(12) United States Patent
`Tilseth et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 8,372,812 B2
`Feb. 12, 2013
`
`US008372812B2
`
`(54) PHOSPHOLIPID AND PROTEIN TABLETS
`(75) Inventors: Snorre Tilseth, Bergen (NO); Nils
`Hoem, Oslo (NO)
`(73) Assignee: Aker Biomarine ASA, Oslo (NO)
`(*) Notice:
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 362 days.
`(21) Appl. No.: 12/711,822
`
`(22) Filed:
`
`Feb. 24, 2010
`
`(65)
`
`Prior Publication Data
`US 2010/0227792 A1
`Sep. 9, 2010
`
`Related U.S. Application Data
`(60) Provisional application No. 61/155,758, filed on Feb.
`26, 2009.
`
`(51) Int. Cl.
`(2006.01)
`A6 IK38/17
`(2006.01)
`A6 IK3I/01
`(2006.01)
`A6 IK9/20
`(2006.01)
`46K9/38
`(2006.01)
`A6 IK9/42
`(52) U.S. Cl. ..................... 514/21.92: 514/762; 424/464;
`424/476; 424/477
`(58) Field of Classification Search ........................ None
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`4,119,619 A 10/1978 Rogozhin et al.
`5,266,564 A 11/1993 Modele11 et al.
`5.434,183 A
`7, 1995 Larsson-Backstrom
`6,537,787 B1
`3/2003 Breton
`6,800,299 B1
`10/2004 Beaudoin et al.
`2003/0044495 A1
`3/2003 Kagan et al.
`2004/0241249 A1 12/2004 Sampalis
`FOREIGN PATENT DOCUMENTS
`870.1265
`3, 1987
`1098.900
`4f1981
`O609078
`8, 1994
`670306
`6, 1995
`1127497
`8, 2001
`1392623
`3, 2004
`1406,641
`4/2004
`1542670
`6, 2005
`O973,532
`9, 2005
`1660071
`5, 2006
`1689413
`8, 2006
`1743531
`1, 2007
`163.1280
`3, 2008
`1123368
`4/2008
`1406,641
`1, 2009
`1419768
`1, 2009
`1292294
`3, 2009
`17O6106
`T 2009
`1385,500
`T 2010
`2097014
`10, 1982
`92.1537
`6, 1999
`61281159
`12, 1986
`
`BR
`CA
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`GB
`GB
`JP
`
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`JP
`SU
`WO
`WO
`WO
`WO
`
`2, 1990
`O2O49091
`8, 1990
`2215351
`1, 1992
`4012665
`2, 1992
`2963152
`T 1994
`3O81692
`8, 1996
`2524217
`7/1997
`3344887
`8, 1997
`3611222
`6, 2001
`2001-158736 A
`1, 2003
`2003-003.192 A
`2, 2003
`2003-048.831 A
`5, 2003
`2003-146883. A
`9, 2003
`3467794
`2003-531857 A 10, 2003
`3486778
`10, 2003
`2004-52S180 A
`8, 2004
`2004-536059 A 12, 2004
`3678317
`5, 2005
`2005-245379 A
`9, 2005
`2006-502196. A
`1, 2006
`2006-069948 A
`3, 2006
`2006-083136. A
`3, 2006
`2006-290784. A 10, 2006
`2006-316073. A 11, 2006
`2006-328O14 A 12/2006
`2006-528233. A 12/2006
`2007-5O2805. A
`2, 2007
`2007-509131 A
`4/2007
`2007-126455. A
`5/2007
`2007-518764. A
`7/2007
`2007-246404 A
`9, 2007
`22O741
`1, 1971
`86,06082
`10, 1986
`90,05765
`5, 1990
`93.24142
`12/1993
`97,38585
`10, 1997
`(Continued)
`OTHER PUBLICATIONS
`Dave “Overview of pharmaceutical excipients used in tablets and
`capsules.” Drug Topics, Oct. 24, 2008.*
`Takaichi et al., 2003, "Fatty Acids of astaxanthin esters in krill deter
`mined by mild mass spectrometry. Comparative Biochemistry and
`Physiology Part B, Biochemistry and Molecular Biology, Elsevier,
`Oxford, vol. 136, Jan. 1, 2003, p. 317-322.
`Tanaka et al., 2004, “Extraction of Phospholipids from Salmon Roe
`with Supercritical Carbon Dioxide and an Entrainer'. J. Oleo Sci.
`53(9):417-424.
`Tanaka et al., 2005. “Extraction of Phospholipids from Unused
`Natrual Resources with Supercritical Carbon Dioxide and an
`Entrainer”, Journal of Oleo Science, vol. 54(11): 569-576.
`Todoric et al., 2006, "Adipose tissue inflammation induced by high
`fat diet in obese diabetic mice is prevented by n-3 polyunsaturated
`fatty acids”. Diabetologia, 49(9): 2109-2119.
`Tou et al., 2007, "Krill for human consumption: nutritional value and
`potential health benefits.”. Nutrition Rev 65 (2): 63-77.
`(Continued)
`Primary Examiner — Christina Bradley
`(74) Attorney, Agent, or Firm — Casimir Jones SC
`(57)
`ABSTRACT
`A new method for krill meal production has been developed
`using a two step cooking process. In the first step the proteins
`and phospholipids are removed from the krill and precipitated
`as a coagulum. In the second stage the krill without phospho
`lipids are cooked. Following this, residual fat and astaxanthin
`are removed from the krill using mechanical separation meth
`ods. A novel krill meal product with superior nutritional and
`technical properties is prepared.
`16 Claims, 7 Drawing Sheets
`RIMFROST EXHIBIT 1140 Page 0001
`
`

`

`US 8,372,812 B2
`Page 2
`
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`WO
`
`FOREIGN PATENT DOCUMENTS
`97/397.59
`10, 1997
`98.34498
`8, 1998
`99.39589
`8, 1999
`OO,23546
`4/2000
`OO,25608
`5, 2000
`OO,387.08
`T 2000
`O1,28526
`4/2001
`O1/82928
`11, 2001
`02/083122
`10, 2002
`O2/O92540
`11, 2002
`02/102394
`12/2002
`O3,O 11873
`2, 2003
`O3,O13497
`2, 2003
`2004/028529
`4/2004
`2004/047554
`6, 2004
`2004/100943
`11, 2004
`2005.004393
`1, 2005
`2005/O18632
`3, 2005
`2005/037848
`4/2005
`2005/038037
`4/2005
`2005, O70411
`8, 2005
`2006/030552
`3, 2006
`2007/080514
`7/2007
`2007/080515
`7/2007
`2007/1087O2
`9, 2007
`2008/0066O7
`1, 2008
`2008/117062
`10, 2008
`2009/027692
`3, 2009
`
`OTHER PUBLICATIONS
`Trayhurnet al., 2004, "Adipokines: inflammation and the pleiotropic
`role of white adipose tissue'. Br. J. Nutrition, 92(3): 347-355.
`Trebble et al., 2003, "Inhibition of tumour necrosis factor-alpha and
`interleukin 6 production by mononuclear cells following dietary
`fish-oil supplementation in healthy men and response to antioxidant
`co-supplementation”. Br. J. Nutrition, 90(2): 405-412.
`Ukkola et al., 2002, "Adiponectin: a link between excess adiposity
`and associated comorbidities?”. J. Mol. Med., 80 (11): 696-702.
`Van Der Veen et al., 1971 "The Lipids of Krill (Euphausia Species)
`and Red Crab (Pleuroncodes planipes)'. Lipids, 6(7): 481-485.
`Virtue, et al. 1996, Reproductive trade-off in male Antarctic krill,
`Euphausia superba, Marine Biology, vol. 126, No. 3, pp. 521-527.
`Yamaguchietal., 1983, "The Composition ofCarotenoid Pigments in
`the Antarctic Krill Euphausia superba', Bulletin of the Japanese
`Society of Scientific Fisheries, 49(9): 141 1-1415.
`Yamaguchi et al., 1986, "Supercritical Carbon Dioxide Extraction of
`Oils From Antarctic Krill.” Journal of Agricultural and Food Chem
`istry, vol. 34, pp. 904-907.
`Yanase M. 1974, “Modification of a Russian method for separation of
`heat-coagulated protein from Antarctic krill'. Database FSTA
`(online); International Food Information Service (IFIS); Frankfurt
`Main, DE.
`Yen et al., 1994, "Effect of dietary omega-3 and omega-6 fatty acid
`Sources on PUVA-induced cutaneous toxicity and tumorogenesis in
`the hairless mouse'. Arch. Dermatol, Res., 286(6): 331-6.
`Database WPI Week 200682, Thomson Scientific, London, GB,
`2006.
`English Abstract; JP 2003-531857; See abstract from corresponding
`WO 2001/082928 filed herewith.
`English Abstract; JP 2004-525180; See abstract from corresponding
`WO 2002/083122 filed herewith.
`English Abstract; JP 2006-528233; See abstract from corresponding
`WO 2004/100943 filed herewith.
`English Abstract; JP 2007-5028.05: See abstract from corresponding
`WO 2005/018632 filed herewith.
`English Abstract; JP 2007-509131; See abstract from corresponding
`WO 2005/037848 filed herewith.
`English Abstract; JP 2007-518764; See abstract from corresponding
`WO 2005/070411 filed herewith.
`English Abstract; JP 2004-536059; See abstract from corresponding
`WO 2002/09254 filed herewith.
`English Abstract; JP 2006-502196. See abstract from corresponding
`WO 2004/028529 filed herewith.
`
`Ando and Hatano, 1988, “Isolation of apolipoproteins from
`carotenoid-carrying lipoprotein in the serum of chum Salmon,
`Oncorhynchus keta'. J. Lipid Research, 29: 1264-1271.
`Aoi et al., 2003, "Astaxanthin limits exercise-induced skeletal and
`cardiac muscle damage in mice'. Antioxidants & Redox Signaling,
`5(1): 139-44.
`Britton, 1985, "General Carotenoid Methods'. Methods in Enzymol
`ogy, vol. 111, pp. 113-149.
`Calder, 2006. “n-3 polyunsaturated fatty acids, inflammation, and
`inflammatory diseases”. Am. J. Clin. Nutr., 83: 1505S.
`Charest et al., 2001, “Astaxanthin Extraction from Crawfish Shells by
`Supercritical CO2 with Ethanolas Cosolvent”. J. Aquatic Food Prod
`uct Technology, 10(3): 79-93.
`Chen and Meyers, 1982, “Extraction of Astaxanthin Pigment from
`Crawfish Waste Using a Soy Oil Process”. J. Food Sci., 47: 892-896.
`Clarke, 1980, “The Biochemical Composition of Krill, Euphausia
`superba dana, from South Georgia'. J. Exp. Mar. Biol. Ecol. 43:
`221-236.
`Czeczuga, 1974, "Comparative Studies of Carotenoids in the Fauna
`of the Gullmar Fjord (Bohuslan, Sweden). II. Crustacea. Eupagurus
`bernhardus, Hyas coarctatus and Upogebia deltaura”. Marine Biol
`ogy, 28:95-98.
`De Ritter and Purcell, 1981, "Carotenoid Analytical Methods',
`Carotenoids as Colorants and Vitamin A Precursors: Technological
`and Nutritional Applications, pp. 815-882.
`Deutch, 1995, “Menstrual pain in Danish women correlated with low
`n-3 polyunsaturated fatty acid intake”, Eur, J. Clin. Nutr., 49(7):
`508-16.
`Diez et al., 2003, “The role of the novel adipocyte-derived hormone
`adiponectin in human disease”, Eur, J. Endocrinol. 148(3): 293-300.
`Ellingsen et al., 1987. “Biochemistry of the autolytic processes in
`Antarctic krill post mortem. Autoproteolysis.” Biochem. J. 246, 295
`305.
`Emodi, 1978, "Carotenoids: Properties and Applications”. Food
`Technology, 32(5): 38.
`Felix-Valenzuela et al., 2001, “Supercritical CO2/Ethanol Extraction
`of Astaxanthin from Blue Crab (Callinecies sapidus) Shell Waste”,
`Journal of Food Process Engineering, 24: 101-112.
`Fox and Scheer, 1941, "Comparative Studies of the Pigments of
`Some Pacific Coast Echinoderms'. The Biological Bulletin, 441
`455.
`Fricke, et al., 1984, "Lipid, Sterol and Fatty Acid Composition of
`Antarctic Krill (Euphausia superba Dana)'. Lipids, 19 (11): 821
`827.
`Geusenset al., 1994, "Long-term effect of omega-3 fatty acid supple
`mentation in active rheumatoid arthritis. A 12-month, double-blind,
`controlled study”. Arthritis Rheum., 37(6): 824-9.
`Gilchrist and Green, 1960, “The Pigments of Artemia', Proceedings
`of the Royal Society, Series B Biological Sciences, vol. 152 No. 946,
`pp. 118-136.
`Goodwin and Srisukh, 1949, "Some Observations on Astaxanthin
`Distribution in Marine Crustacea'. Department of Biochemistry,
`University of Liverpool, pp. 268-270.
`Gulyaev and Bugrova, 1976 “Removing fats from the protein paste
`Okean”. Konservnaya I Ovoshchesushilnaya Promyshlennost, (4),
`37-8.
`Hardardottir and Kinsella, 1988. “Extraction of Lipid and Choles
`terol from Fish Muscle with Supercritical Fluids' Journal of Food
`Science, 53(6): 1656-1658.
`International Aqua Feed, 2006, vol. 9.
`International Search Report and Written Opinion for PCT/GB2008/
`002934, Dated Mar. 11, 2009.
`International Search Report and Written Opinion for PCT/IB2010/
`000512; dated Jun. 24, 2010.
`International Search Report for PCT/IB2007/000098, dated: Jun. 26,
`2007.
`Itoh et al., 2007; "Increased adiponectin secretion by highly purified
`eicosapentaenoic acid in rodent models of obesity and human obese
`Subjects'. Arteriosclerosis, Thrombosis, and Vascular Biology;
`27(9): 1918-1925.
`Johnson et al., 1978, "Simple Method for the Isolation of Astaxanthin
`from the Basidiomycetous Yeast Phafia rhodozyma”. Applied and
`Environmental Microbiology, 35(6): 1155-1159.
`RIMFROST EXHIBIT 1140 Page 0002
`
`

`

`US 8,372,812 B2
`Page 3
`
`Kolakowska, 1989, “Krill lipids after frozen storage of about one year
`in relation to storage time beforefreezing”. Die Nahrung Food,33(3):
`241-244.
`Kris-Etherton et al., 2002, “Fish Consumption, Fish Oil, Omega-3
`Fatty Acids, and Cardiovascular Disease'. Circulation, 106:2747
`2757.
`Kristensen et al., 1989, “Dietary supplementation with n-3 polyun
`Saturated fatty acids and human platelet function: a review with
`particular emphasis on implications for cardiovascular disease'. J.
`Intern. Med. Suppl. 731:141-50.
`Kunesova et al., 2006, "The influence of n-3 polyunsaturated fatty
`acids and very low calorie diet during a short-term weight reducing
`regimen on weight loss and serum fatty acid composition in severely
`obese women”, Physiol Res.; 55 (1):63-72.
`Laight et al., 1999. “F2-isoprostane evidence of oxidant stress in the
`insulin resistant, obese Zucker rat: effects of vitamin E'. Eur, J.
`Pharmacol. 377(1): 89-92.
`Lambertson and Braekkan, 1971, “Method of Analysis of
`Astaxanthin and its Occurrence in some Marine Products.” J. Sci.
`Food. Agr. vol. 22(2): 99-101.
`Libby et al., 2006. “Inflammation and Atherothrombosis: From
`Population Biology and Bench Research to Clinical Practice”. J.
`Amer. Coll. Card. 48 (9, Suppl. A): A33-A46.
`Lopez et al., 2004, “Selective extraction of astaxanthin from crusta
`ceans by use of Supercritical carbon dioxide'. Talanta, 64: 726-731.
`Mandeville, 1991, “Isolation and Identification of Carotenoid Pig
`ments, Lipids and Flavor Active Components from Raw Commercial
`Shrimp Waste”, Food Biotechnology, 5(2): 185-195.
`Meyers and Bligh, 1981, "Characterization of Astaxanthin Pigments
`from Heat-Processed Crawfish Waste”. J. Agric. Food Chem. 29:
`505-508.
`Meyers, 1977. “Using Crustacean Meals and Carotenoid-Fortified
`Diets”. Feedstuffs, vol. 49(19).
`
`Meyers, 1994, “Developments in world aquaculture, feed formula
`tions, and role of carotenoids'. Pure & Appl. Chem, vol. 66(5):
`1069-1076.
`Mills et al., 1989, “Dietary N-6 and N-3 fatty acids and salt-induced
`hypertension in the borderline hypertensive rat'. Lipids, 24(1):
`17-24.
`Moates and Van Bentem, 1990, "Separating out the value'. Food
`Science and Technology Today, 4(4): 213-214.
`Nikolaeva, 1967 "Amino acid composition of protein-coagulate in
`krill”, VNIRO, 63:161-4.
`Phleger, et al. (2002) “Interannual and between species comparison
`in the lipids, fatty acids, and sterols of Antarctic krill from the US
`AMLR Elephant Island Survey area: 1997 and 1998”. Comp
`Biochem Physiol 131B:733-747.
`Popp-Snijders et al., 1987. “Dietary supplementation of omega-3
`polyunsaturated fatty acids improves insulin sensitivity in non-insu
`lin-dependent diabetes', Diabetes Res. 4(3): 141-7.
`Sachindra, 2006, "Recovery of carotenoids from shrimp waste in
`organic solvents', Waste Management, 26: 1092-1098.
`Saether et al., 1986, "Lipids of North Atlantic krill'. J. Lipid Res.,
`27(3):274-85.
`Shahidi et al., 1998, "Carotenoid Pigments in Seafoods and
`Aquaculture” Critical Reviews in Food Science, 38(1): 1-67.
`Sidehu et al., 1970, "Biochmical Composition and NutritiveValue of
`Krill (Euphausia superb dana)”. J. Sci Food Agr. vol. 21, 293-296.
`Simopoulos, 1991, “Omega-3 fatty acids in health and disease and in
`growth and development'. Am. Clin. Nutr. 54:438-63.
`Somiya, 1982, “Yellow lens' eyes of a stomiatoid deep-sea fish,
`Malacosteous niger'. Proc. R. Soc. Lond., 215: 481-489.
`* cited by examiner
`
`RIMFROST EXHIBIT 1140 Page 0003
`
`

`

`U.S. Patent
`
`Feb. 12, 2013
`
`Sheet 1 of 7
`
`US 8,372,812 B2
`
`FIGURE 1
`
`KRELL WATER
`
`WATER
`
`Solid (Ds)
`
`
`
`
`
`Coagulum
`(Coag.)
`
`Liquid (DI)
`
`Coagulated liquid (Cl)
`
`RIMFROST EXHIBIT 1140 Page 0004
`
`

`

`U.S. Patent
`
`Feb. 12, 2013
`
`Sheet 2 of 7
`
`US 8,372,812 B2
`
`
`
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`RIMFROST EXHIBIT 1140 Page 0005
`
`

`

`U.S. Patent
`
`Feb. 12, 2013
`
`Sheet 3 of 7
`
`US 8,372,812 B2
`
`
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`RIMFROST EXHIBIT 1140 Page 0006
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`

`

`U.S. Patent
`
`Feb. 12, 2013
`
`Sheet 4 of 7
`
`US 8,372,812 B2
`
`FIGURE 4
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`RIMFROST EXHIBIT 1140 Page 0007
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`U.S. Patent
`
`Feb. 12, 2013
`
`Sheet 6 of 7
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`US 8,372,812 B2
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`

`

`1.
`PHOSPHOLIPID AND PROTEIN TABLETS
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`
`This application claims priority to U.S. Provisional Appli
`cation Ser. No. 61/155,758, filed: Feb.26, 2009, which is here
`incorporated by reference in its entirety.
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`FIELD OF THE INVENTION
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`The invention relates to tablets comprising phospholipids
`and protein, and in particular to the production of tablets
`containing astaxanthin and phospholipids comprising
`omega-3 fatty acid moieties.
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`BACKGROUND OF THE INVENTION
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`Krill is a small crustacean which lives in all the major
`oceans world-wide. For example, it can be found in the Pacific
`Ocean (Euphausia pacifica), in the Northern Atlantic (Mega
`nyctiphanes norvegica) and in the Southern Ocean off the
`coast of Antarctica (Euphausia superba). Krill is a key spe
`cies in the oceanas it is the food source for many animals such
`as fish, birds, sharks and whales. Krill can be found in large
`quantities in the ocean and the total biomass of Antarctic krill
`(E. superba) is estimated to be in the range of 300-500 million
`metric tons. Antarctic krill feeds on phytoplankton during the
`short Antarctic Summer During winter, however, its food Sup
`ply is limited to ice algae, bacteria, marine detritus as well as
`depleting body protein for energy. Virtue et al., Mar. Biol.
`126,521-527. For this reason, the nutritional values of krill
`vary during the season and to Some extent annually. Phlegeret
`al., Comp. Biochem. Physiol. 131B (2002) 733. In order to
`accommodate variations in food Supply, krill has developed
`an efficient enzymatic digestive apparatus resulting in a rapid
`breakdown of the proteins into amino acids. Ellingsen et al.,
`Biochem. J. (1987) 246, 295-305. This autoproteolysis is
`highly efficient also post mortem, making it a challenge to
`catch and store the krill in a way that preserves the nutritional
`quality of the krill. Therefore, in order to prevent the degra
`dation of krill the enzymatic activity is either reduced by
`storing the krill at low temperatures or the krill is made into a
`krill meal.
`During the krill meal process the krill is cooked so that all
`the active enzymes are denatured in order to eliminate all
`enzymatic activity. Krill is rich in phospholipids which act as
`emulsifiers. Thus it is more difficult to separate water, fat and
`proteins using mechanical separation methods than it is in a
`regular fish meal production line. In addition, krill becomes
`Solid, gains weight and loose liquid more easily when mixed
`with hot water. Eventually this may lead to a gradual buildup
`of coagulated krill proteins in the cooker and a non-continu
`ous operation due to severe clogging problems. In order to
`alleviate this, hot steam must be added directly into the
`cooker. This operation is energy demanding and may also
`result in a degradation of unstable bioactive components in
`the krill Such as omega-3 fatty acids, phospholipids and astax
`anthin. The presence of these compounds, make krill oil an
`attractive source as a food Supplement, a functional food
`products and a pharmaceutical for the animal and human
`applications.
`Omega-3 fatty acids have recently been shown to have
`potential effect of preventing cardiovascular disease, cogni
`tive disorders, joint disease and inflammation related diseases
`Such as rheumatoid arthritis. Astaxanthin is a strong antioxi
`dant and may therefore assist in promoting optimal health.
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`US 8,372,812 B2
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`Hence, there is a need for a method of processing krill into a
`krill meal at more gentle conditions which prevents the deg
`radation of these valuable bioactive compounds.
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`SUMMARY OF THE INVENTION
`
`The invention relates to processing crustaceans such as
`krill to provide oil and meal products, and in particular to the
`production of oils and other lipid extracts containing astax
`anthin and phospholipids comprising omega-3 fatty acid moi
`eties and meal rich in astaxanthin.
`In some embodiments, the present invention provides com
`positions comprising less than about 150, 100, 10, 5, 2 or 1
`mg/kgastaxanthin or from about 0.1 to about 1, 2, 5, 10 or 200
`mg/kg astaxanthin, preferably endogenous, naturally occur
`ring astaxanthin, also in the form of astaxanthin esters, from
`about 20% to about 50%, 15% to 45%, or 25% to 35%
`phospholipids on a w/w basis, and about 15% to 60%, about
`20% to 50%, or about 25% to 40% protein on a w/w basis,
`wherein said phospholipids comprise omega-3 fatty acid resi
`dues. In some embodiments, the composition comprises a
`lipid fraction having an omega-3 fatty acid content of from
`about 5% to about 30%, from 10% to about 30%, or from
`about 12% to about 18% on a w/w basis. In some embodi
`ments, the phospholipids comprise greater than about 60%,
`65%, 80%, 85% or 90% phosphatidylcholine on a w/w basis.
`In some embodiments, the phospholipids comprise less than
`about 15%, 10%, 8% or 5% ethanolamine on a w/w basis. In
`Some embodiments, the compositions comprise from about
`1% to 10%, preferably 2% to 8%, and most preferably about
`2% to 6% alkylacylphosphatidylcholine. In some embodi
`ments, the compositions comprise from about 30% to about
`70% triacylglycerol on a w/w basis. In further embodiments,
`the compositions comprise less than about 1% cholesterol. In
`some embodiments, the protein comprises from about 8% to
`about 14% leucine on awfw basis and from about 5% to 11%
`isoleucine on a W/w basis.
`In some embodiments, the present invention comprises an
`aqueous phase and a Solid phase, said solid phase comprising
`from about 20% to about 50% phospholipids on a w/w basis,
`and about 20% to 50% protein on a w/w basis, wherein said
`phospholipids comprise from about 10% to about 20%
`omega-3 fatty acid residues.
`In other embodiments, the present invention provides krill
`compositions comprising astaxanthin, a protein fraction, and
`a lipid fraction, wherein said lipid fraction comprises less
`than about 10%, 5% or 3% phospholipids on a w/w basis. In
`Some embodiments, the phospholipids comprise less than
`about 15%, 10% or 5% phosphatidylcholine on a w/w basis.
`In some embodiments, the present invention provides a
`krill meal comprising astaxanthin and from about 8% to about
`31% lipids, preferably from about 8% to about 10 or 18%
`lipids, wherein said lipids comprises greater than about 80%
`neutral lipids on a w/w basis. In some embodiments, the krill
`meal comprises less than about 15%, 10%, 5%, 3% or 1%
`phospholipids. In some embodiments, the phospholipids
`comprise less than about 15%, 10% or 5% phosphatidylcho
`line on a w/w basis.
`In some embodiments, the present invention provides
`methods of preparing a phospholipid composition from bio
`logical material or biomass comprising: mixing said biologi
`cal material or biomass with waterata suitable temperature to
`form a solid phase and an aqueous phase comprising phos
`pholipids and proteins; separating said solid phase from said
`aqueous phase; heating said aqueous phase at a temperature
`Sufficient to form a phospholipid-protein precipitate; and
`separating said phospholipid-protein precipitate from said
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`US 8,372,812 B2
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`aqueous phase. In some embodiments, the present invention
`provides a phospholipid-protein precipitate obtained by using
`the foregoing method. In some embodiments, the biological
`material or biomass is krill. In other embodiments, the bio
`logical material or biomass is selected from crabs, shrimp,
`calanus, plankton, crayfish, eggs or other phospholipid con
`taining biological materials or biomass. In some embodi
`ments, the methods further comprise the step of forming a
`meal from said Solid phase. In some embodiments, the step of
`forming a meal comprises: heating the Solid phase in the
`presence of water, separating fat and protein in said Solid
`phase; and drying said protein to form a meal. In some
`embodiments, the processes further comprise the steps of
`pressing and drying the coagulum to form a coagulum meal.
`In some embodiments, the drying is by hot air or steam. In
`Some embodiments, the present invention provides a phos
`pholipid-protein precipitate obtained by using the foregoing
`method. In some embodiments, the present invention pro
`vides a composition comprising a krill Solid phase according
`to the foregoing methods. In some embodiments, the present
`invention provides a krill meal obtained by the foregoing
`methods.
`In some embodiments, the present invention provides pro
`cesses comprising: extracting a first lipid fraction from a krill
`biomass; extracting a second lipid fraction from a krill bio
`mass; and blending said first lipid fraction and said second
`lipid fraction to provide a krill lipid composition having a
`desired composition. In some embodiments, the one or more
`of the extracting steps are performed in the absence of Sub
`stantial amounts of organic Solvents. In some embodiments,
`the first lipid fraction is extracted by: mixing krill with water
`at a suitable temperature to form a solid phase and an aqueous
`phase comprising phospholipids and protein; separating said
`Solid phase from said aqueous phase; heating said aqueous
`phase at a temperature Sufficient to form a phospholipid
`protein precipitate; separating said phospholipid-protein pre
`cipitate from said aqueous phase; and separating said phos
`pholipids from said protein. In some embodiments, the
`second lipid fraction is extracted by: heating the Solid phase in
`the presence of water; and separating fat and protein in said
`solid phase. In some embodiments, the first lipid fraction
`comprises a phospholipid fraction comprising greater than
`about 90% phosphatidylcholine on a w/w basis. In some
`embodiments, the second lipid fraction comprises greater
`than about 80% neutral lipids on a w/w basis.
`In some embodiments, the present invention provides pro
`cesses of producing a phospholipid composition from bio
`logical material or biomass comprising: mixing said biologi
`cal material or biomass with water to increase the temperature
`of said biological material to about 25 to 80°C., preferably to
`about 50 to 75° C., and most preferably to about 60 to 75° C.
`to form a first Solid phase and a first aqueous phase compris
`ing phospholipids and proteins; separating said first Solid
`phase from said first aqueous phase; and separating a protein
`and phospholipid fraction from said first aqueous phase. In
`some embodiments, the biomass is heated to the first tem
`perature for at least 3 minutes, preferably from about 3 min
`utes to 60 minutes, more preferably from about 3 minutes to
`20 minutes, and most preferably from about 3 minutes to 10
`minutes. The present invention is not limited to the use of any
`particular biological materials or biomass. In some embodi
`ments, the biological material is a marine biomass. In some
`preferred embodiments, the biological material or biomass
`comprises krill crabs, shrimp, calanus, plankton, crayfish,
`eggs or other phospholipid containing biological materials or
`biomass. The present invention is not limited to the use of any
`particular type of krill. In some embodiments, the krill is
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`fresh, while in other embodiments, the krill is frozen. In some
`embodiments, the krill is of the species Euphausia superba.
`In some embodiments, the step of separating a protein and
`phospholipid fraction from said first aqueous phase com
`prises heating said first aqueous phase at a temperature Suf
`ficient to form a phospholipid-protein coagulate and separat
`ing said phospholipid-protein coagulate from said aqueous
`phase. In some embodiments, the processes utilize a second
`heating step. In some embodiments, the first aqueous phase is
`heated to over 80°C., preferably to about 80 to 120° C., and
`most preferably to about 90 to 100°C. In some embodiments,
`the krill milk is held at these temperatures for from about 1
`minute to about 60 minutes, preferably about 1 minute to
`about 10 minutes, and most preferably for about 2 minutes to
`8 minutes. In some embodiments, the heating is at atmo
`spheric pressure, while in other embodiments, the pressure is
`greater than atmospheric pressure. In some embodiments, the
`processes further comprise the step of pressing said phospho
`lipid-protein coagulate to form a coagulate liquid phase and a
`coagulate press cake. In some embodiments, the processes
`further comprise drying said coagulate press cake to form a
`coagulate meal. In some embodiments, the processes further
`comprise extracting a coagulate oil from said coagulate meal.
`In some embodiments, the processes further comprise the
`steps of pressing and drying the coagulum to forma coagulum
`meal. In some embodiments, the drying is by hot air or steam.
`In some embodiments, the step of separating a protein and
`phospholipid fraction from said first aqueous phase com
`prises filtration of said aqueous phase to provide a phospho
`lipid-protein retentate comprising proteins and phospholip
`ids. In some embodiments, filtration is via membrane
`filtration. In some embodiments, the filtration comprises fil
`tering said aqueous phase through a microfilter with a pore
`size of from about 50 to 500 nm. In some embodiments, the
`processes further comprise the step of dewatering said phos
`pholipid-protein retentate to form a retentate liquid phase and
`a retentate concentrate. In some embodiments, the processes
`further comprise the step of removing water from said reten
`tate concentrate so that said retentate concentrate is microbi
`ally stable. In some embodiments, the processes further com
`prise the step of extracting a retentate oil from said retentate
`concentrate. In some embodiments, the processes further
`comprise the step of heating said first solid phase and then
`pressing said first solid phase to form a first press cake and a
`second liquid phase. In some embodiments, the processes
`further comprise the step of drying said first press cake to
`provide a first krill meal. In some embodiments, the processes
`further comprise the steps of heating said second liquid phase
`and then separating said second liquid phase to provide a first
`krill oil and stickwater. In some embodiments, the stickwater
`is evaporated and added to said first press cake, and a meal is
`formed from said evaporated Stickwater and said first press
`cake to provide a second krill meal. In some embodiments,
`the second liquid phase is heated to over 80°C., preferably to
`about 80 to 120° C., and most preferably to about 90 to 100°
`C. prior to said separation. In some embodiments, the pro
`cesses further comprise the step of combining the previously
`described coagulate oil or the retentate oil and the first krill oil
`to provide a blended oil. In other embodiments, the coagulate
`oil, retentate oil, or oil pressed from the first solid phase are
`combined with the coagulate meal or retentate. In further
`embodiments, the processes of the present invention com
`prise the further step of Supplementing the meals or oils
`produced as described above with additional proteins, phos
`pholipids, triglycerides, fatty acids, and/or astaxanthin to pro
`duce an oil or meal with a desired defined composition. As
`such, a person of skill in the art will readily recognize that the
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