Lipid extraction method

Abstract

The present invention relates to methods of lipid extraction, and specifically provides improved methods for extracting and preparing lipids for use in pharmaceuticals, nutraceuticals and functional food products from biological sources.

Description

[0001] This application is a divisional application of the invention patent application filed on June 13, 2014, with application number 201480041687.1 and entitled "Lipid Extraction Method". Invention Field

[0002] This invention provides an improved method for extracting and preparing lipids from biological sources for use in pharmaceuticals, nutritional products, and functional foods. Background of the Invention Accumulated evidence suggests that long-chain omega-3 fatty acids, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), found in fish, reduce the risk of heart disease (CHD) and ischemic heart disease. Numerous epidemiological studies, such as the Physicians' Health Study and the Nurses' Health Study, have examined diet and other lifestyle factors that influence health outcomes. The Physicians' Health Study reported that consuming one or more servings of fish per week was associated with a 52% lower risk of sudden cardiac death compared to less than one serving per week. Another epidemiological study (the Nurses' Health Study in the United States) found that consuming five or more servings of fish per week was associated with a 45% lower risk of cardiac death compared to consuming one serving per month. Long-chain omega-3 fatty acids are known to be protective dietary factors against cardiovascular disease. EPA and DHA have been shown to lower triglyceride levels and act as antiarrhythmic agents. The American Heart Association (AHA) has compiled data on fish and fish oil consumption and cardiovascular disease. The AHA report recommends that individuals with and without heart disease and elevated blood triglyceride levels consume fish or take fish oil supplements. A report prepared in 2003 by the Third Task Force of European and Other Societies also recommended fish oil as a standard treatment for myocardial infarction.

[0003] Blood triglyceride levels are positively correlated with increased CHD, as elevated triglyceride levels increase the risk of CHD. Multiple factors influence elevated serum triglyceride levels throughout life, with diet being a major contributor. DHA and EPA, abundant in many marine foods, both appear to support cardiovascular health and lower blood triglyceride levels. Fish oil is known to lower serum triglyceride levels by 20-50%, similar to the effects observed with medications such as statins, niacin, and fibrates. The American Heart Association recommends that individuals with no documented CHD consume two servings of fish (preferably oily fish) per week. Patients with CHD should take 1 gram of EPA and DHA daily (preferably from oily fish or as a supplement, if under the guidance of a physician). For those patients who need to lower their triglyceride levels, the American Heart Association recommends taking 2-4 grams of EPA and DHA daily as a supplement under the guidance of a physician. The prescription form of EPA and DHA, Lovaza (formerly known as Omacor), is a good source of omega-3 fatty acids available to people with high blood triglyceride levels. Each 1-gram capsule of Lovaza contains 465 mg of EPA ethyl ester, 375 mg of DHA ethyl ester, 80 mg of other omega-3 fatty acids, 30 mg of omega-6 fatty acids, and 50 mg of antioxidants. It is prescribed as a dietary supplement to lower very high triglyceride levels in adult patients.

[0004] In medical research, omega-3 fatty acids are being investigated to determine whether they can effectively improve a wide range of disease conditions—including heart disease, diabetes, inflammation, depression, Alzheimer's disease, and attention deficit disorder—making this group of nutrients an exciting and very active area of ​​clinical research. Ensuring that omega-3 fatty acids are part of the diet (as recommended by dietary guidelines) is a proper starting point for achieving better health; therefore, there is a tremendous opportunity to utilize the potential of omega-3 fatty acids to improve human conditions.

[0005] Oral administration of omega-3 compositions to some subjects resulted in undesirable side effects, including hiccups and reflux. The bioavailability of certain forms of omega-3 may also be limited. Therefore, what is needed in the art is an improved omega-3 formulation. Invention Overview This invention provides an improved method for extracting and preparing lipids from biological sources for use in pharmaceuticals, nutritional products, and functional foods.

[0006] In some embodiments, the present invention provides a method for extracting a polar lipid-rich extract from biological material, the method comprising: contacting the biological material with a concentrated protonated solvent under conditions that preferentially extract the polar lipids to form a slurry comprising a polar lipid solution and biological residue material; separating the polar lipid solution from the biological residue material to provide a separated polar lipid solution; adding an aqueous solution to the polar lipid solution to dilute the protonated solvent, such that the polar lipid solution is separated into an upper phase comprising the diluted protonated solvent and a lower polar lipid-rich bottom phase; and separating the polar lipid-rich bottom phase to provide a polar lipid-rich extract. In some embodiments, adding an aqueous solution to the polar lipid solution to dilute the protonated solvent (to separate the polar lipid solution into an upper phase comprising the diluted protonated solvent and a lower polar lipid-rich bottom phase) comprises adding an aqueous solution to dilute the concentration of the protonated solvent to about 50% to 70% w / w, preferably about 55% to 65% w / w, more preferably about 58% to 62% w / w, when combined with water in the biological sample. In some embodiments, the concentration of the proton solvent is diluted to about 60% w / w when combined with water in the biological sample. In some embodiments, contacting the biological material with the concentrated proton solvent under conditions that preferentially extract polar lipids includes mixing the biological material with the concentrated proton solvent at a temperature of about -10°C to about 50°C, such that the solvent concentration is about 70% to about 95% w / w when combined with water in the biological material.

[0007] In some embodiments, the method further includes washing the polar lipid-rich extract with a diluted protonated solvent in which the polar lipids are poorly soluble, to provide an upper phase comprising the diluted protonated solvent and a polar lipid-rich lower phase, and separating the lower phase to provide the washed polar lipid-rich extract. In some embodiments, the washing is repeated 2 to 5 times. In some embodiments, the washing of the polar lipid-rich extract with the diluted protonated solvent under conditions that make the phospholipids poorly soluble further includes mixing the polar lipid-rich extract with the diluted protonated solvent at a ratio of about 0.5:1 to 5:1, wherein the diluted protonated solvent comprises an aqueous solution having about 30% to 70% protonated solvent. In some embodiments, the protonated solvent is selected from the group consisting of concentrated n-butanol, n-propanol, isopropanol, ethanol, and methanol. In some embodiments, the protonated solvent is ethanol.

[0008] In some embodiments, the washed polar lipid-rich extract is characterized by containing at least 50% w / w phospholipids. In some embodiments, the washed polar lipid-rich extract is characterized by containing less than about 3% w / w lysophospholipids. In some embodiments, the washed polar lipid-rich extract is characterized by containing less than about 1% w / w lysophospholipids. In some embodiments, the washed polar lipid-rich extract is characterized by containing less than about 0.5% w / w lysophospholipids. In some embodiments, the washed polar lipid-rich extract is characterized by containing less than about 0.1% lysophospholipids. In some embodiments, the washed polar lipid-rich extract is characterized by having a conductivity of less than 300 μS / cm as measured in a saturated 60% ethanol solution. In some embodiments, the method further includes precipitating phospholipids from the washed polar lipid-rich extract. In some embodiments, the precipitation of phospholipids from the washed, polar lipid-rich extract further includes mixing the washed, polar lipid-rich extract with cold acetone under conditions that allow phospholipid precipitation to provide a precipitate of polar lipid-rich lipids.

[0009] In some embodiments, the method further includes removing residual solvent from the polar lipid-rich extract or a washed polar lipid-rich extract by evaporation to provide a solid polar lipid-rich composition. In some embodiments, the solid polar lipid-rich composition contains at least 90% w / w phospholipids. In some embodiments, the solid polar lipid-rich composition contains less than about 3% w / w lysophospholipids. In some embodiments, the solid polar lipid-rich composition contains less than about 1% w / w lysophospholipids.

[0010] In some embodiments, the method further includes contacting the bioresidue material with a protonated solvent under conditions that allow the extraction of neutral lipids from the bioresidue material to form a slurry comprising a neutral lipid solution and the bioresidue material; separating the neutral lipid solution from the bioresidue material; and A concentrated proton solvent is evaporated from a neutral lipid solution to provide a neutral lipid extract. In some embodiments, the conditions for extracting neutral lipids from biological residues include mixing the biological residues with the concentrated proton solvent at a ratio of about 2:1 to 8:1 at a temperature of about 15°C to about 40°C.

[0011] In some embodiments, the method further includes combining a washed extract rich in polar lipids with a neutral lipid extract to provide a mixed lipid composition. In some embodiments, the mixed lipid composition is characterized by containing less than about 3% lysophosphatidylcholine. In some embodiments, the mixed lipid composition is characterized by containing less than about 1% lysophosphatidylcholine. In some embodiments, the mixed lipid composition is characterized by containing less than about 0.5% lysophosphatidylcholine. In some embodiments, the mixed lipid composition is characterized by containing less than about 0.1% lysophosphatidylcholine.

[0012] In some embodiments, the biological material is selected from the group consisting of algae, marine animal materials, and plant materials. In some embodiments, the marine animal material is selected from fish materials, krill materials, and marine plankton materials. In some embodiments, the krill material is selected from the group consisting of fresh krill, frozen krill, krill powder, wet krill paste, dried krill paste, and krill oil.

[0013] In some embodiments, the washed, polar lipid-rich extract, polar lipid-rich precipitate, solid polar lipid-rich composition, neutral lipid extract, or mixed lipid composition produced as described above is formulated into an oral delivery medium. In some embodiments, the oral delivery medium is selected from the group consisting of tablets, capsules and gel capsules, solutions, suspensions, emulsions, and chewable matrices. In some embodiments, the washed, polar lipid-rich extract, polar lipid-rich precipitate, solid polar lipid-rich composition, neutral lipid extract, or mixed lipid composition produced as described above is formulated into an emulsion for parenteral delivery.

[0014] In some embodiments, the present invention provides a method for producing a gummy candy product, the method comprising: mixing a washed, polar lipid-rich extract, a polar lipid-rich precipitate, a solid polar lipid-rich composition or a mixed lipid composition, or a phospholipid composition as described above, into a gel matrix to provide a mixed gummy candy mixture and to form gummy candies from the mixed gummy candy mixture. In some embodiments, the present invention provides a product made by the above method.

[0015] In some embodiments, the present invention provides compositions or articles comprising, in a solid gel matrix, a washed extract rich in polar lipids, a precipitate rich in polar lipids, a solid composition rich in polar lipids, or a mixed lipid composition, or a phospholipid composition as described above.

[0016] In some embodiments, the present invention provides a method for extracting lipid compositions from biological material, the method comprising: contacting the biological material with a concentrated protonated solvent under conditions that preferentially extract polar lipids to form a slurry comprising a polar lipid solution and biological residue material; separating the polar lipid solution from the biological residue material; evaporating the protonated solvent from the polar lipid solution to provide a polar lipid extract comprising phospholipids; washing the polar lipid extract with a diluted protonated solvent under conditions that make the phospholipids poorly soluble; and evaporating the second protonated solvent to provide a washed phospholipid extract. In some embodiments, contacting the biological material with the concentrated protonated solvent under conditions that preferentially extract polar lipids includes mixing the biological material with the concentrated protonated solvent at a temperature of about -10°C to about 50°C so that the solvent concentration is about 70% w / w to about 95% w / w when combined with water in the biological material. In some embodiments, washing the polar lipid extract with a second proton solvent under conditions that make the phospholipids poorly soluble comprises mixing the polar lipid extract with a diluted proton solvent at a ratio of about 0.5:1 to 5:1, wherein the diluted proton solvent comprises an aqueous solution having about 30% w / w to 70% w / w of the proton solvent.

[0017] In some embodiments, the concentrated proton solvent is selected from the group consisting of concentrated n-butanol, n-propanol, isopropanol, ethanol, and methanol. In some embodiments, the diluted proton solvent is selected from the group consisting of diluted n-butanol, n-propanol, isopropanol, ethanol, and methanol. In some embodiments, the concentrated proton solvent is concentrated ethanol. In some embodiments, the diluted proton solvent is diluted ethanol.

[0018] In some embodiments, the washed phospholipid extract is characterized by containing at least 50%, 60%, 70%, 80%, or 90% phospholipids (i.e., the weight of phospholipids / total weight of the washed phospholipid extract). In some embodiments, the washed phospholipid extract is characterized by containing less than about 3% lysophospholipids (i.e., the weight of lysophospholipids / total weight of the washed phospholipid extract). In some embodiments, the washed phospholipid extract is characterized by containing less than about 1% lysophospholipids. In some embodiments, the washed phospholipid extract is characterized by containing less than about 0.5% lysophospholipids. In some embodiments, the washed phospholipid extract is characterized by containing less than about 0.1% lysophospholipids. In some embodiments, the washed phospholipid extract is characterized by having a conductivity of less than 300 μS / cm measured in a saturated 60% ethanol solution.

[0019] In some embodiments, the method further includes contacting the bioresidue material with a concentrated protonated solvent under conditions that allow for the extraction of neutral lipids from the bioresidue material, to form a slurry comprising a neutral lipid solution and the bioresidue material; separating the neutral lipid solution from the bioresidue material; and evaporating the concentrated protonated solvent from the neutral lipid solution to provide a neutral lipid extract. In some embodiments, the conditions for extracting neutral lipids from the bioresidue material include mixing the bioresidue material with the concentrated protonated solvent at a temperature of about 15°C to about 40°C at a ratio of about 2:1 to 8:1. In some embodiments, the method further includes combining a washed phospholipid extract with the neutral lipid extract to provide a mixed lipid composition.

[0020] In some embodiments, the mixed lipid composition is characterized by containing less than about 3% lysophosphatidylcholine (i.e., the weight of lysophosphatidylcholine / the total weight of the washed phospholipid extract). In some embodiments, the mixed lipid composition is characterized by containing less than about 1% lysophosphatidylcholine. In some embodiments, the mixed lipid composition is characterized by containing less than about 0.5% lysophosphatidylcholine. In some embodiments, the mixed lipid composition is characterized by containing less than about 0.1% lysophosphatidylcholine. In some embodiments, the washed phospholipid extract is characterized by having a conductivity of less than 300 μS / cm measured in a saturated 60% ethanol solution.

[0021] In some embodiments, the method further includes washing the phospholipids with cold acetone to recover a further concentrated phospholipid fraction. In some embodiments, the concentrated phospholipid fraction is at least 80%, 85%, 90%, or 95% w / w phospholipids (i.e., the weight of the phospholipids / the total weight of the concentrated phospholipid fraction).

[0022] In some embodiments, the biological material is selected from the group consisting of algae, marine animal materials, and plant materials. In some embodiments, the marine animal material is selected from fish materials, krill materials, and marine plankton materials. In some embodiments, the krill material is selected from the group consisting of fresh krill, frozen krill, krill powder, wet krill paste, dried krill paste, and krill oil.

[0023] In some embodiments, washed phospholipid extracts, neutral lipid extracts, or mixed lipid compositions are formulated into oral delivery media. In some embodiments, the oral delivery media are selected from the group consisting of tablets, capsules and gel capsules, solutions, suspensions, emulsions, and chewable matrices. In some embodiments, washed phospholipid extracts, neutral lipid extracts, or mixed lipid compositions are formulated into emulsions for parenteral delivery.

[0024] In some embodiments, the present invention provides phospholipid compositions comprising a mixture of phospholipid compounds having the following structures: Wherein R1 and R2 are selected from the group consisting of fatty acid moieties and -H, and R3 is H or selected from choline, ethanolamine, inositol, and serine moieties, the mixture of phospholipid compounds contains more than about 85% or 90% of the choline moieties (i.e., mol%, moles of choline moieties / total moles of phospholipid compounds) and more than about 30% w / w of the ω-3 fatty acid moieties (i.e., weight of the ω-3 fatty acid moieties / total weight of phospholipid compounds) at position R3, wherein more than about 90% w / w of the ω-3 fatty acid moieties (i.e., weight of the ω-3 fatty acid moieties / total weight of fatty acid moieties) is at position R2, the composition is further characterized by containing less than about 3% w / w, preferably less than 1% w / w of lysophospholipids (i.e., weight of lysophospholipids / total weight of the phospholipid composition). In some embodiments, the composition has a conductivity of less than about 300 μS / cm measured in a saturated 60% ethanol solution. In some embodiments, the ω-3 fatty acid portion is selected from the group consisting of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and combinations thereof. In some embodiments, the composition comprises at least 50%, 60%, 70%, 75%, 80%, 90%, or 95% w / w (i.e., weight of the phospholipid compound / total weight of the phospholipid composition) of a phospholipid compound. In some embodiments, the ω-3 portion of the mixture of phospholipid compounds is eicosapentaenoic acid and DHA, and wherein eicosapentaenoic acid and DHA are present in a ratio of eicosapentaenoic acid:DHA of about 1:1 to about 3:1. In some embodiments, the composition further comprises at least 5% w / w and up to about 10%, 20%, 30%, 40%, or 50% w / w of an ethyl ester containing the ω-3 fatty acid portion (i.e., weight of the ethyl ester / total weight of the composition). In some embodiments, the composition comprises at least 10% w / w and up to about 20%, 30%, 40%, or 50% w / w of a glycerol ester compound containing an ω-3 fatty acid moiety (i.e., weight of the glycerol ester compound / total weight of the composition). In some embodiments, the composition comprises astaxanthin. In some embodiments, the composition comprises at least a second antioxidant. In some embodiments, the composition is partially or wholly derived from krill.

[0025] In some embodiments, the composition is provided in formulations selected from the group consisting of capsules, tablets, liquids, powders, emulsions, dietary supplements, nutritional supplements, beverages, and functional foods.

[0026] In some embodiments, the composition is administered orally or intravenously to a subject to lower serum triglycerides, lower serum cholesterol, reduce plaque formation, reduce platelet aggregation, treat atherosclerosis, improve cardiovascular health, reduce inflammation, reduce coronary heart disease, treat depression, treat Alzheimer's disease, treat attention deficit disorder, and treat metabolic syndrome. In some embodiments, the composition is administered at a daily dose of about 0.1 to about 3 grams. In some embodiments, the composition is administered to a subject selected from the group consisting of: humans, non-human primates, domesticated or farm animals, and companion animals.

[0027] In some embodiments, the present invention provides a method for producing a gummy candy product, the method comprising: mixing a washed, polar lipid-rich extract, a polar lipid-rich precipitate, a solid polar lipid-rich composition or a mixed lipid composition, or a phospholipid composition as described above, into a gel matrix to provide a mixed gummy candy mixture and to form gummy candies from the mixed gummy candy mixture. In some embodiments, the present invention provides a product made by the above method.

[0028] In some embodiments, the present invention provides a composition comprising, in a solid gel matrix, a washed extract rich in polar lipids as described above, a precipitate rich in polar lipids, a solid composition rich in polar lipids, a mixed lipid composition, or a phospholipid composition as described above.

[0029] definition As used herein, "phospholipid" refers to organic compounds having the following general formula: Where R1 is a fatty acid moiety or -H, R2 is a fatty acid moiety or -H, and R3 is -H or a phospholipid head moiety such as choline (HOCH2CH2N). + (CH3)3OH - The compound may contain a moiety of ethanolamine (HOCH2CH2NH2), serine, or inositol, such as cyclohexane polyol inositol or a derivative thereof. Preferably, R1 and R2 cannot both be -H. When R3 is -H, the compound is a diacylglycerol phosphate, and when R3 is a nitrogen-containing compound, the compound is a phospholipid such as lecithin, cephalin, phosphatidylserine, or phosphatal acetal.

[0030] As used in this article, the term "long-chain polyunsaturated fatty acid" refers to a fatty acid that has 20 or more carbons and is unsaturated on two or more bonds.

[0031] As used herein, the term ω-3 fatty acid refers to a polyunsaturated fatty acid having a final double bond in a hydrocarbon chain between the third and fourth carbon atoms, beginning at the methyl terminus of the molecule. Non-limiting examples of ω-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).

[0032] As used herein, the term "part" when referring to a fatty acid means a portion of a fatty acid that is bonded to another molecule, such as a glycerol ester or glycerophosphate ester, by bonds such as ester or ether bonds. A fatty acid "part" thus refers to either the fatty acid chain or the fatty acyl group of the fatty acid. In the phospholipid structures defined herein, when the fatty acid portion is an acyl group, the fatty acid chain of the acyl group is linked by an ester bond, and when the fatty acid portion is a fatty acid chain, the fatty chain is linked by an ether bond. When a specific fatty acid is mentioned in connection with the phospholipids of this invention (e.g., EPA or DHA), it should therefore be considered a reference to the relevant acyl group or its fatty acid chain.

[0033] As used herein, the term "physiologically acceptable carrier" refers to any carrier or excipient commonly used with oily pharmaceutical products. Such carriers or excipients include, but are not limited to, oils, starches, sucrose, and lactose.

[0034] As used herein, the term "oral delivery medium" means any device for the oral delivery of a drug, including, but not limited to, capsules, pills, tablets, and syrups.

[0035] As used herein, the term "food" means any food or feed suitable for consumption by humans, non-ruminant animals, or ruminant animals. "Food" can be prepared and packaged food (e.g., mayonnaise, salad dressing, bread, or cheese) or animal feed (e.g., extruded and pelleted animal feed or roughage mix). "Prepared food" means any pre-packaged food approved for human consumption.

[0036] As used in this article, the term "food" refers to any substance suitable for human or animal consumption.

[0037] As used in this article, the term "functional food" refers to food in which bioactive supplements have been added.

[0038] As used in this article, the term "infant food" refers to food formulated for infants, such as baby food.

[0039] As used in this article, the term "food for the elderly" refers to food formulated for the elderly.

[0040] As used in this article, the term "pregnancy food" refers to food formulated for pregnant women.

[0041] As used in this article, the term "nutritional supplement" refers to a food that is formulated as a diet or nutritional supplement for use as part of a diet. Invention Details This invention provides an improved method for extracting and preparing lipids from biological sources for use in pharmaceuticals, nutritional products, and functional foods.

[0042] In some preferred embodiments, the present invention provides a multi-stage extraction process designed to provide neutral and polar lipid extracts from starting biological materials. In some embodiments, the isolated neutral and polar lipid extracts are subsequently combined to provide a lipid composition having the desired neutral and polar lipid content.

[0043] In some embodiments of the invention, lipids are extracted in a single step using a protonated solvent of a target concentration, adjusting it to the water content of the biomaterial. The polarity of the solvent can be balanced or the ratio of polar to neutral lipids optimized. The ratio of polar lipids can be further increased by utilizing a subsequent washing process with the protonated solvent described below. After the single-step extraction and subsequent 3-5 washing steps, when derived from a feedstock in which phospholipids contain 40% w / w lipids (i.e., weight of phospholipids / total weight of feedstock), the extract rich in polar lipids typically contains about 55-60% w / w phospholipids (i.e., weight of phospholipids / total weight of washed phospholipid extract). In other embodiments, the biomaterial is contacted with a stronger protonated solvent to obtain a lipid extract in which phospholipids are highly enriched.

[0044] In a preferred embodiment, the extraction is performed under conditions that allow polar lipids to be extracted into a liquid phase (i.e., a polar lipid solution) and neutral lipids to preferentially remain bound to the biomaterial. Proton solvents include any solvent having hydrogen atoms bound to oxygen (e.g., in hydroxyl groups) or nitrogen (e.g., in amine groups). Generally, solvents containing unstable H atoms are preferred. + Any solvent that readily accepts protons (H+) is called a proton solvent. The molecules of such solvents readily accept protons (H+). +The proton solvent is supplied as a reagent. In a preferred embodiment, the proton solvent is an organic proton solvent. Suitable organic proton solvents include, but are not limited to, n-butanol, n-propanol, isopropanol, nitromethane, ethanol, and methanol. In a particularly preferred embodiment, the proton solvent is ethanol. In a preferred embodiment, the biological material is extracted with the proton solvent at a temperature of about -10°C to about 50°C, preferably about 10°C to 30°C. In a preferred embodiment, the concentration of the proton solvent is at least greater than 90%, 95%, or 98% w / w (i.e., the weight of the proton solvent / total weight of the diluted solvent solution), or is about 100% pure. In some embodiments, the proton solvent is used at a ratio of about 1:1 to 10:1, preferably about 3:1 to 7:1, and most preferably about 5:1, of the biological material. In some preferred embodiments, the proton solvent is added such that when combined with water in the biological material, the concentration of the proton solvent is about 70% to about 95%. This step produces a polar lipid solution enriched with polar lipids (especially phospholipids) and a biological residue containing neutral lipids. The polar lipid solution and biological residue are preferably separated, for example, by centrifugation, filtration, or other suitable methods.

[0045] In some embodiments, an aqueous solution is added to the polar lipid solution to dilute the proton solvent. In a preferred embodiment, the dilution of the proton solvent results in the separation of the solution into an upper phase containing the diluted proton solvent and a lower bottom phase rich in polar lipids. In some embodiments, the concentration of the proton solvent is diluted to about 50% to 70% w / w (i.e., the weight of the proton solvent / the total weight of the diluted solvent solution), preferably to about 60% w / w. In some embodiments, the polar lipid-rich bottom phase is separated to provide a polar lipid-rich extract. The polar lipid-rich extract may then be washed or residual solvent and water may be removed, for example, by evaporation.

[0046] In some embodiments, the extract rich in polar lipids is washed with a proton solvent solution (preferably containing the same proton solvent used in the initial extraction step). The proton solvent is preferably used at a concentration in which the polar lipids (especially phospholipids) are poorly soluble. In a preferred embodiment, the washing step provides an upper phase containing the proton solvent solution and a lower phase rich in polar lipids, which provides the extract rich in polar lipids upon separation. In each washing step, the extract rich in polar lipids separated from the previous step is remixed with a fresh proton solvent solution, and preferably the extract rich in polar lipids is separated again. In a preferred embodiment, the proton solvent solution used in the washing step contains about 30% to about 70% w / w of proton solvent (i.e., the weight of the proton solvent / the total weight of the diluted solvent solution), preferably about 40% to 60% w / w, with the remainder of the solution being water or other suitable diluents for the proton solvent. In a preferred embodiment, the washing step uses a ratio of about 0.5:1 to 5:1 of polar lipid extract to diluted proton solvent. The washing step may be repeated multiple times, for example, at least 2, 3, 4, 5 times or up to about 10 times, preferably about 2 to 5 times. The washing process results in the washing of an extract rich in polar lipids.

[0047] In some embodiments, even purer phospholipid concentrates are provided. These concentrates can be obtained by subjecting an extract rich in polar lipids, as described in this invention, to precipitation processes known in the art. In some embodiments, the precipitation step is carried out with pure acetone at a temperature of about -20°C to about +20°C, more preferably at about 4°C, to provide a precipitate rich in polar lipids. In some embodiments, the precipitation step is repeated, for example, 2 to 5 times, preferably about 3 times, to provide a precipitate rich in polar lipids. This technique removes virtually all remaining neutral lipids. In other embodiments, the phospholipids can be concentrated by chromatography, for example, silica gel chromatography.

[0048] In some embodiments, residual solvents and water are preferably removed from the washed polar lipid-rich extract or precipitate by vacuum evaporation to provide a solid polar lipid-rich composition.

[0049] In some embodiments, the washed polar lipid-rich extract is preferably characterized by containing at least 50%, 60%, 70%, or 75% w / w phospholipids (i.e., weight of phospholipids / total weight of the washed polar lipid-rich extract). In some embodiments, the washed polar lipid-rich extract contains about 30% to 99% w / w, 40% to 60% w / w, or 50% to 70% w / w phospholipids. In some embodiments, the washed polar lipid-rich extract is characterized by containing less than about 3%, 2%, 1%, 0.5%, or 0.1% w / w lysophospholipids (i.e., weight of lysophospholipids / total weight of the washed polar lipid-rich extract). In some embodiments, the washed polar lipid-rich extract is characterized by having a conductivity of less than 500 μS / cm, preferably less than 300 μS / cm, and most preferably less than 200 μS / cm (measured in a saturated 60% ethanol solution).

[0050] In some embodiments, the polar lipid-rich precipitate is preferably characterized by comprising at least 75%, 80%, 85%, or 80% w / w phospholipids (i.e., weight of phospholipids / total weight of the polar lipid-rich precipitate). In some embodiments, the washed polar lipid-rich extract is characterized by containing less than about 3%, 2%, 1%, 0.5%, or 0.1% w / w lysophospholipids (i.e., weight of lysophospholipids / total weight of the polar lipid-rich precipitate).

[0051] The krill oil compositions rich in polar lipids obtained from the method differ from naturally occurring krill oil. However, the lysophospholipid content is preferably low, and the present invention provides for an increase in lysophospholipid content (compared to available krill oil). In some embodiments, the compositions contain a lower limit of phospholipids greater than 0.01%, 0.05%, 0.1%, or 0.2% w / w (i.e., weight of lysophospholipids / total weight of the polar lipid-rich precipitate). Regarding the lysophospholipid content, 2-lysophospholipids are generally dominant. Free fatty acids indicate the presence of C-14, C-16, and C-18 fatty acids, rather than EPA and DHA, as expected after hydrolysis at the SN-2 position. It is conceivable that this degradation pathway involves hydrolysis at the SN-1 position followed by in vitro trans-esterification to produce 2-lysophospholipids. The krill oil compositions rich in polar lipids obtained from the method of the present invention are also oxidized to a certain extent, as evidenced by the presence of low levels of polyphospholipids not found in nature and certain aldehydes.

[0052] The krill oil compositions rich in polar lipids obtained from the method of the present invention also exhibit altered astaxanthin content. Astaxanthin is degraded by oxidation and heating, and the astaxanthin diester:monoester ratio shifts during processing due to aprotic solvent washing. The diester form is predominant in krill. This shift in astaxanthin content can be determined by measuring the ratio between the absorbance at 487 nm and 390 nm. In some embodiments, the astaxanthin monoester:astaxanthin diester ratio is greater than about 2:1, 3:1, 4:1, 5:1, or 10:1. In other embodiments, the range of the astaxanthin monoester:astaxanthin diester ratio is preferably about 2:1 to 100:1, 3:1 to 50:1, 4:1 to 20:1, 5:1 to 20:1, or 10:1 to 50:1. In some embodiments, the polar lipid-rich compositions contain less than about 50, 40, 30, 20, 10, or 5 mg / kg (ppm) of astaxanthin diester. The use of aprotic solvents in the extraction and washing methods described herein can also lead to the formation of fatty acid esters, such as ethyl fatty acids, not found in krill. An advantage of the methods described herein is that these fatty acid esters are typically removed to extremely low levels by the washing step. Therefore, in some embodiments, the compositions of the present invention contain less than about 1.0%, 0.5%, 0.1%, 0.05%, or 0.01% w / w of fatty acid esters, preferably ethyl esters (i.e., the weight of ethyl esters or other esters / the total weight of the composition rich in polar lipids). In some embodiments, the compositions have a lower limit of trace fatty acid esters (preferably ethyl esters), and in some embodiments, contain more than about 0.001% or 0.005% of fatty acid esters, such as ethyl fatty acids.

[0053] In some embodiments, the polar lipid-rich composition is preferably characterized by containing at least 70%, 80%, 85%, 90%, or 95% w / w phospholipids (i.e., weight of phospholipids / total weight of the solid polar lipid-rich composition). In some embodiments, the solid polar lipid-rich composition is characterized by containing less than about 3%, 2%, 1%, 0.5%, or 0.1% w / w lysophospholipids (i.e., weight of lysophospholipids / total weight of the solid polar lipid-rich composition).

[0054] In some embodiments, the washed, polar lipid-rich extract, polar lipid-rich precipitate, and solid polar lipid-rich composition are further characterized by comprising a mixture of phospholipid compounds having the following structure: Wherein R1 and R2 are selected from the group consisting of fatty acid moieties and -H, and R3 is H or selected from choline, ethanolamine, inositol, or serine moieties, the mixture of phospholipid compounds contains more than about 85% or 90% of the choline moieties (i.e., mol%, moles of choline moieties / total moles of phospholipid compounds) and more than about 30% w / w of the ω-3 fatty acid moieties (i.e., weight of the ω-3 fatty acid moieties / weight of the phospholipid compounds) at position R3, wherein more than about 90% w / w of the ω-3 fatty acid moieties (i.e., weight of the ω-3 fatty acid moieties / total weight of fatty acid moieties) is at position R2. In some embodiments, the composition is characterized by containing less than about 3%, 2%, 1%, 0.5%, or 0.1% w / w of lysophospholipids (i.e., weight of lysophospholipids / total weight of the solid, polar lipid-rich composition).

[0055] In some embodiments, the ω-3 fatty acid portion is preferably selected from the group consisting of eicosapentaenoic acid, docosahexaenoic acid, and combinations thereof. In some embodiments, the ω-3 fatty acid moiety is selected from the group consisting of: eicosatrienoic acid (ETE; 20:3 (n−3); all-cis-11,14,17-eicosatrienoic acid); eicosatraenoic acid (ETA; 20:4 (n−3); all-cis-8,11,14,17-eicosatraenoic acid); eicosapentaenoic acid (EPA; 20:5 (n−3); all-cis-5,8,11,14,17-eicosapentaenoic acid); docosapentaenoic acid (HPA; 21:5 (n−3); all-cis-6,9,12,15,18-docosapentaenoic acid); docosapentaenoic acid (DPA; 22:5 (n−3); all-cis-7,10,13,16,19-docosapentaenoic acid); docosahexaenoic acid (DHA; 22:6) (n−3); all-cis-4,7,10,13,16,19-docosahexaenoic acid); tetradocosapentaenoic acid (24:5 (n−3); all-cis-9,12,15,18,21-docosapentaenoic acid; and tetradocosahexaenoic acid (24:6 (n−3); all-cis-6,9,12,15,18,21-docosahexaenoic acid). In some embodiments, the ω-3 fatty acid moiety is bonded to the R1 or R2 position via an ester bond (to provide an acylphospholipid), while in other embodiments, the ω-3 fatty acid moiety is bonded via an ether bond or a vinyl ether bond (to provide an ether phospholipid, an alkyl acylphospholipid, or an alkenyl acylphospholipid).

[0056] In some embodiments, the washed, polar lipid-rich extract comprises at least 50% w / w, 60% w / w, 75% w / w, or 90% w / w of a phospholipid compound, or about 30% to 99%, 40% to 60%, or 50% to 70% of a phospholipid compound (i.e., weight of the phospholipid compound / total weight of the phospholipid composition). In some embodiments, the polar lipid-rich precipitate comprises at least 75% w / w, 80% w / w, 85% w / w, or 90% w / w of a phospholipid compound, or about 60% to 99%, 70% to 95%, or 80% to 95% of a phospholipid compound (i.e., weight of the phospholipid compound / total weight of the polar lipid-rich precipitate). In some embodiments, the solid polar lipid-rich composition comprises at least 80% w / w, 85% w / w, 90% or 95% w / w of phospholipid compounds, or about 60% to 99%, 80% to 99%, or 85% to 99% of phospholipid compounds (i.e., weight of phospholipid compounds / total weight of the solid polar lipid-rich composition).

[0057] In some embodiments, the ω-3 portion of the mixture of phospholipid compounds is eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and wherein the EPA and DHA are present in a ratio of EPA:DHA of about 1:1 to about 3:1. In some embodiments, the composition is partially or entirely derived from krill.

[0058] In other embodiments, the fatty acid content of the extract, precipitate, and composition is, by weight / weight (w / w; calculated as the weight of the ω-3 fatty acid fraction in the phospholipid fraction divided by the total weight of the fatty acids in the phospholipid fraction) or by molar ratio (the number of moles of the ω-3 fatty acid fraction in the composition as a percentage of the total number of moles of fatty acids), from about 1% to about 99% of the ω-3 fatty acid fraction, 10% to 40% w / w or molar ratio of the ω-3 fatty acid fraction, 20% to 40% w / w or molar ratio of the ω-3 fatty acid fraction, 20% to 50% w / w or molar ratio of the ω-3 fatty acid fraction, 40% to 60% w / w or molar ratio of the ω-3 fatty acid fraction, 40% to 99% w / w or molar ratio of the ω-3 fatty acid fraction, 60% to 99% w / w or molar ratio of the ω-3 fatty acid fraction, or 80% to 99% w / w or molar ratio of the ω-3 fatty acid fraction. The w / w% can preferably be determined by an analytical method selected from the group consisting of gas chromatography (GC), high performance liquid chromatography (HPLC), GC-mass spectrometry (GC-MS), nuclear magnetic resonance (NMR), or other suitable methods known in the art. In some preferred embodiments, the ω-3 fatty acid fraction is preferably selected from DHA, EPA, and combinations thereof. In some embodiments, more than 90% w / w of the ω-3 fatty acid fraction, preferably more than 95% w / w, and most preferably more than about 98% w / w, of the ω-3 fatty acid fraction is distributed at the R2 position. In some preferred embodiments, the ω-3 fatty acid fraction is greater than 50%, 60%, 70%, 80%, 90%, or 95% w / w of EPA and / or DHA (i.e., the weight of EPA and / or DHA / the total weight of the ω-3 fatty acid fraction). In some embodiments, the EPA:DPA ratio is about 10:1 to 1:10, 3:1 to 1:3, 5:1 to 1:1, 3:1 to 1:1, 2:1 to 1:1, 1:1 to 1:3, or 1:1 to 1:5 on a molar basis. In some embodiments, the extract and composition comprise greater than about 40%, 50%, 60%, 70%, 80%, 90%, or 95% w / w of a phospholipid compound (i.e., the weight of the phospholipid compound / the total weight of the phospholipid composition).

[0059] In some embodiments, the bioresidue from the initial extraction step is extracted to provide a neutral lipid extract. This extraction step preferably uses a protonated solvent, most preferably the same protonated solvent used in the initial extraction step. In a preferred embodiment, the bioresidue is extracted with a protonated solvent at a temperature of about 15°C to about 40°C, preferably about 20°C to about 30°C. In a preferred embodiment, the concentration of the protonated solvent is at least 90%, 95%, or 98% w / w (i.e., volume of protonated solvent / total volume of diluted solvent) or close to 100% pure. In some embodiments, the protonated solvent is used at a ratio of about 1:1 to 10:1, preferably about 3:1 to 7:1, and most preferably about 5:1, of the bioresidue. This step produces a neutral lipid-enriched neutral lipid extract and lipophilic substances such as astaxanthin and vitamin E (if present in the bioresidue).

[0060] In some embodiments, the neutral lipid extract preferably comprises a mixture of triacylglycerols, diacylglycerols, and free fatty acids. In a phospholipid concentrate comprising about 60% by weight of phospholipids (i.e., the weight of phospholipids / total weight of the concentrate), the neutral phase preferably comprises about 70-80% w / w triacylglycerols (i.e., the weight of triacylglycerols / total weight of the concentrate), about 10-15% w / w free fatty acids (i.e., the weight of free fatty acids / total weight of the concentrate), about 2-5% w / w diacylglycerols (i.e., the weight of diacylglycerols / total weight of the concentrate), and about 1-2% w / w minor components (i.e., the weight of minor components / total weight of the concentrate), such as cholesterol and astaxanthin. In a phospholipid concentrate containing more than about 80-90% w / w phospholipids (i.e., the weight of phospholipids / total weight of the concentrate) from an extract obtained from krill coagulants, the neutral phase preferably contains about 70-80% w / w diacylglycerols (i.e., the weight of diacylglycerols / total weight of the concentrate), about 10-25% w / w triacylglycerols (i.e., the weight of triacylglycerols / total weight of the concentrate), and about 3-6% w / w free fatty acids (i.e., the weight of free fatty acids / total weight of the concentrate).

[0061] Washed extracts rich in polar lipids, precipitates rich in polar lipids, solid compositions rich in polar lipids, and neutral lipid extracts can be used as is, or they can be combined to provide a mixed lipid composition with desired polar and neutral lipid contents. In a preferred embodiment, the mixed lipid composition has a defined phospholipid and triglyceride content. In some preferred embodiments, the mixed lipid composition has a phospholipid content of about 30% to 60% w / w, 35% to 50% w / w, or 36% to 44% w / w (i.e., weight of phospholipids / total weight of the mixed lipid composition), and a triglyceride content of about 40% to 70% w / w, 50% to 65% w / w, or 56% to 64% w / w (i.e., total weight of triglycerides / total weight of the mixed lipid composition). In some embodiments, the mixed lipid composition contains astaxanthin, preferably neutral astaxanthin provided from a neutral lipid extract. In some embodiments, the lipid composition provides at least a second antioxidant such as vitamin E. In some embodiments, the lipid composition may be supplemented with additional ω-3 fatty acid derivatives such as EPA or DHA ethyl ester, preferably EPA and / or DHA ethyl ester concentrates, or triglycerides or diglycerides containing EPA and DHA residues. In these embodiments, the additional ω-3 fatty acid derivatives are added to provide the desired ω-3 fatty acid content in the lipid composition.

[0062] This invention is not limited to the use of any particular biological material. The biological material can preferably be algal biomass, plant biomass, or marine animal biomass or produced therefrom. In a preferred embodiment, marine animal biomass is used as the raw material. Suitable marine animal biomass includes, but is not limited to, krill, crab, Daphnia, plankton, eggs, crayfish, shrimp, fish, especially herring, and marine algae. The biological material can be fresh or frozen, or can be material derived from algae, plants, or marine animal biomass, such as powder, concentrate, hydrolysate, or coagulate (paste). The paste can be a wet paste or a dry paste. In some preferred embodiments, the biological material is krill material, such as krill coagulate, krill powder, krill hydrolysate, or fresh or frozen krill. Any species of krill can be used. In a preferred embodiment, the krill is Antarctic krill or Pacific krill.

[0063] In some particularly preferred embodiments, the biomaterial is krill paste or coagulant, and may be wet or dry. Suitable krill paste is described, for example, in WO 09 / 027692 (which is incorporated herein by reference in its entirety). In some embodiments, the biomass (preferably krill, freshly harvested or frozen) is heated to a temperature in the range of 25 to 80°C, preferably 40 to 75°C, and most preferably 60 to 75°C, to dissolve / disperse lipids and proteins from the krill into the aqueous phase. In some embodiments, proteins and phospholipids are precipitated from the aqueous phase by heating water (after removing the krill) to a temperature above about 80°C, preferably 80 to 120°C, and most preferably 95 to 100°C. The aqueous phase may be heated at atmospheric pressure or in a closed system under elevated pressure so that the temperature can be raised above 100°C. The formed precipitate (hereinafter referred to as coagulant) can be separated and characterized. In other embodiments, the aqueous phase is microfiltered. The solid phase produced by microfiltration (called the residue) is similar to the solid phase of the coagulated material. Public data show that many phospholipids in krill are transferred from krill to the coagulated material or residue.

[0064] The krill paste composition is characterized as a protein-lipid mixture. In a preferred embodiment, the composition comprises about 10% to about 30% phospholipids on a dry w / w basis and about 20% to 50% protein on a dry w / w basis, wherein the phospholipids contain ω-3 fatty acid residues. In some embodiments, the composition comprises a lipid fraction having an ω-3 fatty acid content of about 10% to about 25% on a dry w / w basis. In some embodiments, the phospholipids comprise more than about 90% phosphatidylcholine on a dry w / w basis. In some embodiments, the phospholipids comprise less than about 10% ethanolamine on a dry w / w basis. In some embodiments, the paste composition comprises about 20% to about 45% triacylglycerols on a dry w / w basis. In some embodiments, the composition comprises less than about 1% w / w cholesterol (i.e., weight of cholesterol / total weight of krill paste). In some embodiments, the composition comprises about 0.01 to about 200 mg / kg of naturally occurring astaxanthin. It should be understood that the astaxanthin content of the composition can be increased by adding astaxanthin from other (exogenous) sources (natural and non-natural). Similarly, the composition can be supplemented during processing with exogenous triglycerides, phospholipids, and fatty acids such as omega-3 fatty acids. In some preferred embodiments, the krill paste is a wet krill paste containing a concentration of about 50% to 80%, 60% to 70%, or about 65% w / w of water (i.e., weight of water / total weight of krill paste).

[0065] In some embodiments, the present invention provides a composition comprising the above-described extract, mixed lipid composition, or concentrate rich in polar lipids and one or more additional ω-3 fatty acid derivatives or free fatty acids. The ω-3 fatty acid derivatives or free fatty acids may be derived from neutral lipid extracts or other sources, such as fish oil or ω-3 ester concentrates. In some embodiments, the one or more additional ω-3 fatty acid derivatives are selected from ω-3 esters and glycerides. For example, in some embodiments, the composition may comprise about 1% to about 60% w / w phospholipids (i.e., the weight of the phospholipid compound / total weight of the composition), with the remaining 99% to 40% w / w of the composition being ω-3 glycerides, esters, or free fatty acids or combinations thereof (i.e., the weight of ω-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 phospholipids, with the remaining 95% to 40% w / w of the composition being ω-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 composition being ω-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 composition being ω-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 composition being ω-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 composition being ω-3 glycerides, esters, or free fatty acids or combinations thereof.

[0066] In some embodiments, administration of the above-described compounds or compositions to a subject in need is intended to treat diseases or conditions related to red blood cells and cell membranes, particularly those related to abnormalities in the cell membranes of red blood cells. In some embodiments, the condition or disease is sickle cell disease, sickle cell anemia, or sickle cell phenotype. In some embodiments, the condition or disease is thalassemia (α-, β-, or Δ-), thalassemia combined with hemoglobinopathies (hemoglobin E, hemoglobin S, or hemoglobin C), splenomegaly, or membrane abnormalities such as acanthocytes or spike cells, target cells, serrated cells, oval and ovoid red blood cells, spheroids, stomatocytes (oral cells), and bite cells (“bite cells”).

[0067] In some embodiments, an effective amount of the above-described compound or composition is administered to a subject in need to treat or prevent cardiovascular disease or metabolic syndrome. In some embodiments, the cardiovascular disease is selected from atherosclerosis, arteriosclerosis, coronary heart disease (carotid artery) (CHD or CAD), acute coronary syndrome (or ACS), valvular heart disease, aortic and mitral valve disease, arrhythmia / atrial fibrillation, cardiomyopathy and heart failure, angina pectoris, acute myocardial infarction (or AMI), hypertension, orthostatic hypotension, shock, embolism (pulmonary and venous), endocarditis, diseases of the arteries, aorta and its branches, diseases of the peripheral vascular system (peripheral artery disease or PAD), Kawasaki disease, congenital heart disease (cardiovascular defects) and stroke (cerebrovascular disease), dyslipidemia, hypertriglyceridemia, hypertension, heart failure, arrhythmia, low HDL levels, high LDL levels. Stable angina, coronary artery disease, acute myocardial infarction, secondary prevention of myocardial infarction, cardiomyopathy, endocarditis, type 2 diabetes, insulin resistance, impaired glucose tolerance, hypercholesterolemia, stroke, hyperlipidemia, hyperlipoproteinemia, chronic kidney disease, intermittent claudication, hyperphosphatemia, omega-3 deficiency, phospholipid deficiency, carotid atherosclerosis, peripheral artery 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 arteriosclerosis, arteriosclerosis, cerebrovascular disease, myocardial ischemia, coagulopathy leading to thrombosis in blood vessels, and diabetic autonomic neuropathy.

[0068] In some embodiments, an effective amount of the above-described compound or composition is administered to a subject in need to treat, prevent, or improve cognitive and / or cognitive disorders, conditions, or impairments (memory, attention, learning (deficiencies)), or to treat or prevent neurodegenerative conditions. In some embodiments, the cognitive disorder, condition, or impairment is selected from Attention Deficit Disorder (ADD), Attention Deficit Hyperactivity Disorder (ADHD), Autism Spectrum Disorder (ASD), (dyslexia, age-related memory and learning disabilities, amnesia, mild cognitive impairment, cognitive impairment without dementia, pre-Alzheimer's disease, Alzheimer's disease, epilepsy, Pick's disease, Huntington's disease, Parkinson's disease, Luggage disease, pre-dementia syndrome, Lewy body dementia, dentate nucleus-rubella-lewy body atrophy, Freidreich ataxia, multiple system atrophy, etc. Type 1, 2, 3, 6, and 7 spinocerebellar ataxia, amyotrophic lateral sclerosis (ALS), familial spastic hemiparesis, spinal muscular atrophy, spinal cord and medullary muscular atrophy, age-related cognitive decline, cognitive decline, moderate mental disorders, age-related mental decline, conditions affecting EEG intensity and / or brain glucose utilization, stress, anxiety, impaired concentration, mood deterioration, general cognitive and mental health status, neurodevelopmental disorders, neurodegenerative diseases, hormonal imbalances, neurological disorders, or any combination thereof. In the specific implementation plan, cognitive impairment is memory impairment.

[0069] In some embodiments, an effective amount of the above-described compound or composition is administered to a subject in need to inhibit, prevent, or treat inflammation or inflammatory diseases. In some embodiments, the inflammation or inflammatory disease is selected from organ transplant rejection; reoxygenation injury resulting from organ transplantation (including, but not limited to, transplantation of the heart, lungs, liver, and kidneys) (see Grupp et al., J. Mol. Cell. Cardiol. 31: 297-303 (1999)); chronic inflammatory diseases of the joints, including arthritis, rheumatoid arthritis, osteoarthritis, and bone diseases associated with increased bone resorption; inflammatory bowel diseases (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, including corneal dystrophy, trachoma, onchocerciasis, uveitis, sympathetic ophthalmia, and endophthalmitis; and chronic inflammatory diseases of the gingiva. Inflammatory diseases include gingivitis and periodontitis; inflammatory kidney diseases, including uremia complications, glomerulonephritis, and nephropathy; inflammatory skin diseases, including sclerosing dermatitis, psoriasis, and eczema; inflammatory diseases of the central nervous system, including 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. Inflammatory diseases can also be systemic inflammation of the body, such as Gram-positive or Gram-negative shock, hemorrhagic or anaphylactic shock, or shock induced by cancer chemotherapy in response to pro-inflammatory cytokines, such as shock associated with pro-inflammatory cytokines. Such shock can be induced, for example, by chemotherapeutic agents administered as a treatment for cancer. Other conditions include depression, obesity, allergic diseases, acute cardiovascular events, wasting diseases, and cancer cachexia. Concentrated therapeutic phospholipid compositions can also be used to treat inflammation caused by surgery and trauma.

[0070] In some embodiments, the effective amount comprises about 0.1 to about 5 grams of a washed phospholipid composition or a mixed lipid composition, preferably about 0.2 to about 3 grams of a washed phospholipid composition or a mixed lipid composition, and most preferably about 0.5 to about 1.5 grams of a washed phospholipid composition or a mixed lipid composition.

[0071] The washed phospholipid extract compositions or mixed lipid compositions (i.e., lipid compositions) of the present invention can be used to treat a variety of subjects. Suitable subjects include humans as well as domestic animals, non-human primates, and companion animals such as dogs, cats, and birds.

[0072] The lipid compositions of the present invention are preferably administered intravenously or orally. Therefore, in some embodiments, the compositions of the present invention (such as those described in the preceding sections) are contained in acceptable excipients and / or carriers for oral consumption or intravenous administration. The actual form of the carrier and thus the composition itself is not critical. The carrier can be a liquid, gel, capsule, powder, solid tablet (coated or uncoated), tea, etc. The composition is preferably in tablet or capsule form, most preferably in soft gel capsule form. Suitable excipients and / or carriers include vegetable oils, fish oil, krill oil, maltodextrin, calcium carbonate, dicalcium phosphate, tricalcium phosphate, microcrystalline cellulose, glucose, rice flour, magnesium stearate, stearic acid, croscarmellose sodium, sodium hydroxyethyl starch, crospovidone, sucrose, plant gum, lactose, methylcellulose, povidone, carboxymethyl cellulose, corn starch, etc. (including mixtures thereof). Preferred carriers include calcium carbonate, magnesium stearate, maltodextrin, and mixtures thereof. Various ingredients and excipients and / or carriers are mixed and formed into the desired form using conventional techniques. The tablets or capsules 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 regarding the techniques used for formulation and administration can be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA). For intravenous or oral administration, the ω-3 compounds and compositions of the present invention are preferably provided as emulsions.

[0073] In some embodiments, the lipid composition is formulated for oral administration with a flavoring agent or sweetener. Examples of available flavoring agents include, but are not limited to, pure star anise extract, banana-like extract, cherry-like extract, chocolate extract, pure lemon extract, pure orange extract, pure peppermint extract, pineapple-like extract, rum-like extract, strawberry-like extract, or pure vanilla extract; or volatile oils such as mesona chinensis oil, laurel oil, bergamot oil, cypress oil, walnut oil, cherry oil, cinnamon oil, clove oil, or peppermint oil; peanut butter, chocolate flavoring, vanilla biscuit crumbs, caramel, or toffee. In one embodiment, the dietary supplement contains cocoa or chocolate. Emulsifiers may be added to stabilize the stability of the finished product. Examples of suitable emulsifiers include, but are not limited to, lecithin (e.g., from eggs or soy), and / or mono- and di-glycerides. Other emulsifiers will readily apparent to those skilled in the art, and the selection of a suitable emulsifier will depend in part on the formulation and the finished product. In addition to the carbohydrates mentioned above, nutritional supplements may also contain natural or artificial (preferably low-calorie) sweeteners, such as sugars, cyclamate, aspartamine, aspartame, acesulfame potassium, and / or sorbitol.

[0074] The lipid compositions of the present invention can also be delivered as dietary supplements, nutritional supplements or functional foods.

[0075] The dietary supplement may contain one or more inert ingredients, particularly when it is desirable to limit the number of calories added to the diet by the dietary supplement. For example, the dietary supplement of the present invention may also contain optional ingredients, including, for example, herbs, vitamins, minerals, enhancers, colorants, sweeteners, flavorings, inert ingredients, etc. For example, the dietary supplement of the present invention may contain one or more of the following substances: ascorbic acid salts (ascorbic acid, ascorbic acid mineral salts, rosehip, golden sesame, etc.), dehydroepiandrosterone (DHEA), green tea (polyphenols), inositol, kelp, red algae, bioflavonoids, maltodextrin, nettle, niacin, nicotinamide, rosemary, selenium, silica (silica, silica gel, horsetail, horsetail grass, etc.), spirulina, zinc, etc. Such optional ingredients may be naturally occurring or in concentrated form.

[0076] In some implementations, the dietary supplement also contains vitamins and minerals, including, but not limited to, tricalcium phosphate or acetate; dipotassium phosphate; magnesium sulfate or magnesium oxide; salt (sodium chloride); potassium chloride or potassium acetate; ascorbic acid; ferric orthophosphate; nicotinamide; zinc sulfate or zinc oxide; calcium pantothenate; copper gluconate; riboflavin; beta-carotene; pyridoxine hydrochloride; thiamine mononitrate; folic acid; biotin; chromium chloride or chromium picolinate; potassium iodide; sodium selenate; sodium molybdate; phylloquinone; vitamin D3; cyanocobalamin; sodium selenite; copper sulfate; vitamin A; vitamin C; inositol; and potassium iodide. Appropriate dosages of vitamins and minerals can be obtained, for example, by consulting the U.S. RDA guidelines.

[0077] In other embodiments, the present invention provides nutritional supplements (e.g., energy bars or meal replacement bars or beverages) composed of the lipid compositions of the present invention. In a preferred embodiment, the nutritional supplement contains an effective amount of the components described above. The nutritional supplement can be used as a dietary or snack substitute and typically provides nutritional calories. Preferably, the nutritional supplement provides carbohydrates, protein, and fat in balanced amounts. The nutritional supplement may also contain carbohydrates, monosaccharides, medium-chain sugars, or polysaccharides, or combinations thereof. The desired sensory properties of the monosaccharides can be selected. Uncooked corn starch is an example of a complex carbohydrate. If it is desired that it should retain its high molecular weight structure, it should only be included in food preparations or its uncooked or heat-treated portions, as heat will break down complex carbohydrates into simpler carbohydrates, which are monosaccharides or disaccharides. In one embodiment, the nutritional supplement contains a combination of carbohydrate sources having tertiary chain lengths (simple, moderate, and complex; e.g., sucrose, maltodextrin, and uncooked corn starch).

[0078] In other embodiments, the present invention provides foods, pre-prepared foods, or grains (i.e., functional foods) comprising the lipid compositions of the present invention. In preferred embodiments, the foods comprise effective amounts of the components as described above. For example, in some embodiments, beverages and solid or semi-solid foods containing fatty acids or derivatives thereof are provided. These forms may include, but are not limited to, beverages (e.g., soft drinks, milk and other dairy beverages and fat-reducing drinks), baked goods, puddings, dairy products, confectionery, snacks or frozen desserts or novelty foods (e.g., ice cream, milkshakes), pre-prepared frozen foods, confectionery, snack products (e.g., potato chips), soups, pastes, sauces, salad dressings, pre-prepared meat products, cheese, yogurt, and any other foods containing fat or oil, as well as food ingredients (e.g., wheat flour).

[0079] In some preferred embodiments, the lipid composition is incorporated into a chewable matrix. Preferred chewable matrices are gummies and gelatin-based gummy candies. Exemplary gummy candies include gummy bears, worms, frogs, hamburgers, cherry gummy candies, soda bottle gummy candies, shark gummy candies, soldier gummy candies, hippo gummy candies, lobster gummy candies, watermelon gummy candies, octopus gummy candies, apple gummy candies, peach gummy candies, and orange gummy candies. The terms "gummy candy" and "gummy-like" are used interchangeably herein.

[0080] In some particularly preferred embodiments, the chewable matrix material is a sweetener commonly referred to as gummy candy or jelly material. Gummy candy or jelly candy is a broad, general type of gelatin-based chewy candy. Rubber bears are the most popular and well-known gummy candy. Other shapes are also provided, and sometimes gummy candy is combined with other forms of candy such as marshmallows and chocolate and fermented.

[0081] In a preferred embodiment, the chewable matrix material comprises a gelling agent, which can be any physiologically tolerable gelling agent (preferably a sugar (e.g., oligosaccharides or polysaccharides), protein, or glycoprotein) or combination thereof capable of forming a soft, chewable, self-supporting chewing gum. Many such materials are known from the food and pharmaceutical industries and are discussed, for example, in Handbook of Hydrocolloids, edited by GO Phillips and PA Williams, Woodhead Publishing, Cambridge, UK, 2000. The gelling agent is preferably a material capable of undergoing a sol-gel transition, for example, under the influence of changes in physicochemical parameters such as temperature, pH, and the presence of metal ions (e.g., group 1 or 2 metal ions). Preferred gelling agents include gelatin, alginate, and carrageenan. However, the use of gelatin is particularly preferred because it ensures that the captured fragments are broken down in the throat, and because the nucleus with the desired properties can be readily produced using gelatin.

[0082] Gelatin, which can be produced from collagen of any mammal or any aquatic species and used as a gelling agent in the chewable matrix of the present invention, is preferred, however, the use of gelatin from saltwater fish, particularly cold-water and warm-water fish. Gelatin having an amino acid content of 5 to 25% by weight is preferred, more particularly those having an amino acid content of 10 to 25% by weight. Gelatin typically has a weight-average molecular weight in the range of 10 to 250 kDa, preferably 75 to 220 kDa, particularly 80 to 200 kDa. Gelatin with no Bloom value or a low Bloom value of 60-300, 150-300, particularly 90-200 is preferred. When using gelatin with no Bloom value, such as cold-water fish gelatin, it is usually used in conjunction with another gelatin or other gelling agent. A combination of cold-water fish gelatin and warm-water fish gelatin is particularly preferred. Gelatin is typically present in the aqueous phase at a concentration of 1 to 50% by weight, preferably 2 to 35% by weight, and preferably 5 to 25% by weight. In the case of a mixture of gelatin and polysaccharides, the weight ratio of gelatin to polysaccharides in the aqueous phase is typically 50:1 to 5:1, preferably 40:1 to 9:1, and especially 20:1 to 10:1.

[0083] When polysaccharides, or mixtures of polysaccharides and gelatin, are used as gelling agents, natural, synthetic, or semi-synthetic polysaccharides are preferred, such as polysaccharides and their derivatives and fragmentation products derived from plants, fish, terrestrial mammals, algae, bacteria. Typical marine polysaccharides include carrageenan, alginate, agar, and chitosan.

[0084] Typical plant polysaccharides include pectin. Typical microbial polysaccharides include gelling sugars and stearans. The use of charged, such as electrostatically charged and / or sulfated polysaccharides is preferred, as is the use of marine polysaccharides, particularly carrageenan and alginate, especially carrageenan. The carrageenan family, including ι- and κ-carrageenan, is a family of linear sulfated polysaccharides derived from red algae. The repeating disaccharide units in κ-carrageenan are β-D-galactose-4-sulfate and 3,6-ahydro-α-D-galactose, while the repeating disaccharide units in ι-carrageenan are β-D-galactose-4-sulfate and 3,6-ahydro-α-D-galactose-2-sulfate. Both κ- and ι-carrageenan are used in food preparation. Carrageenan is used as a stabilizer, emulsifier, gelling agent, and fat substitute.

[0085] Both ι and κ carrageenan form salt- or cold-curing reversible gels in aqueous environments. The coil-helical transition and helical aggregation form a gelatin network. κ carrageenan possesses specific binding sites for monovalent cations, leading to gel formation, and its shear modulus and elastic modulus follow a Cs-like relationship. + >K + >>Na + >Li +The order decreases. Generally, increasing salt concentration increases the elastic modulus of κ carrageenan as well as its solidification and melting temperatures. When κ carrageenan is used according to the invention, for example at concentrations up to 100 mM, more particularly up to 50 mM, the use of water-soluble potassium, rubidium, or cesium compounds, especially potassium compounds, particularly naturally occurring compounds (e.g., salts), is preferred. Salt-dependent conformational changes have also been observed for ι carrageenan. It is also known that the molecule in polyvalent cations such as Ca... 2+ In its presence, it undergoes a coil-helix transition with strong helical stability. When carrageenan is used according to the invention, for example at a concentration up to 100 mM, the use of water-soluble calcium, strontium, barium, iron, or aluminum compounds, especially calcium compounds, particularly naturally occurring compounds (e.g., salts), is preferred.

[0086] The polysaccharide gelling agents used according to the invention typically have a weight-average molecular weight of 5 kDa to 2 MDa, preferably 10 kDa to 1 MDa, most preferably 100 kDa to 900 kDa, and particularly 200 to 800 kDa. They are typically used in the aqueous phase at a concentration of 0.01 to 5% by weight, preferably 0.1 to 1.5% by weight, and particularly 0.2 to 1% by weight. When monovalent or polyvalent cations (typically group I or group II metal ions) are contained in the aqueous phase, this is typically at a concentration in the range of 2.5 to 100 mM, particularly 5 to 50 mM.

[0087] In addition to gelling agents and water, and any desired gelling initiators, other physiologically tolerable materials may also be present in the chewable matrix, such as emulsifiers, emulsion stabilizers, pH adjusters, viscosity adjusters, sweeteners, fillers, vitamins (e.g., vitamin C, thiamine, riboflavin, niacin, vitamin B6, vitamin B12, folic acid, pantothenic acid), minerals, flavorings, seasonings, manufacturing agents, pigments, bioactive agents, etc., as described above in detail regarding additives that may be included in oxidizable fatty acid compositions.

[0088] The chewable matrix preferably has a gelation temperature in the range of 10 to 30°C, more preferably 15 to 28°C, and a melting temperature in the range of 20 to 80°C, more preferably 24 to 60°C, particularly 28 to 50°C.

[0089] When sweeteners are incorporated into a chewable matrix, they are typically selected from natural sweeteners such as sucrose, fructose, glucose, reduced glucose, maltose, xylitol, maltitol, sorbitol, mannitol, lactitol, isomaltitol, erythritol, hydrogenated glucose, polyglucanol, glycerin, stevia, agave nectar, invert sugar syrup, and artificial sweeteners such as aspartame, acetylsupanitate, neotame, saccharin, and sucralose. The use of non-cariogenic sweeteners is preferred, and the use of xylitol is particularly preferred. Preferred flavorings include orange, raspberry, cherry, lemon, blood orange, grapefruit, strawberry, blueberry, blackberry, and combinations thereof, especially orange and raspberry.

[0090] Mass production of gummy candies (such as gummy bears) involves mixing the gummy candy ingredients and pouring the resulting mixture into numerous scraper-lined (e.g., cornstarch-lined) trays / molds. The cornstarch prevents the gummy bears from sticking to the molds and makes them easy to remove once solidified. First, the desired lettering is produced and, if necessary, replicated by machine. Optionally, starch powder is applied to the lettering. The gummy candy ingredients, such as sugar, glucose syrup, gelatin, and water, are mixed together and heated. In one aspect, the ingredients are mixed with pigments and flavorings that give the bears their characteristic appearance and flavor. The molten gelatin mixture is poured into molds and allowed to cool and solidify before packaging or consumption. Preferably, the gummy candy is then heated and placed in a large drum mixer to apply a composition of isolated Bacillus coagulans and a sweetener (e.g., sugar).

[0091] In some preferred embodiments, the production of gummy candies includes the following: forming a colloid batch and a fruit puree batch, and combining them with corn syrup and sugar to form a matrix slurry. The colloid batch contains a solution of gelling agent in water at a level of 5 to 15% by weight of gelling agent, more preferably 7 to 12% by weight of gelling agent in total weight of the colloid batch. The colloid batch is maintained at a temperature of 170 to 190°F. The fruit puree batch preferably contains water, fruit puree and / or high-fructose corn syrup or other sweeteners, low-viscosity modified starch, and sodium citrate. It is maintained at a temperature of 65 to 75°F. Preferably, the fruit puree has a Brix value of 10 to 45, more preferably 25 to 40. Optionally, the fruit puree batch includes a variety of fruit purees. Fruit purees include typical fruit purees, juices, or fruit powders. The fruit puree batch comprises 30 to 40% water by weight, 0 to 40% fruit puree by weight, 0 to 40% high-fructose corn syrup by weight, 25% to 35% low-viscosity modified starch by weight, and 0.0 to 2.0% sodium citrate by weight. In a mixing vessel, 25 to 40% by weight of the additional corn syrup is combined with 15 to 40% by weight of fine granulated sugar, 10% to 15% by weight of colloid batch, and 20 to 30% by weight of fruit puree batch to form a matrix slurry. Preferably, the corn syrup is about 42 DE; however, other DE corn syrups may be used, as will be understood by those skilled in the art. The matrix slurry components are thoroughly mixed in a storage tank and maintained at 130 to 150°F.

[0092] The matrix slurry is then cooked to increase the Brix level from 70 to 85, more preferably to 75 to 80. In one embodiment, the matrix slurry is passed through a coil cooker and heated to a temperature of 250 to 325°F for cooking. Other cooking methods may be used, as will be understood by those skilled in the art. Preferably, the cooked matrix slurry is subjected to a vacuum to further increase the Brix level to the desired range. The cooked matrix slurry is held at about 200°F until use. Preferably, an acidifying agent solution is added to the cooked matrix slurry along with the pigments and flavorings, and then immediately deposited into a starch mold. In one aspect, the acidifying agent solution contains ascorbic acid in an amount of 15 to 20% by weight, citric acid in an amount of 10 to 20% by weight, and malic acid in an amount of 5 to 10% by weight, the remainder being water. As will be understood by those skilled in the art, other edible acids may be used in place of those listed, or other edible acids may be used besides those listed. In one aspect, 95 to 97% by weight of a cooked matrix slurry is combined with 2 to 3% by weight of an acidifying agent solution, and the remainder comprises flavoring agents and pigments. Optionally, the acidifying agent solution is used to raise the pH of the matrix slurry from 2.6 to 3.2. Suitable pigments and flavoring agents will be readily selected by those skilled in the art. The combined mixture is then deposited in a starch mold, for example, using a Mogul starch molding machine. Such starch molding machines are well known to those skilled in the art. In one aspect, 0.3 to 3 grams of matrix slurry is deposited in each mold cavity. In some preferred embodiments, the starch molding machine (“Mogul”) for forming rubber bears includes two nozzles per mold and means for delivering small soft capsules. Before placing a capsule in the mold, the first nozzle provides approximately 40% of the mold's volume. Finally, the second nozzle fills the mold. The rubber bears containing the capsules are then rapidly cooled. The starch trays with the deposited matrix slurry are transferred to a drying chamber, where they are held for 12 to 48 hours. Optionally, the tray is first kept at 130 to 150°F for 10 to 15 hours, then cooled to 70 to 80°F and kept at that temperature for 6 to 12 hours. The food sheets molded from the gelled starch are then removed from the tray, and the starch is recovered.

[0093] In some embodiments of the invention, it is intended that the oil within the capsule be protected from hydrolysis by the water present in the product. Gummy candies and other jelly candies contain a relatively high water content and, furthermore, have low acidity to counteract bacterial growth.

[0094] Example 1 This embodiment describes the formation of krill coagulants (paste-like substances).

[0095] Materials and methods Raw materials. Frozen krill was obtained from Aker Biomarine, with 10 tons stored in Norway Pelagic, Bergen, and retrieved as needed. The krill was packaged in plastic bags at 2 x 12.5 kg krill per box, placed in cardboard boxes. The boxes were stamped with FRAL and C05S or A06S postmarks. The boxes containing the krill were placed in a single layer on the bottom plate of the processing unit the day before processing. At processing time, the krill temperature was varied from +3°C to -3°C.

[0096] Processing and equipment Heating is performed in a steam-heated autoclave (200 l) or in a scraped heat exchanger (Contherm 6×4, Alfa Laval AB).

[0097] Krill and hot liquid (typically 95±2°C) are mixed in a single-screw pump (PCM 4L IVA, PCMS.A., Vanves, France) with a screw feed. The total flow rate of the pump is calibrated with water, and the water flow is reduced when krill is added.

[0098] The mixture of krill and hot liquid is maintained at a specified temperature (70±5°C) by transport via separate stainless steel tubing (6 x 2" tubes á 6 m and 5 x 180° bends á 0.3 m, total length 37.5 m) or via an Archimedes-style hose pump (Matcon, Herslev, tube 3” x 26 m).

[0099] First, the krill and liquid are separated on a metal sieve.

[0100] For most experiments, krill and condensate were removed using single or dual belt filters (Sobye Miljøfilter AS, Skogsvåg, synthetic fiber filter with an air opening of 0.8 mm). Membrane filtration of the boiled liquid was performed at 70°C using a (P19-40 100 nm ZrO2 membrane, Ceramic MF plant, Downstream Processing, Bergen). As a pre-filtration step before membrane filtration, a rotary fluid sieve (Jesma vs 20 / 65, Hans Jessens Maskinbyggeri AS, Vejle) was used (air opening 100 µm).

[0101] The discharged condensate was dehydrated in a twin-screw press (P9, Stord Bartz, Bergen, volume ratio 1:4.2).

[0102] The discharged krill were dehydrated in a twin-screw press (P13, Stord Bartz, Bergen, volume ratio 1:5).

[0103] The press liquor was separated in a three-phase horizontal screw centrifuge (Z23, Flottweg GmbH&Co., Vilsbiburg, Germany) or in a combination of a rotary fluid screen (Jesma vs 20 / 65, Hans Jessens Maskinbyggeri AS, Vejle) (100 µm air opening) and an oil separator (SA-1, Westfalia Separator AG, Oelde, Germany).

[0104] The press liquor was concentrated in a 4-stage falling film evaporator (Anhydro, Søborg, Denmark).

[0105] Drying is carried out in a hot air dryer that supports mechanical fluidization (FT200, Forberg, Larvik) or in a steam-heated Rotadisc dryer (TST 0, 3R, Stord Bartz, Bergen).

[0106] Analytical methods Proteins, Kjeldahl method: Nitrogen in the sample is converted to ammonium by dissolving in concentrated sulfuric acid (with copper as a catalyst). Ammonia is released in an alkaline distillate and determined by titration (ISO 5983:1997(E), Method A 01). Uncertainty: 1%.

[0107] Protein, Combustion: Nitrogen release is achieved by burning the sample in pure oxygen at high temperature. Detection is by thermal conductivity. The percentage of protein in the sample is calculated by multiplying the percentage of nitrogen analyzed by a given protein factor (AOAC Official Method 990.03, 16th Edition. 1996, Method A 25).

[0108] Moisture content: Determined by mass loss after drying at 103°C for 4 hours (ISO 6496 (1999). Method A 04). Uncertainty: 4%.

[0109] Ash content: The ash content of a sample is defined as the residue remaining after combustion of organic matter at 550°C. (ISO 5984:2002. Method A 02). Uncertainty: 3%.

[0110] Fat, ethyl acetate extraction: Moisture in the wet sample was absorbed by sodium sulfate, followed by extraction of fat with ethyl acetate (NS 9402, 1994 (corrected calculation). Method A 29).

[0111] Fat, Soxhlet extraction: Fat is extracted using petroleum ether. The main determination is the triglyceride content (AOCSOfficial Method Ba 3-38 Reapproved 1993. Method A 03).

[0112] Lipids, Bligh and Dyer: Lipids were extracted using a mixture of chloroform, methanol, and water in a 1:2:0.8 ratio, forming a single-phase system. The addition of chloroform and water produced a chloroform phase and a water / methanol phase containing lipids. Lipids were determined in aliquots of the chloroform phase after evaporation and weighing. The extract included triglycerides and phospholipids. (EG Bligh & W.J. Dyer: A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. Vol. 37 (1959). Metode A 56).

[0113] Astaxanthin: Extracted using ethanol and dichloromethane. Polar products were removed by open silica gel column chromatography. Isomers were separated by normal-phase HPLC on a Si 60 column and detected at 470 nm. (Schierle J. & Härdi W. 1994. Determination of stabilized astaxanthin in Carophyll) ® Pink, premixes and fish feeds. 3rd edition. Revised Supplement to: Hoffman P, Keller HE, SchierleJ., Schuep W. Analytical methods for vitamins and carotenoids in feed. Basel: Department of Vitamin Research and Development, Roche. Method A 23).

[0114] Water content in oils: The actual water content of fats and oils was determined by titration with Karl Fischer reagent, which reacts quantitatively with water (AOCS Official Method CA 2e-84. Reapproved 1993. Method A 13).

[0115] Krill and preheated water were mixed in a cooker and heated to 70°C. The heated krill and hot water were then separated by filtration to provide krill milk. The krill milk was then coagulated by heating to 95°C and separated from the liquid using a Soby Miljø filter. The coagulated material was then pressed in a P-9 press. Tables 1 and 2 show the analysis of the coagulated material on wet and dry basis. The dry matter content of the coagulated material ranged from 12.8% to 16.7%. On a dry basis, the fat content was approximately 60%, and the TMAO was 340 mg N / 100 g. Pressing increased the dry matter content of the coagulated material to 34-38%. The fat content also increased on a dry basis (Table 3), but the TMAO decreased to 145 mg N / 100 g. After washing the filter cake (1 part water: 1 part coagulated cake) and pressing again, the TMAO decreased to 45 mg N / 100 g on a dry basis (Table 4).

[0116] Table 1. Analysis of agglomerates on wet basis (wb)

[0117] Table 2. Analysis of condensate on dry basis (db)

[0118] Table 3 Analysis of filter cake from condensates on a wet basis

[0119] Table 4. Analysis of filter cake from condensate on a dry basis.

[0120] Example 2 This embodiment describes the washing steps of an oil extract obtained from dried krill meal via ethanol extraction. After initial removal of most of the ethanol, the material containing 60.5% dry matter and 2.4% water is separated from the ethanol. 24.8 kg of water and 35.0 kg of ethanol are added to 94.5 kg of this material. The mixture is stirred and separated into a bottom layer containing mostly phospholipids, an intermediate layer rich in triglycerides, and a top layer containing less than about 3% lipids (mainly lysophospholipids). The conductivity of the bottom layer is 722 μS / cm. The two top layers, containing 81.7 kg, are removed, and 67 kg of a 60% (w / w) aqueous solution of ethanol is added to the bottom layers, and the mixture is vigorously stirred for about 10 minutes. After sedimentation, the top layer, containing 82.2 kg, is removed, and 76.0 kg of a 60% ethanol solution (with a conductivity now of 384 μS / cm) is added to the bottom layer, and the mixture is vigorously stirred for about 10 minutes. After sedimentation, the top phase, containing 82.0 kg, is removed. The conductivity of the substrate phase was 183 uS / cm. Evaporation of ethanol / water from the substrate phase yielded 31.0 kg of lipid product containing 16.4% EPA and 7.9% DHA (starting from 12% EPA and 6% DHA) and 55.21% phospholipids (starting from 40%). The product showed further significant improvement in odor and taste.

[0121] The composition of polar lipid fractions is as follows:

[0122] Example 3 This embodiment describes the extraction of oil from a wet material. As described in WO 2009 / 027692 (incorporated herein by reference in its entirety), a coagulant containing approximately 70% water, 15% lipids, and approximately 15% other dry matter (mainly proteins) was obtained from krill. The material underwent the following extraction steps: 3500 g of pure ethanol was added to 1004 g of the coagulant, and the mixture was stirred for 45 minutes. The mixture was then filtered through filter paper under vacuum on a receiving flask, yielding 3854 g of filtrate. 1179 g of the filtrate was evaporated on a rotary evaporator, followed by washing the obtained dry matter four times with a 60% ethanol solution, and finally evaporating the solvent on a rotary evaporator. The resulting oil (23.7 g) was solid at room temperature and contained 76.8% phospholipids. Water was removed by freeze-drying.

[0123] The EPA content is 200 mg / g, and the DHA content is 87 mg / g oil. The composition of the phospholipid fraction is as follows:

[0124] Example 4 This embodiment describes an alternative method for extracting oil from wet krill material, starting with frozen krill paste and subjecting it to the extraction steps described below. Unlike Example 3, all steps are performed under a nitrogen atmosphere.

[0125] The paste comprises approximately 65% ​​water (assessed by dry matter), 17% lipids (approximately equal weights of phospholipids and neutral lipids), and approximately 18% other dry matter (primarily protein). Within the lipids, certain fatty acids are present in the following proportions by weight: C16:0 approximately 15-17%; C14:0 approximately 6-10%; C18:3n-3 approximately 1.4-3.1%; and C18:4n-3 approximately 3.5-7%.

[0126] 100 kg of frozen condensate (-20°C) was added to the container. Based on the water content of the condensate, 350 kg of pure ethanol (99.8% w / w, room temperature) was then added to the container, resulting in a final ethanol concentration of approximately 84% w / w in the liquid phase (~350 kg of ethanol in 415 kg of liquid solvent). Ethanol was added to approximate the desired final concentration, and the water content was then checked by Karl Fischer titration, with additional ethanol added as needed to obtain the correct final amount.

[0127] The mixture was stirred in a container for 45 minutes, with gentle heating if necessary. Four final temperatures were studied in individual batches: a) 2°C, b) 10°C, c) 15°C, and d) 20°C. After stirring, the mixtures were allowed to stand, each containing a red liquid phase and a wet slurry containing shell fragments and other insoluble substances. To remove the liquid phase from the slurry, the mixture was decanted, and the liquid material was passed through a coarse filter, followed by a series of filtrations through 75µm and 5µm cartridge filters to obtain filtrates of a) 345 kg, b) 366 kg, c) 372 kg, or d) 374 kg, with the remaining material remaining in the filter cake. Smaller cartridge filters (e.g., 1.2µm) were also used.

[0128] The filtrate was then subjected to a series of washes. First, deionized water was added to obtain a ~60% w / w ethanol solution (a: 137 kg water; b: 149 kg; c: 152 kg; d: 155 kg). The mixture was stirred for 10–15 minutes and allowed to settle at room temperature (15–20 °C) in a container with a valve at the bottom for 12–24 hours. The bottom phase was separated by draining it through the valve, yielding a lipid-rich fraction between 5.4 and 9.0 kg. The lipid-rich fraction was washed 2 to 5 times with 60% w / w ethanol at room temperature to obtain a final material containing approximately 80% phospholipids and 20% neutral lipids by weight. Even in the first wash, 85% of TMAO was removed, and further washing resulted in a material with undetectable TMAO (less than 1 mgN / 100 g, i.e., at most 1 / 20 of that reported in Table X of WO 2013 / 102792).

[0129] The lipid-rich material is treated at least once by cold precipitation with acetone. Three parts w / w acetone are added, and the lipid-rich material is dissolved by gentle heating and slow stirring. Stirring is stopped, and the mixture is cooled to 4°C to allow precipitation. When precipitation is complete, the upper solvent phase is removed. This cold precipitation step is performed a total of three times, redissolving the material in fresh acetone each time after the first precipitation.

[0130] The precipitate was then subjected to evaporation and freeze-drying to remove residual acetone and water. Batch c (i.e., extracted at 15°C, followed by washing three times with 60% EtOH, and then cold acetone precipitation) yielded 1.9 kg of solid material (orange wax) consisting of 98% phospholipids / 1.7% neutral lipids and 3% water. The EPA content was 19.2 g / 100 g and the DHA content was 11.0 g / 100 g of solid material. 31 The composition of the phospholipid fraction measured by P NMR is as follows:

[0131] Therefore, based on the total weight of the material analyzed by NMR, nearly 93% of the final material was phospholipids. After compensating for residual water (approximately 3%), residual organic solvents, and salts / minerals present after ignition, the overall purity was 98%. Thus, this method provides phospholipids with a higher purity than that seen using Example 1.

[0132] Further analysis of the lipid composition was performed by HPLC, and the results are shown below (g / 100g oil): (1) Based on the techniques in Winther et al. (2011) Lipids 46:25-36; Homan R et al. (1998) J ChromatogrB Biomed Sci Appl 708:21-26 and Moreau et al. (2006) Lipids 41:727-734.

[0133] (2) It was calculated to be a fatty acid methyl ester by AOCS Ce 1b-89.

[0134] Looking at specific fatty acids, the proportions are as follows, measured in multiple batches:

[0135] Purified phospholipids contain both ether-linked and ester-linked fatty acids, but 10% or less are ether-linked. NMR shows that the ether-linked fatty acid moieties are located at position sn1 rather than sn2, and that the ether-linked fatty acids are either fully saturated or monounsaturated. When the phospholipid is phosphatidylcholine, approximately 10% of the molecules contain ether-linked fatty acids; when the phospholipid is phosphatidylethanolamine (with or without N-acetylation), approximately 40% of the molecules contain ether-linked fatty acids. PUFAs were observed to contain only ester bonds. 30-40% by weight of the fatty acids in purified phospholipids are ω-3, and these fatty acids are distributed at both sn1 and sn2 positions (primarily sn2). The majority of the ω-3 fatty acids are EPA and / or DHA, with EPA being approximately twice as abundant as DHA.

[0136] The phosphatidylethanolamine content using this method is higher (approximately twice as high) than that seen using the method in Example 1.

[0137] The lysophosphatidylcholine content (0.2-0.4 mol%) was very low in the purified phospholipids compared to the amounts observed using the method of Example 1 (approximately 1%) and the amounts in the starting wet material (approximately 1.2-1.4 mol%). No molecules in which both the fatty acid chains at the sn1 and sn2 positions were lost were detected. Lysophosphatidylethanolamine (with or without N-acetylation) and lysophosphatidylinositol were also not observed.

[0138] When compared to the material obtained in Example 3, the astaxanthin level in the purified phospholipids was significantly lower. This reduction was even visible due to the weaker red color. Amino acids, TMAO, and lobster myoline were all below the LOQ according to standard analytical methods.

[0139] Therefore, very pure krill phospholipids can be obtained by extraction in 84% ethanol, followed by washing in 60% ethanol, and then performing multiple cold acetone precipitation steps.

[0140] Example 5 This embodiment describes a method for producing gummy candies containing krill oil extract, preferably using a 60% w / w krill phospholipid composition prepared from a washed, polar lipid-rich extract. Gelatin is dissolved in water (1:2), and the protein is gelled at 80-100°C (gelatin melts at 68°C). Simultaneously, in another drum, sugar is mixed with syrup and cooked at 100-120°C. The gelatin and sugar are mixed in another drum by cooking and stirring. After 2 hours, the final product typically contains 15-17% water. The gelatin and sugar mixture is automatically cooled to 70-80°C outside a vacuum drum. Different flavorings (e.g., a combination of raspberry and orange) are added to the gummy candy mixture. The gelatin (pork gelatin, 250 bloom with a pH of 3-4) is then acidified or alkali-treated at pI. Krill phospholipid extract oil is then added to the gelatin and sugar mixture. This process takes approximately 5-10 minutes. Before addition, the krill extract is preferably diluted with water or sorbitol syrup (e.g., the extract is a liquid of oil:water in a 1:4 ratio). Pour the homogeneous mixture of gummy candy material into a mold. The mold can be made of corn or wheat and is reusable. After 1-3 days at RT, the gummy candies form and are removed from the mold. Transfer the gummy candies to a chamber where they are waxed and oiled. A second coating with sugar may be applied.

[0141] All publications and patents mentioned in the foregoing specification are incorporated herein by reference. Various modifications and variations to the compounds, compositions, methods, and uses described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications to the modes of the invention will be apparent to those skilled in the medical, biological, and chemical sciences and are intended to be included within the scope of the following claims.

Claims

1. A method for extracting an extract rich in polar lipids from biological material, wherein the biological material is selected from krill material, the method comprising: The biomaterial is contacted with a concentrated proton solvent under conditions that allow for preferential extraction of polar lipids to form a slurry containing a polar lipid solution and residual biomaterial. The polar lipid solution is separated from the biological residue material to provide a separated polar lipid solution; An aqueous solution is added to the polar lipid solution to dilute the proton solvent, so that the polar lipid solution separates into an upper phase containing the diluted proton solvent and a lower bottom phase rich in polar lipids. as well as The polar lipid-rich substrate is separated to provide the polar lipid-rich extract. The addition of the aqueous solution to the polar lipid solution to dilute the proton solvent so that the polar lipid solution separates into an upper phase containing the diluted proton solvent and a lower bottom phase rich in polar lipids includes adding the aqueous solution to dilute the concentration of the proton solvent to about 50% to 70% w / w when combined with water in the biological sample. The method further includes washing the polar lipid-rich extract with a diluted protonated solvent in which the polar lipids are poorly soluble, to provide an upper phase comprising the diluted protonated solvent and a polar lipid-rich lower phase, and separating the lower phase to provide the washed polar lipid-rich extract; The washing process is repeated 2 to 5 times. The method further includes removing residual solvent from the polar lipid-rich extract or the washed polar lipid-rich extract by evaporation to provide a solid polar lipid-rich composition.

2. The method of claim 1, wherein the concentration of the proton solvent is diluted to about 60% w / w when combined with water in the biological sample.

3. The method according to claim 1 or 2, wherein contacting the biomaterial with a concentrated proton solvent under conditions that allow for preferential extraction of polar lipids includes mixing the biomaterial with the concentrated proton solvent at a temperature of about -10°C to about 50°C, such that the concentration of the solvent is about 70% to about 95% w / w when combined with water in the biomaterial.

4. The method of claim 1, wherein washing the polar lipid-rich extract with a diluted proton solvent under conditions that make the phospholipids poorly soluble further comprises mixing the polar lipid-rich extract with the diluted proton solvent at a ratio of about 0.5:1 to 5:1, wherein the diluted proton solvent comprises an aqueous solution having about 30% to 70% of the proton solvent.

5. A method for producing a gummy candy product, the method comprising: The washed, polar lipid-rich extract, polar lipid-rich precipitate, solid polar lipid-rich composition, or mixed lipid composition produced as described in any one of claims 1 to 4 are mixed into a gel matrix to provide a mixed gummy candy mixture and Gummy candies are formed from the mixed gummy candy mixture.

6. A product produced by the method according to claim 5.

7. A composition comprising, in a solid gel matrix, a washed polar lipid-rich extract, a polar lipid-rich precipitate, a solid polar lipid-rich composition, or a mixed lipid composition produced as described in any one of claims 1 to 4.

8. A phospholipid composition comprising a mixture of phospholipid compounds having the following structure: Wherein R1 and R2 are selected from the group consisting of: fatty acid moieties and -H, and R3 is H or choline, ethanolamine, inositol or serine moieties, wherein the mixture of phospholipid compounds contains more than about 85% (mol%) of choline moieties and more than about 30% w / w of ω-3 fatty acid moieties at position R3, wherein more than about 90% w / w of the ω-3 fatty acid moieties are at position R2, and the composition is further characterized by containing less than about 3% w / w of lysophospholipids.

9. The composition according to claim 8, wherein the ω-3 fatty acid portion is selected from the group consisting of eicosapentaenoic acid, docosahexaenoic acid, and combinations thereof.

10. The composition according to any one of claims 8 or 9, wherein the composition comprises at least 50% w / w of the phospholipid compound.

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