation rum extract, imitation strawberry extract, or pure vanilla extract; or volatile oils, such as balm oil, bay oil, bergamot oil, cedarwood oil, walnut oil, cherry oil, cinnamon oil, clove oil, or peppermint oil; peanut butter, chocolate flavor, vanilla cookie crumb, butterscotch, or toffee. In one embodiment, the dietary supplement comprises cocoa or chocolate. An emulsifier may be added for the stability of the final product. Examples of suitable emulsifiers include, but are not limited to, lecithin (e.g., from egg or soybean), and / or mono- and diglycerides. Other emulsifiers will be readily apparent to one of skill in the art, and the selection of an appropriate emulsifier will depend, in part, on the formulation and final product. In addition to the carbohydrates listed above, the dietary supplement may also contain natural or artificial (preferably low calorie) sweeteners, such as sugars, cyclamate, aspartamine, aspartame, acesulfame K, and / or sorbitol.

[0083] The LPC compositions of the present invention may also be delivered as a dietary supplement, nutraceutical, or functional food.

[0084] Dietary supplements may contain one or more inactive ingredients, especially when it is desirable to limit the number of calories added to the diet by dietary supplements.For example, dietary supplements of the present invention may also contain optional ingredients, including, for example, herbs, vitamins, minerals, enhancers, colorants, sweeteners, flavorings, inactive ingredients, etc.For example, dietary supplements of the present invention may contain one or more of the following: ascorbates (ascorbic acid, inorganic ascorbates, rosehip, acerola, etc.), dehydroepiandrosterone (DHEA), green tea (polyphenols), inositol, kelp, dulse, bioflavonoids, maltodextrin, nettle, niacin, niacinamide, rosemary, selenium, silica (silicon dioxide, silica gel, horsetail, shaved grass, etc.), spirulina, zinc, etc.Such optional ingredients may be in either naturally occurring or concentrated form.

[0085] In some embodiments, the dietary supplement further comprises vitamins and minerals, including but not limited to calcium phosphate or calcium acetate, tribasic; potassium phosphate, dibasic; magnesium sulfate or oxide; salt (sodium chloride); potassium chloride or potassium acetate; ascorbic acid; ferric orthophosphate; niacinamide; zinc sulfate or oxide; calcium pantothenate; copper gluconate; riboflavin; beta-carotene; pyridoxine hydrochloride; thiamine mononitrate; folic acid; biotin; chromium chloride or picolinate; potassium iodide; sodium selenate; sodium molybdate; phylloquinone; vitamin D3; cyanocobalamin; sodium selenate; copper sulfate; vitamin A; vitamin C; inositol; potassium iodide. The appropriate dosage of vitamins and minerals can be obtained, for example, by referring to the US RDA guidelines.

[0086] In another embodiment, the present invention provides a nutritional supplement (e.g., an energy bar or meal replacement bar or beverage) comprising the LPC composition of the present invention. In a preferred embodiment, the nutritional supplement comprises an effective amount of the ingredients as described above. The nutritional supplement functions as a meal or snack replacement and generally provides nutritional calories. Preferably, the nutritional supplement provides carbohydrates, proteins, and fats in balanced amounts. The nutritional supplement may further comprise carbohydrates, simple carbohydrates, medium chain length, or polysaccharides, or a combination thereof. Simple sugars may be selected for desirable organoleptic properties. Uncooked cornstarch is an example of a complex carbohydrate. If it is desired to maintain a high molecular weight structure, complex carbohydrates need only be included in food formulations or portions thereof that are not cooked or heat treated, since heating breaks them down into simple carbohydrates (where simple carbohydrates are monosaccharides or disaccharides). The nutritional supplement, in one embodiment, comprises a combination of carbohydrate sources of three levels of chain length (simple, medium, and complex; e.g., sucrose, maltodextrin, and uncooked cornstarch).

[0087] In yet another embodiment, the present invention provides a food, prepared food, or food (i.e., functional food) comprising the LPC composition of the present invention. In a preferred embodiment, the food comprises an effective amount of the ingredients as described above. For example, in some embodiments, beverages and solid or semi-solid foods comprising fatty acids or their derivatives are provided. These forms may include, but are not limited to, beverages (e.g., soft drinks, milk and other dairy drinks, and diet drinks), baked goods, puddings, dairy products, confectionery, snack foods, or frozen confectionery or novelty items (e.g., ice cream, milkshakes), prepared frozen foods, candies, snack products (such as chips), soups, spreads, sauces, salad dressings, cooked meat products, cheese, yogurt, and other fat- or oil-containing foods, and food ingredients (e.g., flour).

[0088] In some preferred embodiments, the LPC composition is incorporated into a chewable matrix. Preferred chewable matrices are jelly candies and gelatin-based gummy candies. Exemplary gummy candies include gummy bears, gummy worms, gummy frogs, gummy burgers, gummy cherries, gummy soda bottles, gummy sharks, gummy army men, gummy hippopotami, gummy lobsters, gummy watermelons, gummy octopuses, gummy apples, gummy peaches, and gummy oranges. The terms "gummy" and "gummy" are used interchangeably herein.

[0089] In some embodiments, the present invention provides a composition comprising the LPC composition described above and one or more additional omega-3 fatty acid derivatives or free fatty acids. The omega-3 fatty acid derivatives or free fatty acids may be derived from a neutral lipid extract or may be derived from an additional source such as a fish oil or an omega-3 ester composition. In some embodiments, the one or more additional omega-3 fatty acid derivatives are selected from omega-3 esters and glycerides. For example, in some embodiments, the composition may comprise about 1% to about 60% (w / w) of the krill oil composition (i.e., weight of phospholipid compounds / total weight of the composition), with the remaining 99% to 40% (w / w) of the composition being omega-3 glycerides, esters, or free fatty acids, or combinations thereof (i.e., omega-3 glycerides, esters, or free fatty acids, or combinations thereof / total weight of the composition). In some embodiments, the composition may comprise about 5% to about 60% (w / w) of phospholipids, with the remaining 95% to 40% (w / w) of the composition being omega-3 glycerides, esters, or free fatty acids, or combinations thereof. In some embodiments, the composition may comprise about 20% to about 60% (w / w) phospholipids, with the remaining 80% to 40% (w / w) of the composition being omega-3 glycerides, esters, or free fatty acids, or combinations thereof. In some embodiments, the composition may comprise about 30% to about 60% (w / w) phospholipids, with the remaining 70% to 40% (w / w) of the composition being omega-3 glycerides, esters, or free fatty acids, or combinations thereof. In some embodiments, the composition may comprise about 40% to about 60% (w / w) phospholipids, with the remaining 60% to 40% (w / w) of the composition being omega-3 glycerides, esters, or free fatty acids, or combinations thereof. In some embodiments, the composition may comprise about 50% to about 60% (w / w) phospholipids, with the remaining 50% to 40% (w / w) of the composition being omega-3 glycerides, esters, or free fatty acids, or combinations thereof.

[0090] The LPC composition of the present invention may further be incorporated into animal and fish feeds and rations. Many different feed rations can be formulated for animals and fish from many different feed ingredients. Rations are generally formulated to provide nutrients according to National Research Council standards. The feeds used in the ration are selected according to market prices and availability. Thus, some components of the ration may change over time. In the feed of the present invention, the ration always contains the LPC composition of the present invention in an amount preferably between 0.1% and 50%, between 0.5% and 50%, between 1.0% and 40%, between 1.0% and 30%, between 1.0% and 20%, between 1.0% and 10%, between 0.1% and 10%, or between 0.5% and 5% of the total fat of the ration. For a discussion of feed ration formulations, actual rations, and NRC guidelines, see Church, Livestock Feeds and Feeding, O&B Books, Inc., Corvallis, Oreg. (1984) and Feeds and Nutrition Digest, Ensminger, Oldfield and Heineman eds., Ensminger Publishing Corporation, Clovis, Calif. (1990), which are incorporated herein by reference.

[0091] The animal feed rations of the present invention may be characterized according to NRC requirements, which may be found in Church, Livestock Feeds and Feeding, O&B Books, Inc., Corvallis, Oreg. (1984), or other nutritional standards. Animal and fish rations are traditionally balanced using protein and energy requirements, and then adjusted as necessary to meet other requirements. The animal and fish feeds of the present invention contain about 0.05% to 5% lipids and other feed materials necessary to balance the diet to meet the NRC requirements (or other recognized requirements) for the various stages of growth and maintenance.

[0092] The relative amounts of protein and energy are adjusted to reflect standard requirements. The amounts of feed ingredients vary depending on the stage of the animal being fed. Rations for growing young animals and fish have higher protein levels, while final rations for finishing animals for market have higher energy values, supplied by carbohydrates. For example, pre-starter, starter, and grower-finisher rations for various animals typically contain about 20-24% protein, 18-20% protein, and 13-17% protein, respectively. In some feeding situations, care must be taken to provide adequate amino acid and overall protein content. Energy requirements may also be met by adding fat to the ration. In the present invention, the lysophospholipid composition provides a portion of the energy requirement.

[0093] A typical salmon ration of the invention will contain up to about 5%-65% fish meal and / or krill meal, 5%-30% vegetable oil and 5%-15% fish oil (expressed as a % of weight of ingredients / weight of ration (% w / w)) and 0.5%-5% of the LPC composition of the invention, resulting in a total fat content of the ration of 10%-45% (w / w). In some embodiments, the ration has a crude protein content of about 32%-46%, preferably about 36%-42%, a crude lipid content of about 26%-42%, preferably about 28%-38%, a carbohydrate (NFE) content of about 11%-18%, preferably about 13%-15%, a fiber content of about 1%-5%, preferably about 1.5%-2.5%, an ash content of about 4%-7%, preferably about 4.5%-6.5%, a total phosphorus content (P) of about 0.5%-1.1%, preferably about 0.6%-1.0%, a total energy content of about 20-30 MJ / kg, preferably about 23-28 MJ / kg, and a digestible energy content of about 20-24 MJ / kg.

[0094] Other ingredients may be added to the feed ration. These ingredients include, but are not limited to, mineral supplements such as calcium, phosphorus, salt, selenium, and zinc; vitamin supplements such as vitamins A, B, D, E, and K; amino acid supplements such as lysine; coccidiostats (except for swine feed) or growth promoters such as bacitracin or virginamycin; and other active drugs such as chlortetracycline, sulfathiozole, and penicillin. For formulations of vitamin, mineral, and antibiotic supplements, see Church, Livestock Feeds and Feeding, O&B Books, Inc., Corvallis, Oreg. (1984).

[0095] In a preferred embodiment, the lysophospholipid composition is incorporated into pelleted feed for administration to livestock. Pelleted feed is made by first mixing the feed ingredients, then compressing and extruding the feed ingredients through a die under heat and pressure. The feed is pelleted by methods known in the art, as described in MacBain, Pelleting Animal Feed, American Feed Manufacturers Association, Arlington, Va. (1974), which is incorporated herein by reference. When incorporating additional fat into pelleted feed, care must be taken not to create chalky pellets. Generally, only about 2% of fat is added during pelleting, and the rest is added after the pellets have cooled.

[0096] Oils and feeds containing oils can be stabilized by the addition of antioxidants. Antioxidants may therefore be added as chemical preservatives in accordance with FDA regulations such as those listed in the 1997 Official Publication, Association of Feed Control Officials Incorporated (1997), which is incorporated herein by reference. Suitable antioxidants include, but are not limited to: lecithin, tocopherol, ascorbate, ascorbyl palmitate, and spice extracts such as rosemary extract.

[0097] Feed is formulated as above and adjusted to the requirements of the animal being fed according to NRC guidelines. For example, feed may be formulated for dogs, cats, poultry, cattle, shrimp, and fish such as salmon, trout, catfish, and tilapia. Various feed formulations, balancing methods, and requirements for these animals are discussed in Church, Livestock Feeds and Feeding, O&B Books, Inc., Corvallis, Oreg. (1984) and Feeds and Nutrition Digest, Ensminger, Oldfield and Heineman eds., Ensminger Publishing Corporation, Clovis, Calif. (1990), which are incorporated herein by reference.

[0098] 6. Uses of Krill Phospholipid Compositions In some embodiments, the lysophospholipid compositions of the present invention are provided for use in increasing the amount of EPA and / or DHA in a target tissue or organ by oral administration of the lysophospholipid composition. Preferred target tissues and organs according to the present invention are the adrenal gland, blood, bone, bone marrow, brain, adipose (white), kidney (whole), large intestine mucosa, liver, lung, muscle, cardiac muscle, pancreas, pituitary gland, prostate, skin, small intestine mucosa, spleen, gastric mucosa, testis, thymus, and / or thyroid gland.

[0099] In some embodiments, an effective amount of the compound or composition is administered to a subject in need thereof to treat, prevent, or improve cognitive and / or cognitive diseases, disorders, or deficiencies (memory, concentration, learning (deficits)), or to treat or prevent neurodegenerative disorders. In some embodiments, the cognitive disease, disorder, or deficiency is attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), autism / autism spectrum disorder (ASD), (dyslexia, age-related memory and learning disorders, amnesia, mild cognitive impairment, cognitively impaired non-dementia, pre-Alzheimer's disease, Alzheimer's disease, epilepsy, Pick's disease, Huntington's disease, Parkinson's disease, Lou Gehrig's disease, pre-dementia, dementia with Lewy bodies, dentatorubral-pallidoluysian atrophy, Friedrich's ataxia, multiple system atrophy, types 1, 2, 3, 6,7 Spinocerebellar ataxia, amyotrophic lateral sclerosis, familial spastic paraplegia, spinal muscular atrophy, spinal and bulbar muscular atrophy, age-related cognitive decline, cognitive decline, moderate psychiatric disorder, mental deterioration as a result of aging, conditions affecting brainwave intensity and / or cerebral glucose utilization, stress, anxiety, concentration and attention disorders, mood deterioration, general cognitive and mental health, neurodevelopmental disorders, neurodegenerative disorders, hormonal disorders, neurological imbalances, or any combination thereof. In certain embodiments, the cognitive disorder is a memory disorder.

[0100] In some embodiments, an effective amount of the compound or composition is administered to a subject in need thereof to treat or prevent cardiovascular disorder or metabolic syndrome.In some embodiments, cardiovascular disorder is atherosclerosis, arteriosclerosis, coronary heart (carotid) disease (CHD or CAD), acute coronary syndrome (or ACS), valvular heart disease, aortic and mitral valve disorders, arrhythmia / atrial fibrillation, cardiomyopathy and heart failure, angina, acute myocardial infarction (or AMI), hypertension, orthostatic hypotension, shock, embolism (pulmonary and venous), endocarditis, diseases of the arteries, aorta and its branches, disorders of the peripheral vascular system (peripheral arterial disease or PAD), Kawasaki disease, congenital heart disease (cardiovascular disease) and stroke (cerebrovascular disease), dyslipidemia, hypertriglyceridemia, hypertension, heart failure, cardiac arrhythmia, low HDL level, high LDL level, stable angina, The indication is selected from coronary heart disease, acute myocardial infarction, secondary prevention of myocardial infarction, cardiomyopathy, endocarditis, type 2 diabetes mellitus, insulin resistance, impaired glucose tolerance, hypercholesterolemia, stroke, hyperlipidemia, hyperlipoproteinemia, chronic kidney disease, intermittent claudication, hyperphosphatemia, omega-3 deficiency, phospholipid deficiency, carotid artery sclerosis, peripheral arterial disease, diabetic nephropathy, hypercholesterolemia in HIV infection, acute coronary syndrome (ACS), non-alcoholic fatty liver disease / non-alcoholic steatohepatitis (NAFLD / NASH), arterial occlusive disease, cerebral atherosclerosis, arteriosclerosis, cerebrovascular disease, myocardial ischemia, coagulation disorders leading to blood clot formation in blood vessels, and diabetic autonomic neuropathy.

[0101] In some embodiments, an effective amount of the compound or composition is administered to a subject in need thereof to inhibit, prevent, or treat inflammation or an inflammatory disease. In some embodiments, the inflammation or inflammatory disease is selected from organ transplant rejection; reoxygenation injury resulting from organ transplantation (see Grupp et al., J. Mol. Cell. Cardiol. 31:297-303 (1999)), including, but not limited to, the following organs: heart, lung, liver, and kidney transplants; chronic inflammatory diseases of the joints, such as arthritis, rheumatoid arthritis, osteoarthritis, and bone diseases associated with increased bone resorption; inflammatory bowel disease (IBD), such as ileitis, ulcerative colitis (UC), Barrett's syndrome, and Crohn's disease (CD); inflammatory lung diseases, such as asthma, acute respiratory distress syndrome (ARDS), and chronic obstructive pulmonary disease (COPD); inflammatory diseases of the eye, such as corneal dystrophy, trachoma, oncolytic pulmonary syndrome, and pulmonary fibrosis. Cercaemia, uveitis, sympathetic ophthalmitis and endophthalmitis; chronic inflammatory diseases of the gums, such as gingivitis and periodontitis; inflammatory diseases of the kidney, such as uremic complications, glomerulonephritis and nephrosis; inflammatory diseases of the skin, such as scleroderma, psoriasis and eczema; inflammatory diseases of the central nervous system, such as chronic demyelinating diseases of the nervous system, multiple sclerosis, AIDS-related neurodegeneration and Alzheimer's disease, infectious meningitis, encephalomyelitis, Parkinson's disease, Huntington's disease, epilepsy, amyotrophic lateral sclerosis, and viral or autoimmune encephalitis, preeclampsia; chronic liver failure, brain and spinal cord trauma, and cancer. The inflammatory disease may also be a systemic inflammation of the whole body, as exemplified by gram-positive or gram-negative shock, hemorrhagic or anaphylactic shock, or shock induced by cancer chemotherapy in response to inflammatory cytokines, such as shock associated with inflammatory cytokines. Such shock may be induced, for example, by chemotherapy administered as a treatment for cancer.Other disorders include depression, obesity, allergic diseases, acute cardiovascular events, muscle wasting diseases, and cancer cachexia.Inflammation resulting from surgery and trauma may also be treated with the phospholipid composition.

[0102] In some embodiments, the LPC compositions described above are administered to a subject in need thereof to treat a disease or condition associated with red blood cells and cell membranes, particularly a disease or condition associated with red blood cell abnormalities in the cell membrane. In some embodiments, the condition or disease is sickle cell disease, sickle cell anemia, or sickle cell trait. In some embodiments, the condition or disease is thalassemia (alpha, beta, or delta), thalassemia in combination with hemoglobinopathy (hemoglobin E, hemoglobin S, or hemoglobin C), splenomegaly, or membrane abnormalities such as epidermal or spinocytic cells (spool cells) / spike cells, target red blood cells (target cells), echinocytes (acanthocytes), ellipsocytes and ovoid red blood cells, spherocytes, stomatocytes (mouth cells), and degmacytes ("bite cells").

[0103] In some embodiments, the effective amount comprises from about 0.1 to about 5 grams of the krill phospholipid composition, preferably from about 0.2 to about 3 grams of the krill phospholipid composition, and most preferably from about 0.5 to about 1.5 grams of the krill phospholipid composition.

[0104] The krill myrizophospholipid composition of the present invention may be used to treat a variety of subjects. Suitable subjects include humans as well as livestock (e.g., cows, horses, sheep, pigs, goats, fish, and shrimp, etc.), non-human primates, and companion animals (e.g., dogs, cats, birds). In some preferred embodiments, the subject is a human subject less than 10 years old, more preferably less than 1 year old, even more preferably less than 1 month old, and most preferably a newborn. In some preferred embodiments, the human subject is about 10-20 years old. In some preferred embodiments, the human subject is about 20-50 years old. In some preferred embodiments, the human subject is about 50-100 years old. In some preferred embodiments, the human subject is about 60-100 years old. In some preferred embodiments, the human subject is about 70-100 years old. EXAMPLES

[0105] experiment Example 1 - SUPERBA™ BOOST™ This example describes the production of an LPC composition using SUPERBA™ BOOST™ (Aker Biomarine AS, Lysaker, NO) as the starting phospholipid source. SUPERBA™ BOOST™ is preferably produced by the SMB process described elsewhere herein. SUPERBA™ BOOST™ is a krill phospholipid composition, 1000 mg contains 560 mg phospholipids, 150 mg EPA and 70 mg DHA. Briefly, 9 grams of SUPERBA™ BOOST™ is mixed with 90 ml of EtOH, then 450 ml of water is added to a nitrogen-purged 1 liter round-bottom reaction flask. 0.40 ml of LECITASE™ Ultra is then added and the reaction is allowed to proceed for 30 minutes with stirring. 500 ml of EtOH is then added to inactivate the enzyme, and the mixture is evaporated to dryness using a rotovap. A solvent-based separation is then performed. The enzyme-treated krill lipid sample is mixed with a solvent bath containing heptane and methanol. LPC and PC species migrate to the polar solvent (methanol) and non-polar lipids partition into heptane. The heptane phase is decanted, leaving a polar phase with a high amount of polar lipids, including LPC and PC. 250 ml each of heptane and MeOH are added to the dried lipid composition in the flask. The flask is shaken vigorously to separate the phases. The upper non-polar heptane phase is decanted and another 250 ml heptane is added to the polar MeOH phase and the separation is repeated. 10 grams of silica gel is then added to the MeOH extract and the solution is evaporated to dryness on a rotovap. LPC is then purified from the dried lipid composition by flash chromatography (100 g silica gel in a 5 cm diameter column). The LPC is eluted with a series of mobile phases: MPA (heptane); MPB (toluene:methanol:triethylamine, 60:40:1); and methanol:triethylamine, 95:5). The fractions are collected and evaporated to dryness.

[0106] The LPC composition produced by the method of the invention may optionally be 1 H-, 31 The product was analyzed by P-, 2D-NMR, TLC, GC and HPLC. 1 The results of H-NMR and HPLC are shown in FIG. 1 and FIG. 2, respectively, and are designated AKB6444-1.

[0107] 31 The phospholipid composition was determined using P- and 2D-NMR, and the results are shown in Tables 1, 2 and 3, designated AKB6444-1.

[0108] Example 2 - SUPERBA™ BOOST™ Pre-wash & FLASH This example describes the preparation of an LPC composition using SUPERBA™ BOOST™ (Aker Biomarine AS, Lysaker, NO) as the starting phospholipid source. SUPERBA™ BOOST™ is preferably produced by the SMB process described elsewhere herein. SUPERBA™ BOOST™ is a krill phospholipid composition, where 1000 mg contains 560 mg phospholipids, 150 mg EPA, and 70 mg DHA. Briefly, 10 grams of SUPERBA™ BOOST™ is mixed with 100 ml of EtOH, then 450 ml of water is added to a nitrogen-purged 1 liter round-bottom reaction flask. 0.40 ml of LECITASE™ Ultra is then added, and the reaction is allowed to proceed with stirring for 40 minutes. 500 ml of EtOH is then added to inactivate the enzyme, and the mixture is evaporated to dryness using a rotovap. Solvent-based separation is then performed. The enzyme-treated krill lipid sample is mixed with a solvent bath containing heptane and methanol. LPC and PC species migrate to the polar solvent (methanol) and non-polar lipids partition into heptane. Specifically, methanol (250 ml) and heptane (250 ml) are added to the dried sample. After vigorous shaking, the phases are separated and the methanol phase is extracted with another portion of heptane (250 ml). The heptane fractions are analyzed by TLC and evaporated separately. Silica gel 60 (20 g) is added to the methanol residue and the solution is concentrated to dryness. LPC is then purified from the dried lipid composition by flash chromatography. Silica gel is loaded onto a column (diameter 5 cm) together with silica gel (130 g) and the column is eluted. Fraction volume 25 ml. Eluent: 500ml EtOAc, 250ml EtOAc:MeOH 80:20, 250ml EtOAc:MeOH 50:50, 250ml MeOH, 500ml MeOH:Et3N 95:5, 500ml MeOH:Et3N 90:10. Fractions were analyzed by TLC and evaporated according to findings. The LPC containing fraction after chromatographic separation gave a final mass of 3.28 grams.

[0109] The LPC composition produced by the method of the invention may optionally be 1 H-, 31 The product was analyzed by P-, 2D-NMR, TLC, GC and HPLC. 1 The H-NMR and HPLC results are shown in Figures 7 and 8, respectively, and are designated AKB70005-4.

[0110] 31 The phospholipid composition was determined using P- and 2D-NMR and the results are shown in Tables 5, 9 and 10, designated AKB70005-4.

[0111] Example 3 - SUPERBA™ BOOST™ FLASH This example describes the preparation of an LPC composition using SUPERBA™ BOOST™ (Aker Biomarine AS, Lysaker, NO) as the starting phospholipid source. SUPERBA™ BOOST™ is preferably produced by the SMB process described elsewhere herein. SUPERBA™ BOOST™ is a krill phospholipid composition, where 1000 mg contains 560 mg phospholipids, 150 mg EPA, and 70 mg DHA. Briefly, 10 grams of SUPERBA™ BOOST™ is mixed with 100 ml of EtOH, then 450 ml of water is added to a nitrogen-purged 1 liter round-bottom reaction flask. 0.40 ml of LECITASE™ Ultra is then added, and the reaction is allowed to proceed for 40 minutes with stirring. 500 ml of EtOH is then added to inactivate the enzyme, and the mixture is evaporated to dryness using a rotovap. The LPC is then purified by flash chromatography from the dried lipid composition as follows: the residue is redissolved in methanol (200 ml), silica gel 60 (20 g) is added and the suspension is concentrated to dryness on a rotary evaporator. The silica gel is loaded onto a column (diameter 5 cm) together with silica gel (130 g) and the column is eluted. Fraction volume 25 ml. Eluent: 500 ml EtOAc, 250 ml EtOAc:MeOH 80:20, 250 ml EtOAc:MeOH 50:50, 250 ml MeOH, 500 ml MeOH:Et3N 90:10. The fractions were analyzed by TLC and evaporated according to the findings. The LPC-containing fraction after chromatographic separation gave a final mass of 3.73 grams.

[0112] The LPC composition produced by the method of the invention may optionally be 1 H-, 31 The product was analyzed by P-, 2D-NMR, TLC, GC and HPLC. 1 The H-NMR and HPLC results are shown in Figures 9 and 10, respectively, and are designated AKB70005-5.

[0113] 31The phospholipid composition was determined using P- and 2D-NMR and the results are shown in Tables 6, 9 and 10, designated AKB70005-5.

[0114] Example 4 - ROMEGA™ Pre-wash & FLASH This example describes the production of an LPC composition using ROMEGA™ (Arctic Nutrition AS, Orsta, NO) as the starting phospholipid source. ROMEGA™ is preferably produced by the process described elsewhere herein. ROMEGA™ is a herring egg oil and fish oil composition, 1000mg / 3000mg contains 340mg / 1020mg phospholipids, 100mg / 300mg EPA and 320mg / 960mg DHA. Briefly, 9 grams of ROMEGA™ is mixed with 90ml of EtOH, then 450ml of water is added to a nitrogen-purged 1 liter round-bottom reaction flask. 0.40ml of LECITASE™ Ultra is then added and the reaction is allowed to proceed with stirring for 40 minutes. 500ml of EtOH is then added to inactivate the enzyme and the mixture is evaporated to dryness using a rotovap. A solvent-based separation is then performed. The enzyme-treated herring egg / fish lipid sample is mixed with a solvent bath containing heptane and methanol. LPC and PC species migrate to the polar solvent (methanol) and non-polar lipids partition into heptane. Specifically, methanol (250 ml) and heptane (250 ml) were added to the dried sample. After vigorous shaking, the phases were separated and the methanol phase was extracted with another portion of heptane (250 ml). The heptane fractions were analyzed by TLC and evaporated separately. Silica gel 60 (20 g) was added to the methanol residue and the solution was concentrated to dryness. LPC is then purified from the dried lipid composition by flash chromatography. The silica gel was loaded onto a column (diameter 5 cm) together with silica gel (130 g) and the column was eluted. Fraction volume 25 ml. Eluent: 500ml EtOAc, 250ml EtOAc:MeOH 80:20, 250ml EtOAc:MeOH 50:50, 250ml MeOH, 500ml MeOH:Et3N 95:5, 500ml MeOH:Et3N 90:10. Fractions were analyzed by TLC and evaporated according to findings. The LPC containing fraction after chromatographic separation gave a final mass of 0.84 grams.

[0115] The LPC composition produced by the method of the invention may optionally be 1 H-, 31 The product was analyzed by P-, 2D-NMR, TLC, GC and HPLC. 1 The H-NMR and HPLC results are shown in Figures 5 and 6, respectively, and are designated AKB70005-3. 31 The phospholipid composition was determined using P- and 2D-NMR and the results are shown in Tables 5, 7 and 8, designated AKB70005-3.

[0116] Example 5 - ROMEGA™ FLASH This example describes the preparation of an LPC composition using ROMEGA™ (Arctic Nutrition AS, Orsta, NO) as the starting phospholipid source. ROMEGA™ is preferably produced by the process described elsewhere herein. ROMEGA™ is a herring egg oil and fish oil composition, 1000mg / 3000mg contains 340mg / 1020mg phospholipids, 100mg / 300mg EPA and 320mg / 960mg DHA. Briefly, 9 grams of ROMEGA™ is mixed with 90ml of EtOH, then 450ml of water is added to a nitrogen-purged 1 liter round-bottom reaction flask. 0.40ml of LECITASE™ Ultra is then added and the reaction is allowed to proceed with stirring for 40 minutes. 500ml of EtOH is then added to inactivate the enzyme and the mixture is evaporated to dryness using a rotovap. The LPC is then purified by flash chromatography from the dried lipid composition as follows: the residue is redissolved in methanol (200 ml), silica gel 60 (20 g) is added and the suspension is concentrated to dryness on a rotary evaporator. The silica gel is loaded onto a column (diameter 5 cm) together with silica gel (130 g) and the column is eluted. Fraction volume 25 ml. Eluent: 500 ml EtOAc, 250 ml EtOAc:MeOH 80:20, 250 ml EtOAc:MeOH 50:50, 250 ml MeOH, 500 ml MeOH:Et3N 90:10. The fractions were analyzed by TLC and evaporated according to the findings. The LPC-containing fraction after chromatographic separation gave a final mass of 0.76 grams.

[0117] The LPC composition produced by the method of the invention may optionally be 1 H-, 31 The product was analyzed by P-, 2D-NMR, TLC, GC and HPLC. 1 The H-NMR and HPLC results are shown in Figures 5 and 6, respectively, and are designated AKB70005-2. 31 The phospholipid composition was determined using P- and 2D-NMR and the results are shown in Tables 4, 7 and 8, designated AKB70005-2. Table 1: Summary of results for sample AKB69444-1, 2D and 31P-NMR (PL) [Table 1] Table 2: Results (summary), 2D and 31P-NMR (ether PL) of sample AKB69444-1 [Table 2] Table 3: LPC composition of sample AKB69444-1 1 The composition determined by H-NMR contained 38.74% (w / w) omega-3 fatty acids: [Table 3] Table 4: Samples AKB70005-1 and AKB70005-2 1 Omega-3 fatty acids (w / w) of LPC composition determined by H-NMR. [Table 4] Table 5: Samples AKB70005-3 and AKB70005-4 1 Omega-3 fatty acids (w / w) of LPC composition determined by H-NMR. [Table 5] Table 6: Sample AKB70005-5 1 Omega-3 fatty acids (w / w) of LPC composition determined by H-NMR. [Table 6] Table 7: Summary of results, 2D and 31P-NMR (PL) for AKB70005-1, AKB70005-2, and AKB70005-3. [Table 7] Table 8: Summary of results, 2D and 31P-NMR (PL) for AKB70005-1, AKB70005-2, and AKB70005-3. [Table 8] Table 9: Summary of results, 2D and 31P-NMR (PL) for AKB70005-4 and AKB70005-5. [Table 9] Table 10: Summary of results, 2D and 31P-NMR (PL) for AKB70005-4 and AKB70005-5. [Table 10] Table 11: Legend (abbreviations). [Table 11]

[0118] Example 6 The LPC composition was made using SUPERBA™ krill oil as the starting point. 31 The results are shown in Table 12. Table 12. [Table 12]

[0119] Example 7 This example provides a summary of the production of additional lysophospholipid compositions of the present invention.

[0120] A method for synthesizing LPC species from krill oil. The method used to synthesize LPC from krill oil can be divided into four steps depending on the target composition of the final mixture / sample: 1. CRUDE: A process to achieve an LPC concentration of 14-27.3% by weight in the final composition (Tables 13.1-1 and 14.1-1). 2. POLAR-1 / POLAR-2: Post-CRUDE processes to achieve a concentration of LPC of 35.2-65.7% by weight in the final composition (Tables 13.1-2 and 14.1-2). 3. FLASH: A post-CRUDE process that achieves a concentration of LPC of 56.6-81.1% by weight in the final composition (Table 13.1-3). 4. FORMULATION: Post-POLAR-2 process (14.1-3) to achieve an LPC concentration of 39.3-41.1 wt% with 5-14 wt% PEG400 in the final composition.

[0121] FLASH and POLAR are alternative processes to achieve LPC enrichment of CRUDE-1 and CRUDE-2 LPC mixtures / samples, while FORMULATION is a process that allows formulation of POLAR-2 LPC mixtures / samples with PEG. Flow diagrams of the various processes are shown in Figures 11 (Process 1) and 12 (Process 2).

[0122] 1.CRUDE sample: CRUDE-1 & CRUDE-2 LPC mixtures / samples follow the same process. Superba Boost (10 g) was dissolved in EtOH (2-10 g) and diluted with pH stabilized water (2-45 g, pH 6-12) to achieve a mixture pH 5.2-6.2, 40 μL of Lecithase Ultra was added, capped and stirred at room temperature for 120-1440 min. Samples may be flushed with N2 to prevent oxidation. The reaction mixture was quenched by adding EtOH (25-50 g) and concentrated under reduced pressure at 50 °C to obtain CRUDE-1 and CRUDE-2 LPC-mixtures / samples (Table 13 and Table 16, respectively).

[0123] 2.POLAR-1 / POLAR-2 Sample: POLAR-2: The POLAR process was started immediately after the quench in the CRUDE process. Therefore, the CRUDE samples were not concentrated under reduced pressure at 50° C. to obtain CRUDE-1 and CRUDE-2 LPC mixtures / batches, but rather, 25-100 g of heptane was added to the CRUDE-1 or CRUDE-2 mixtures to achieve phase separation, thereby achieving enrichment of polar lipids in the EtOH and water-rich phase (referred to as the polar phase). The heptane phase was decanted to leave only the polar phase. The polar phase was concentrated under reduced pressure to obtain POLAR-1 and POLAR-2 LPC-mixtures / samples (Tables 14 and 17, respectively).

[0124] 3.FLASH sample: CRUDE samples were redissolved in ethanol (10-100 g) by adding 25-50 g of Silicagel 60 and evaporated to dryness. Flash chromatography (125 g Silicagel 60, 5 cm column diameter) was performed. Elution with 500 ml 80:20 MPA:MPB, 500 ml 50:50 MPA:MPB, 500 ml MP B, 500 ml MP C was performed. The column was subsequently pushed with EtOH:Et3N (80:20, 300 mL). Based on the results, different fractions were combined and evaporated to obtain the FLASH LPC-mixtures / samples (Table 15).

[0125] 4. Formulation samples: Formulations containing PEG400 were made from POLAR mixtures / samples. For this, EtOH and PEG400 were added to the POLAR mixtures and the mixtures (EtOH, PEG400 and POLAR LPC mixtures) were concentrated under reduced pressure at 50 °C to obtain FORMULATION LPC-mixtures / samples (Table 18) with final concentrations of 5-14 wt% PEG400 and 4-7 wt% EtOH (Table 18). Table 13. Analysis results of six CRUDE-1 krill LPC composition batches from Process 1 [Table 13] JPEG0007625416000014.jpg129169 Table 14. Analytical results from six POLAR-1 krill LPC composition batches from Process 1 [Table 14] TIFF0007625416000016.tif130169 Table 15. Analytical results from six FLASH krill LPC composition batches from Process 1 [Table 15] JPEG0007625416000018.jpg127170 Table 16. Analytical results from two CRUDE-2 krill LPC composition batches from Process 2 [Table 16] TIFF0007625416000020.tif108169 Table 17. Analytical results from eight POLAR-2 krill LPC composition batches from Process 2 [Table 17] TIFF0007625416000022.tif174169 Table 18. Analytical results from two formulation krill LPC composition batches from process 2 [Table 18] TIFF0007625416000024.tif85169 Table 19 shows additional analytical data for the various batches. Table 19 [Table 19]

[0126] Example 9 This example provides data regarding the uptake of the lysophospholipid composition of the present invention in biological tissue. Briefly, the LS:ABM-9C0 lysophospholipid composition described above was administered in a 30-mL aliquot of 100 mL ... 14 Spiked with C-labeled lysoPC-EPA or lysoPC-DHA 14 The results were compared to purified krill PC compositions (98% PC formulated with PEG) spiked with C-labeled PC-EPA or DHA. The spiked compositions were orally administered to rats and uptake into various tissues was measured by quantitative whole body autoradiography. Data was collected on both the timing of uptake into various organs and tissues as well as the amount of uptake into tissues. The results are shown in Tables 20-23. Surprisingly, these data show that the uptake of both the lysoPC-EPA spiked lysophospholipid compositions and the lysoPC-DHA spiked lysophospholipid compositions was faster in time and greater in both total amounts of EPA and DHA incorporated compared to samples prepared with intact phospholipids (i.e., samples without significant amounts of lysophospholipids). This result was observed in all organs / tissues examined, except for the eye. Table 20 [Table 20] TIFF0007625416000027.tif28169 Table 21 [Table 21] Table 22 [Table 22] Table 23 [Table 23]

Claims

1. 1. A lysophosphatidylcholine (LPC) composition comprising LPC from about 10% to about 100% (w / w) of the composition, an omega-3 fatty acid content of 5% to 50% (w / w) of the composition, said omega-3 fatty acids being linked to phospholipid and glycerol molecules in said composition by ester or ether bonds, said omega-3 fatty acids comprising EPA and DHA in an EPA:DHA ratio of 1:1 to 3:1, and having a 2-LPC:1-LPC ratio of 1:8 to 18:1 on a w / w basis.

2. 2. The lysophosphatidylcholine (LPC) composition of claim 1, wherein said LPC composition is prepared from krill oil.

3. 2. The lysophosphatidylcholine (LPC) composition of claim 1, wherein the composition comprises 20% to 100% (w / w) LPC of the composition, such as 60% to 100% (w / w) LPC of the composition, 20% to 90% (w / w) LPC of the composition, or 70% to 90% (w / w) LPC of the composition.

4. The lysophosphatidylcholine (LPC) composition of claim 1, wherein the composition comprises 20% to 100% (w / w) LPC of the composition.

5. 2. The lysophosphatidylcholine (LPC) composition of claim 1, wherein the composition has an omega-3 fatty acid content of 30% to 50% (w / w) of the composition.

6. 10. A pharmaceutical or dietary supplement composition comprising the composition of claim 1 and a physiologically acceptable carrier.

7. 10. An oral delivery vehicle comprising the LPC composition of claim 1, the pharmaceutical composition of claim 6, or the dietary supplement composition of claim 6.

8. 1. A method for producing a lysophosphatidylcholine (LPC) composition having a high content of EPA and DHA from a marine or other starting material containing phospholipids, comprising: treating a marine or other starting material containing said phospholipids with a phospholipase that is not native to the marine or other starting material containing said phospholipids to provide a phospholipase-treated material; and fractionating the phospholipase-treated feedstock to provide a lipid composition having a higher lysophosphatidylcholine content than a marine or other starting feedstock containing the phospholipids. Including, the phospholipase-treated material is fractionated by phase separation in a polar / non-polar solvent system to provide a lipid composition having a higher lysophosphatidylcholine content than the starting material; The method of claim 1, wherein the lipid composition comprises LPC at about 10% to about 100% (w / w) of the composition, an omega-3 fatty acid content of 5% to 50% (w / w) of the composition, the omega-3 fatty acids being linked to phospholipids and glycerol molecules in the composition by ester or ether bonds, the omega-3 fatty acids comprising EPA and DHA in an EPA:DHA ratio of 1:1 to 3:1, and having a ratio of 2-LPC:1-LPC of 1:8 to 18:1 on a w / w basis.

9. 9. The method of claim 8, wherein the raw material is a krill lipid preparation.

10. 9. The method of claim 8, wherein the lipid composition having a higher lysophosphatidylcholine content than the phospholipid-containing marine or other starting material is further enriched by a chromatographic separation procedure.

11. The method of claim 8, wherein the method is a method for producing the lysophosphatidylcholine (LPC) composition of claim 1.

12. To increase the amount of EPA and / or DHA in a target tissue or organ, the lysophosphatidylcholine (LPC) composition is administered by oral administration; The lysophosphatidylcholine (LPC) composition of claim 1; the pharmaceutical or dietary supplement composition of claim 6; or the oral delivery vehicle of claim 7.

13. For increasing the amount of EPA and / or DHA in a target tissue or organ, and for use in treating, inhibiting or ameliorating a condition selected from the group consisting of cognitive diseases, disorders or deficiencies, neurodegenerative disorders, cardiovascular disorders, metabolic syndromes, inflammation, inflammatory diseases, and diseases or conditions associated with abnormalities of red blood cells of cell membranes. The lysophosphatidylcholine (LPC) composition of claim 1; the pharmaceutical or dietary supplement composition of claim 6; or the oral delivery vehicle of claim 7.

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