Attorney Docket No. AKBM-14409/US-18/CON
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`BIOEFFECTIVE KRILL OIL COMPOSITIONS
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`CROSS-REFERENCE TO RELATED APPLICATIONS
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`This application is a continuation of U.S. Patent Application No. 15/589,605, filed May8,
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`2017, which is a continuation of U.S. Patent Application No. 15/180,439, filed June 13, 2016,
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`now U.S. Patent No. 9,644,170, which is a continuation of U.S. Patent Application No.
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`14/020,162, filed September 6, 2013, now U.S. Patent No. 9,375,453, which is a continuation of
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`U.S. Patent Application No. 12/057,775, filed March 28, 2008, now U.S. Patent No. 9,034,388,
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`which claims the benefit of expired U.S. Provisional Patent Application No. 60/920,483, filed
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`March 28, 2007, expired U.S. Provisional Patent Application No. 60/975,058, filed September
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`25, 2007, expired U.S. Provisional Patent Application No. 60/983,446,filed October 29, 2007,
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`and expired U.S. Provisional Patent Application No. 61/024,072, filed January 28, 2008, all of
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`which are incorporated by reference herein in their entirety.
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`FIELD OF THE INVENTION
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`This invention relates to extracts from Antarctic krill that comprise bioactive fatty acids.
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`BACKGROUNDOF THE INVENTION
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`In the Southern Ocean,off the coast of Antarctica, Antarctic krill (Kuphausia superba)
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`can be found in large quantities, ranging from 300-500 million metric tons of biomass. It feeds on
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`phytoplankton during the short Antarctic summer. During winter, however, its food supply is
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`limited to ice algae, bacteria, marine detritus as well as depleting body protein for energy.
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`In orderto isolate the krill oil from the krill, solvent extraction methods have beenused.
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`See, e.g., WO 00/23546. Krill lipids have been extracted by placing the material in a ketone
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`solvent (e.g. acetone) in order to extract the lipid soluble fraction. This method involves
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`separating the liquid and solid contents and recoveringa lipid rich fraction from the liquid
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`fraction by evaporation. Further processing steps include extracting and recovering by
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`evaporation the remaining soluble lipid fraction from the solid contents by using a solvent such as
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`ethanol. See, e.g., WO 00/23546. The compositions produced by these methodsare characterized
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`by containing at least 75 ug/g astaxanthin, preferably 90 ug/g astaxanthin. Anotherkrill lipid
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`extract disclosed contained at least 250 ug/g canastaxanthin, preferably 270 ug/g canastaxanthin.
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`Attorney Docket No. AKBM-14409/US-18/CON
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`Krill oil compositions have been described as being effective for decreasing cholesterol,
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`inhibiting platelet adhesion, inhibiting artery plaque formation, preventing hypertension,
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`controlling arthritis symptoms, preventing skin cancer, enhancing transdermal transport, reducing
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`the symptoms of premenstrual symptomsor controlling blood glucose levels in a patient. See,
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`e.g., WO 02/102394. In yet another application, a krill oil composition has been disclosed
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`comprising a phospholipid and/or a flavonoid. The phospholipid contentin the krill lipid extract
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`could be as high as 60% w/w and the EPA/DHAcontentas high as 35% (w/w). See, e.g., WO
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`03/011873.
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`Furthermore, nutraceuticals, pharmaceuticals and cosmetics comprising the phospholipid
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`extract were disclosed. Previously, it was also shown that supercritical fluid extraction using neat
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`CO) could be used to prevent the extraction of phospholipids in order to extract the neutral lipid
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`fraction from krill, which comprised of esterified and free astaxanthin. See, e.g., Yamaguchiet
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`al., J. Agric. Food Chem. (1986), 34(5), 904-7. Supercritical fluid extraction with solvent
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`modifier has previously been used to extract marine phospholipids from salmonroe, but has not
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`been previously used to extract phospholipids from krill meal. See, e.g., Tanakaet al., J. Oleo
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`Sci. (2004), 53(9), 417-424.
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`The methodsdescribed above rely on the processing of frozen krill that are transported
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`from the Southern Ocean to the processing site. This transportation is both expensive and can
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`result in degradation ofthe krill starting material. Data in the literature showing a rapid
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`decomposition of the oil in krill explains why somekrill oil currently offered as an omega-3
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`supplement in the marketplace contains very high amounts of partly decomposed
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`phosphatidylcholine and also partly decomposed glycerides. Saether et al., Comp. Biochem
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`Phys. B 83B(1): 51-55 (1986). The products offered also contain high levels of free fatty acids.
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`Whatis neededin the art are methods for processing krill that do not require transport of
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`frozen krill material over long distances and the products produced by those methods.
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`SUMMARYOF THE INVENTION
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`In a first aspect of the invention is a composition characterized by comprising at least 65%
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`(w/w) phospholipids.
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`In another aspect of the invention is a composition obtained from aquatic or marine
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`sources, characterized by comprising 65% (w/w)phospholipids.
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`In yet another aspect of the invention is a composition obtained from krill, characterized
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`by comprising at least 65% (w/w) phospholipids.
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`In another aspect of the invention is a composition obtained from krill, characterized by
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`comprising at least 65% (w/w) phospholipids and at least 39% omega-3 fatty acids (w/w).
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`In yet another aspect of the invention is a composition obtained from krill, characterized
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`by comprising at least 65% (w/w) phospholipids, at least 39% omega-3 fatty acids (w/w) and at
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`least 580 mg/kg astaxanthinesters.
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`In another aspect of the invention is a composition obtained from krill, characterized by
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`comprising at least 39% omega-3 fatty acids (w/w) and at least 580 mg/kg astaxanthinesters.
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`In yet another aspect of the invention is a composition obtained from krill, characterized
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`by comprising at least 65% (w/w) phospholipids and at least 580mg/kg astaxanthin esters.
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`In yet another aspect, the present invention provides a krill oil effective for reducing
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`insulin resistance, improving bloodlipid profile, reducing inflammation or reducing oxidative
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`stress.
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`In some embodiments, the present invention provides compositions comprising: from
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`about 3% to 10% ether phospholipids on a w/w basis; from about 35% to 50% non-ether
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`phospholipids on w/w basis, so that the total amount of ether phospholipids and non-ether
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`phospholipids in the composition is from about 48% to 60% on a w/w basis;
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`from about 20% to 45% triglycerides on a w/w basis; and from about 400 to about 2500 mg/kg
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`astaxanthin. In some embodiments, the ether phospholipids are selected from the group
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`consisting of alkylacylphosphatidylcholine, lyso-alkylacylphosphatidylcholine,
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`alkylacylphosphatidylethanolamine, and combinations thereof. In some embodiments, the ether
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`lipids are greater than 90% alkylacylphosphatidylcholine. In some embodiments, the non-ether
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`phospholipids are selected from the group consisting of phosphatidylcholine, phosphatidylserine,
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`phosphatidylethanolamine and combinations thereof. In some embodiments, krill oil composition
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`comprises a blend oflipid fractions obtained from krill. In somepreferred embodiments,krill is
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`Euphausia superba, although otherkrill species also find use in the present invention. Otherkrill
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`species include, but are not limited to E. pacifica, FE. frigida, E. longirostris, E. triacantha, E.
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`vallentini, Meganyctiphanes norvegica, Thysanoessa raschii and Thysanoessa inermis. In some
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`embodiments, the compositions comprise from about 25% to 30% omega-3 fatty acids as a
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`percentage of total fatty acids and wherein from about 80% to 90% of said omega-3 fatty acids
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`are attached to said phospholipids. In some embodiments, the present invention provides a
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`capsule containing the foregoing compositions.
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`In further embodiments, the present inventions provide compositions comprising: from
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`about 3% to 10% ether phospholipids on a w/w basis; and from about 400 to about 2500 mg/kg
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`astaxanthin. In some embodiments, the compositions further comprise from about 35% to 50%
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`non-ether phospholipids on w/w basis, so that the total amount of ether phospholipids and non-
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`ether phospholipids in the composition is from about 38% to 60% on a w/w basis. In some
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`embodiments, the compositions further comprise from about 20% to 45% triglycerides on a w/w
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`basis. In some embodiments, the ether phospholipids are selected from the group consisting of
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`alkylacylphosphatidylcholine, lyso-alkylacylphosphatidylcholine,
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`alkylacylphosphatidylethanolamine, and combinations thereof. In some embodiments, the ether
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`lipids are greater than 90% alkylacylphosphatidylcholine. In some embodiments, the non-ether
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`phospholipids are selected from the group consisting of phosphatidylcholine, phosphatidylserine,
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`phosphatidylethanolamine and combinations thereof. In some embodiments, krill oil composition
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`comprises a blend oflipid fractions obtained from krill. In somepreferred embodiments,krill is
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`Euphausia superba, although otherkrill species also find use in the present invention. Otherkrill
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`species include, but are not limited to E. pacifica, FE. frigida, E. longirostris, E. triacantha, E.
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`vallentini, Meganyctiphanes norvegica, Thysanoessa raschii and Thysanoessa inermis. In some
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`embodiments, the compositions comprise about 25% to 30% omega-3 fatty acids as a percentage
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`of total fatty acids and wherein from about 80% to 90% of said omega-3 fatty acids are attached
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`to said phospholipids. In some embodiments, the present invention provides a capsule containing
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`the foregoing compositions.
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`In some embodiments, the present invention provides a composition comprising at least
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`65% (w/w) of phospholipids, said phospholipids characterized in containing at least 35% omega-
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`3 fatty acid residues. In some preferred embodiments, the composition is derived from a marine
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`or aquatic biomass. In somefurther preferred embodiments, the composition is derived from
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`krill. In some embodiments, the composition comprises less than 2% free fatty acids. In some
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`embodiments, composition comprisesless than 10% triglycerides. In some preferred
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`embodiments, the phospholipids comprise greater than 50% phosphatidylcholine. In some
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`embodiments, the composition comprises at least 500 mg/kg astaxanthin esters. In some
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`embodiments, the composition comprises at least 500 mg/kg astaxanthin esters and at least 36%
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`(w/w) omega-3 fatty acids. In some embodiments, the composition comprises less than about
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`0.5g/100g total cholesterol. In some embodiments, the composition comprises less than about
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`0.45% arachidonic acid (w/w).
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`In some embodiments, the present invention providesa krill lipid extract comprising at
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`least 500, 100, 1500, 2000, 2100, or 2200 mg/kg astaxanthin esters and at least 36% (w/w)
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`omega-3 fatty acids. In further embodiments, the present invention provides a krill lipid extract
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`comprising at least 100 mg/kg astaxanthin esters, at least 20% (w/w) omega-3 fatty acids, and
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`less than about 0.45% arachidonic acid (w/w).
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`In some embodiments, the present invention provides methods comprising administering
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`the foregoing compositions to a subject in an amounteffective for reducing insulin resistance,
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`reducing inflammation, improving blood lipid profile and reducing oxidativestress.
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`In some embodiments, the present invention providesa krill lipid extract comprising
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`greater than about 80% triglycerides and greater than about 90, 100, 500, 1000, 1500, 200, 2100
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`or 2200 mg/kg astaxanthin esters. In some embodiments, the krill lipid extract is characterized in
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`containing from about 5% to about 15% omega-3 fatty acid residues. In some embodiments, the
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`krill lipid extract is characterized in containing less than about 5% phospholipids. In some
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`embodiments, the krill lipid extract is characterized in comprising from about 5% to about 10%
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`cholesterol.
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`In some embodiments, the present invention provides a krill meal composition comprising
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`less than about 50g/kg total fat. In some embodiments, the krill meal composition comprises
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`from about 5 to about 20 mg/kg astaxanthin esters. In some embodiments,the krill meal
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`composition comprises greater than about 65% protein. In some embodiments, the krill meal
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`composition of comprises greater than about 70% protein. In some further embodiments, the
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`present invention provides an animal feed comprising the krill meal composition.
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`In some embodiments, the present invention provides methodsof increasing flesh
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`coloration in an aquatic species comprising feeding said aquatic species a composition
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`comprising the krill meal described above. In some embodiments, the present invention provides
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`methods of increasing growth and overall survival rate of aquatic species by feeding the krill
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`meal described above.
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`In some embodiments, the present invention provides methods of producing krill oil
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`comprising: a) providing krill meal; and b) extracting oil from said krill meal. In some
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`embodiments, the krill meal is produced by heat-treating krill. In some embodiments,the krill
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`mealis stored prior to the extraction step. In some embodiments, the extracting step comprises
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`extraction by supercritical fluid extraction. In some embodiments,the supercritical fluid
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`extraction is a two step process comprising a first extraction step with carbon dioxide and alow
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`concentration of a co-solvent(e.g., from about 1-10% co-solvent) and a second extraction step
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`with carbon dioxide and a high concentration of a co-solvent (e.g., from about 10-30% co-
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`solvent). In preferred embodiments, the co-solvent is a Ci-C3 monohydric alcohol, preferably
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`ethanol. In some embodiments, the present invention provides oil produced by the foregoing
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`method.
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`In some embodiments, the present invention provides methods of production of krill oil
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`comprising: a) providing fresh krill; b) treating said fresh krill to denature lipases and
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`phospholipasesin said fresh krill to provide a denatured krill product; and c) extracting oil from
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`said denatured krill product. In some embodiments, the denaturation step comprises heating of
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`said fresh krill. In some embodiments, the denaturation step comprises heating said fresh krill
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`after grinding. In some embodiments, the methods further comprise storing said denatured krill
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`product at room temperature or below between the denaturation step and the extraction step. In
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`some embodiments, the enzyme denaturation step is achieved by application of heat. In some
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`embodiments, the extraction step comprises use of supercritical carbon dioxide, with or without
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`use of a polar modifier. In some embodiments, the extraction step comprises use of ethanol. In
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`some embodiments, the extraction step is comprises ethanol extraction followed by acetone to
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`precipitation of phospholipids. In some embodiments, the denaturedkrill product is a meal. In
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`some embodiments, the present invention provides oil produced by the foregoing method.
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`In some embodiments, the present invention provides a composition comprising oil
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`extracted from krill having a phosphatidylcholine content of greater then about 50% (w/w). In
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`some embodiments, the oil has a phosphatidylcholine content of greater then about 70% (w/w).
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`In some embodiments, the oil has a phosphatidylcholine content of greater then about 80% (w/w).
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`In some embodiments, the composition comprises less than 2% free fatty acids. In some
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`embodiments, the composition comprises less than 10% triglycerides. In some embodiments, the
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`composition comprises at least 500 mg/kg astaxanthin esters. In some embodiments,the
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`composition comprises less than about 0.45% arachidonic acid (w/w).
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`In some embodiments, the present invention provides composition comprising odorless
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`krill oil. In some embodiments, the odorless krill oil comprises less than about 10 mg/kg (w/w)
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`trimethylamine. In some further embodiments, the present invention provides an odorless krill
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`oil produced by the method comprising: extracting a neutral krill oil from a krill oil containing
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`material by supercritical fluid extraction to provide a deodorized krill material, wherein said
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`neutral krill oil contains odor causing compoundsand extracting a polarkrill oil from said
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`deodorized krill material by supercritical fluid extraction with a polar entrainer to provide an
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`essentially odorless krill oil.
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`In some embodiments, the present invention provides a composition comprising krill oil
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`containing less than about 70 micrograms/kilogram (w/w) astaxanthin esters. In some
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`embodiments, the compositions comprise less than about 50 micrograms/kilogram (w/w)
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`astaxanthin esters. In some embodiments, the compositions comprise less than about 20
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`micrograms/kilogram (w/w) astaxanthin esters. In some embodiments, the compositions
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`comprise less than about 5 micrograms/kilogram (w/w) astaxanthinesters.
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`In some embodiments, the present invention providesa krill oil produced by the process
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`comprising: pumping fresh krill from a trawl onto a ship, heating the krill to provide a krill
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`material, and extracting oil from the krill material.
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`In further embodiments, the present invention provides a blended krill oil composition
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`comprising: from about 45% to 55% w/w phospholipids; from about 20% to 45% w/w
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`triglycerides; and from about 400 to about 2500 mg/kg astaxanthin. In some embodiments, the
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`blended krill oil product comprises a blendoflipid fractions obtained from Euphausia superba.
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`In some embodiments, the composition comprises from about 25% to 30% omega-3 fatty acids as
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`a percentage oftotal fatty acids and wherein from about 80% to 90% of said omega-3 fatty acids
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`are attached to said phospholipids.
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`In still other embodiments, the present invention provides a Euphausia superba krill oil
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`composition comprising: from about 30% to 60% w/w phospholipids; from about 20% to 50%
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`triglycerides; from about 400 to about 2500 mg/kg astaxanthin; and from about 20% to 35%
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`omega-3 fatty acids as a percentage oftotal fatty acids in said composition, wherein from about
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`70% to 95% of said omega-3 fatty acids are attached to said phospholipids.
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`In still further embodiments, the present invention provides a dietary supplement
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`comprising encapsulated Euphausia superba krill oil comprising from about 30% to 60% w/w
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`Attorney Docket No. AKBM-14409/US-18/CON
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`phospholipids; from about 20% to 50% triglycerides; from about 400 to about 2500 mg/kg
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`astaxanthin; and from about 20% to 35% omega-3 fatty acids as a percentage oftotal fatty acids
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`in said composition, wherein from about 70% to 95% of said omega-3 fatty acids are attached to
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`said phospholipids.
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`In some embodiments, the present invention provides methods of making a Euphausia
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`superba krill oil composition comprising: contacting Euphausia superba with a polar solvent to
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`provide a polar extract comprising phospholipids; contacting Euphausia superba with a neutral
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`solvent to provide a neutral extract comprising triglycerides and astaxanthin; combining said
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`polar extract and said neutral extract to provide Euphausia superba krill oil comprising from
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`about 30% to 60% w/w phospholipids; from about 20% to 50% triglycerides; from about 400 to
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`about 2500 mg/kg astaxanthin; and from about 20% to 35% omega-3 fatty acids as a percentage
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`of total fatty acids in said composition, wherein from about 70% to 95% of said omega-3 fatty
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`acids are attached to said phospholipids. In some embodiments, the methods further comprise the
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`step of encapsulating the Euphausia superbakrill oil. In some embodiments, the present
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`invention provides a Euphausia superba krill oil produced by the methods described above.
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`In some embodiments, the present invention provides methods of producing a dietary
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`supplement comprising; contacting Kuphausia superba with a polar solvent to provide an polar
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`extract comprising phospholipids; contacting Huphausia superba with a neutral solvent to provide
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`a neutral extract comprising triglycerides and astaxanthin; combining said polar extract and said
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`neutral extract to provide Euphausia superba krill oil comprising from about 30% to 60% w/w
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`phospholipids; from about 20% to 50% triglycerides; from about 400 to about 2500 mg/kg
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`astaxanthin; and from about 20% to 35% omega-3 fatty acids as a percentage oftotal fatty acids
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`in said composition, wherein from about 70% to 95% of said omega-3 fatty acids are attached to
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`said phospholipids; and encapsulating said Euphausia superba krill oil.
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`In some embodiments, the present invention provides methods of reducing diet-induced
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`hyperinsulinemia, insulin insensitivity, muscle mass hypertrophy, serum adiponectin reduction or
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`hepatic steatosis comprising in a subject exposed to a high fat diet: administering to said subject
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`exposedto a high fat diet an effective amountof a krill oil composition under conditions such that
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`a condition selected from the group consisting of diet-induced hyperinsulinemia, insulin
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`insensitivity, muscle mass hypertrophy, serum adiponectin reduction and hepatic steatosis is
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`reduced. The present invention is not limited to any particular krill oil composition. In some
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`embodiments, the krill oil composition is a Euphausia superba krill oil composition. The present
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`invention is not limited to any particular formulation of krill oil. In some embodiments, the krill
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`oil composition is encapsulated. In some preferred embodiments, the effective amount of a krill
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`oil composition is from 0.2 grams to 10 grams ofsaid krill oil composition. In some
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`embodiments, the krill oil composition comprises: from about 45% to 55% w/w phospholipids;
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`from about 20% to 45% w/w triglycerides; and from about 400 to about 2500 mg/kg astaxanthin.
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`In some embodiments, the krill oil composition comprises a blend oflipid fractions obtained
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`from Euphausia superba. In some embodiments,the krill oil composition comprises from about
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`25% to 30% omega-3 fatty acids as a percentage oftotal fatty acids and wherein from about 80%
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`to 90% of said omega-3 fatty acids are attached to said phospholipids. In some embodiments, the
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`krill oil composition comprises from about 30% to 60% w/w phospholipids; from about 20% to
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`50% triglycerides; from about 400 to about 2500 mg/kg astaxanthin; and from about 20% to 35%
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`omega-3 fatty acids as a percentage oftotal fatty acids in said composition, and wherein from
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`about 70% to 95% of said omega-3 fatty acids are attached to said phospholipids.
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`In some embodiments, the present invention provides methods of reducing diet-induced
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`hyperinsulinemia, insulin insensitivity, muscle mass hypertrophy, serum adiponectin reduction or
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`hepatic steatosis comprising in a subject consuming a high fat diet or a normalfat diet:
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`administering to said subject consuming a high fat diet or a normalfat diet an effective amount
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`of a krill oil composition under conditions such that a condition selected from the group
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`consisting of diet-induced hyperinsulinemia, insulin insensitivity, muscle mass hypertrophy,
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`serum adiponectin reduction and hepatic steatosis is reduced. The present invention is not limited
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`to any particular krill oil composition. In some embodiments,the krill oil composition is a
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`Euphausia superba krill oil composition. The present invention is not limited to any particular
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`formulation of krill oil. In some embodiments, the krill oil composition is encapsulated. In some
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`preferred embodiments, the effective amountofa krill oil composition is from 0.2 grams to 10
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`gramsofsaid krill oil composition. In some embodiments, the krill oil composition comprises:
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`from about 45% to 55% w/w phospholipids; from about 20% to 45% w/w triglycerides; and from
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`about 400 to about 2500 mg/kg astaxanthin. In some embodiments, the krill oil composition
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`comprises a blendoflipid fractions obtained from Euphausia superba. In some embodiments,
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`the krill oil composition comprises from about 25% to 30% omega-3 fatty acids as a percentage
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`of total fatty acids and wherein from about 80% to 90% of said omega-3 fatty acids are attached
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`to said phospholipids. In some embodiments, the krill oil composition comprises from about
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`30% to 60% w/w phospholipids; from about 20% to 50% triglycerides; from about 400 to about
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`2500 mg/kg astaxanthin; and from about 20% to 35% omega-3 fatty acids as a percentageoftotal
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`fatty acids in said composition, and wherein from about 70% to 95% of said omega-3 fatty acids
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`are attached to said phospholipids.
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`In some embodiments, the present invention provides methodsof inducing diuresis in a
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`subject comprising: administering to said subject an effective amountofa krill oil composition
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`under conditions such that diuresis is induced. In some embodiments, the present invention
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`provides methods of increasing muscle massin a subject, comprising:
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`administering to said subject an effective amountof a krill oil composition under conditions such
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`that muscle mass is increased. In some embodiments, the present invention provides methods of
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`decreasing protein catabolism in a subject, comprising: administering to said subject an effective
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`amountof a krill oil composition under conditions such that protein catabolism is decreased. In
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`some embodiments, the present invention provides methods of decreasing lipid content in the
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`heart of a subject, comprising: administering to said subject an effective amountofa krill oil
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`composition under conditions such that lipid content in the heart of the subject is decreased. In
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`some embodiments, the present invention provides methods of decreasing lipid content in the
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`liver of a subject, comprising: administering to said subject an effective amountofa krill oil
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`composition under conditions such that lipid content in the liver of the subject is decreased.
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`DESCRIPTION OF THE FIGURES
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`Figure 1. 31P NMRanalysis of polarlipids in krill oil.
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`Figure 2. Bloodlipid profiles in Zuckerrats fed different forms of omega-3 fatty acids
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`(TAG = FO, PL1 = NKOand PL2 = Superba).
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`Figure 3. Plasma glucose concentration in Zuckerrats fed different forms of omega-3
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`fatty acids.
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`Figure 4. Plasma insulin concentration in Zucker rats fed different forms of omega-3 fatty
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`acids.
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`acids.
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`Figure 5. Estimated HOMA-IR values in Zuckerrats fed different forms of omega-3 fatty
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`Figure 6. The effect of dietary omega-3 fatty acids on TNFL
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`production by peritoneal
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`Attorney Docket No. AKBM-14409/US-18/CON
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`macrophages.
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`Figure 7. The effect of dietary omega-3 fatty acids on lipid accumulation in theliver.
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`Figure 8. The effect of dietary omega-3 fatty acids on lipid accumulation in the muscle.
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`Figure 9. The effect of dietary omega-3 fatty acids on lipid accumulation in the heart.
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`Figure 10. Relative concentrations of DHAin the brain in Zucker rats supplemented with
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`omega-3 fatty acids.
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`Figure 11. Mean group body weights (g) in the collagen-induced male DBA/1 arthritic
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`mice. B - PL2is the krill oil group. * p<0.05, significantly different from Group A (Positive
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`Control - Fish Oil) and Group C (Control).
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`Figure 12.
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`Body weightfor the various treatment groups.
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`Figure 13.
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`Muscle weight for the various treatment groups.
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`Figure 14.
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`Muscle to body weight ratio for the various treatment groups.
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`Figure 15.
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`Serum adiopnectin levels (ng/ml) for the various treatment groups.
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`Figure 16.
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`Serum insulin levels for the various treatment groups.
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`Figure 17.
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`Blood glucose (mmol/l) levels in the various treatment groups.
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`Figure 18.
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`HOMA-IRvaluesfor the various treatment groups.
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`Figure 19.
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`Livertriglyceride levels (umol/g) for the various treatment groups.
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`
`DEFINITIONS
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`As used herein, "phospholipid" refers to an organic compound having the following
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`generalstructure:
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`Attorney Docket No. AKBM-14409/US-18/CON
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`l
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`O——C——R'
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`fl
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`——O——C—— RR?
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`wherein R1 is a fatty acid residue, R2 is a fatty acid residue or -OH, and R3 is a —-H ornitrogen
`containing compound choline (HOCH)CH)N(CH3)30H), ethanolamine (HOCH)CH)NH)),
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`inositol or serine. R1 and R2 cannot simultaneously be OH. When R3is an —OH, the compound
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`is a diacylglycerophosphate, while when R3is a nitrogen-containing compound, the compoundis
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`a phosphatide such aslecithin, cephalin, phosphatidyl serine or plasmalogen.
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`An “ether phospholipid” as used herein refers to a phospholipid having an ether bond at
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`position 1 the glycerol backbone. Examples of ether phospholipids include, but are not limited
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`to, alkylacylphosphatidylcholine (AAPC), lyso-alkylacylphosphatidylcholine (LAAPC), and
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`alkylacylphosphatidylethanolamine (AAPE). A “non-ether phospholipid” is a phospholipid that
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`does not have an ether bond at position 1 of the glycerol backbone.
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`Asused herein, the term omega-3 fatty acid refers to polyunsaturated fatty acids that have
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`the final double bond in the hydrocarbon chain between the third and fourth carbon atoms from
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`the methyl end of the molecule. Non-limiting examples of omega-3 fatty acids include,
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`5,8,11,14,17-eicosapentaenoic acid (EPA), 4,7,10,13,16,19-docosahexanoic acid (DHA) and
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`7,10,13,16,19-docosapentanoic acid (DPA).
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`Asused herein, astaxanthin refers to the following chemical structure:
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`Attorney Docket No. AKBM-14409/US-18/CON
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`
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`Asused herein, astaxanthin esters refer to the fatty acids esterified to OH groupin the
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`astaxanthin molecule.
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`Asused herein, the term w/w (weight/weight) refers to the amount of a given substance in
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`a composition on weight basis. For example, a composition comprising 50% w/w phospholipids
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`meansthat the mass of the phospholipids is 50% of the total mass of the composition (1.¢e., 50
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`gramsof phospholipids in 100 grams of the composition, such as an oil).
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`DETAILED DESCRIPTION OF THE INVENTION
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`This invention discloses novel krill oil compositions characterized by containing high
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`levels of astaxanthin, phospholipids, included an enriched quantities of ether phospholipids, and
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`omega-3 fatty acids. The krill oils compositions are extracted from krill meal using supercritical
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`fluid extraction (SFE) with a co-solvent modifier. The krill meal has been processed on board a
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`ship in Antarctica using live krill as starting material in order to ensure the highest possible
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`quality of the krill meal. The krill oils are extracted from the krill meal in two stages, in step 1 the
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`neutral fraction is extracted using neat supercritical CO» or in combination with 5% ethanol. The
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`neutral fraction consisted mostly of triglycerides and cholesterol. In stage 2, the polar lipids
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`(phospholipids) are extracted by adding at least 20% ethanol to the supercritical CO, extraction
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`medium.
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`The present invention provides methods to avoid decomposition of glycerides and
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`phospholipids in krill oil and compositions produced by those methods. The product obtained by
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`these new methodsis virtually free of enzymatically decomposedoil constituents. The solution to
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`the problem is to incorporate a protein denaturation step on fresh krill prior to use of any
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`extraction technology. Denaturation can be achieved by thermal stress or by other means. After
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`denaturation, the oil can be extracted by an optional selection of nonpolar and polar solvents
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`Attorney Docket No. AKBM-14409/US-18/CON
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`including use of supercritical carbon dioxide. Krill is adapted to a very efficient nutrient digestion
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`at very low temperatures. Therefore the enzymesare sensitive to heat and the step of applying
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`thermal denaturation of lipases and phospholipases does not imply use of very high temperatures.
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`Surprisingly, it has been found that the use of mild denaturation conditions can greatly enhance
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`the quality of krill oil.
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`Additionally, a major obstacle of several processes of extraction is the cost of removing
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`water. This is particularly true for methods feasible for extraction of highly unsaturated lipids
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`where freeze drying has been regarded as the method of choice to avoid oxidative breakdown of
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`lipids. However, the lipids in krill are surprisingly stable against oxidative deterioration.
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`Therefore, a process including moderate use of heat in the water removing processis feasible
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`provided that the enzymes havebeen inactivated.
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`A.
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`Krill Processing
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`The present invention provides methods for processing freshly caughtkrill at the site of
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`capture and preferably on b