`
`APPLICATION
`NUMBER
`61/024,072
`
`FILING or
`37l(c)DATE
`01/28/2008
`
`GRPART
`UNIT
`
`FIL FEE REC'D
`210
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`72960
`Casimir Jones, S.C.
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`UKITED STATES DEPARTME.\IT OF COMMERCE
`United States Patent and Trademark Office
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`\V\\lw.uspto.gov
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`TOT CLAIMS IND CLAIMS
`
`ATTY.DOCKET.NO
`NA INUT-14409/US-4/PRO
`CONFIRMATION NO. 3084
`FILING RECEIPT
`111111111111111111111111]~!1~~~1~~1~~1~~H~~~~~111111111111111111111111
`
`Date Mailed: 02/26/2008
`
`Receipt is acknowledged of this provisional patent application. It will not be examined for patentability and will
`become abandoned not later than twelve months after its filing date. Any correspondence concerning the application
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`Applicant(s)
`
`Inge Bruheim, Valda, NORWAY;
`Asegir Saebo, Eidsnes, NORWAY;
`Snorre Tilseth, Bergen, NORWAY;
`Mikko Griinari, Espoo, FINLAND;
`Power of Attorney: The patent practitioners associated with Customer Number 72960
`
`If Required, Foreign Filing License Granted: 02/25/2008
`The country code and number of your priority application, to be used for filing abroad under the Paris Convention,
`is US 61 /024,072
`Projected Publication Date: None, application is not eligible for pre-grant publication
`Non-Publication Request: No
`Early Publication Request: No
`Title
`
`Bioeffective krill oil compositions
`
`PROTECTING YOUR INVENTION OUTSIDE THE UNITED STATES
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`Since the rights granted by a U.S. patent extend only throughout the territory of the United States and have no
`effect in a foreign country, an inventor who wishes patent protection in another country must apply for a patent
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`application under the Patent Cooperation Treaty (PCT). An international (PCT) application generally has the same
`effect as a regular national patent application in each PCT-member country. The PCT process simplifies the filing
`of patent applications on the same invention in member countries, but does not result in a grant of "an international
`page 1of3
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`RIMFROST EXHIBIT 1002 page 0001
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`
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`RIMFROST EXHIBIT 1002 page 0002
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`
`page 3 of 3
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`RIMFROST EXHIBIT 1002 page 0003
`
`
`
`Bioeffective krill oil compositions
`
`Inventors: Inge Bruheim (Volda, Norway), Asgeir Srebe (Eidsnes, Norway), Snorre Tilseth
`
`(Bergen , Norway) and Mikko Griinari (Espoo, Finland).
`
`5
`
`FIELD OF THE INVENTION
`
`This invention relates to extracts from Antarctic krill that comprise bioactive fatty acids.
`
`BACKGROUND OF THE INVENTION
`
`10
`
`In the Southern Ocean, off the coast of Antarctica, Antarctic krill (Euphausia superba) can be
`
`found in large quantities, ranging from 300-500 million metric tons of biomass. It feeds on
`
`phytoplankton during the short Antarctic summer. During winter, however, its food supply is
`
`limited to ice algae, bacteria, marine detritus as well as depleting body protein for energy [ 1].
`
`15
`
`In order to isolate the krill oil from the krill, solvent extraction methods have been used [7-8].
`
`Krill lipids have been extracted by placing the material in a ketone solvent (e.g. acetone) in
`
`order to extract the lipid soluble fraction. This method involves separating the liquid and solid
`
`contents and recovering a lipid rich fraction from the liquid fraction by evaporation. Further
`
`processing steps include extracting and recovering by evaporation the remaining soluble lipid
`
`20
`
`fraction from the solid contents by using a solvent such as ethanol [8]. The compositions
`
`produced by these methods are characterized by containing at least 75 µgig astaxanthin,
`
`preferably 90 µgig astaxanthin. Another krill lipid extract disclosed contained at least 250
`
`~tg/g canastaxanthin [8], preferably 270 µg/g canastaxanthin.
`
`25 Krill oil compositions have been described as being effective for decreasing cholesterol,
`
`inhibiting platelet adhesion, inhibiting artery plaque formation, preventing hypertension,
`
`controlling athritis symptoms, preventing skin cancer, enhancing transdermal transport,
`
`reducing the symptoms of premenstrual symptoms or controlling blood glucose levels in a
`
`patient.
`
`[9] In yet another application, a krill oil composition has been disclosed [4]
`
`30
`
`comprising a phospholipid and/or a flavanoid. The phospholipid content in the krill lipid
`
`extract could be as high as 60% w/w and the EPA/DHA content as high as 35% (w/w).
`
`1
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`RIMFROST EXHIBIT 1002 page 0004
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`
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`Furthermore, nutraceuticals, pharmaceuticals and cosmetics compnsmg the phospholipid
`
`extract were disclosed. Previously, it was also shown that supercritical fluid extraction using
`
`neat C02 could be used to prevent the extraction of phospholipids in order to extract the
`
`neutral lipid fraction from krill, which comprised of esterified and free astaxanthin [10].
`
`5
`
`Supercritical fluid extraction with solvent modifier has previously been used to extract marine
`
`phospholipids from salmon roe [11], but has not been previously used to extract phospholipids
`
`from krill meal.
`
`The methods described above rely on the processing of frozen krill that are transported from
`
`10
`
`the Southern Ocean to the processing site. This transportation is both expensive and can
`
`result in degradation of the krill starting material. Data in the literature showing a rapid
`
`decomposition of the oil in krill explains why some krill oil currently offered as an omega-3
`
`supplement in the marketplace contains very high amounts of partly decomposed
`
`phosphatidylcholine and also partly decomposed glycerides. Saether et al., Comp. Biochem
`
`15
`
`Phys. B 83B(l): 51-55 (1986). The products offered also contain high levels of free fatty
`
`acids.
`
`What is needed in the art are methods for processing krill that do not require transport of
`
`frozen krill material over long distances and the products produced by those methods.
`
`20
`
`SUMMARY OF THE INVENTION
`
`In a first aspect of the invention is a composition characterized by comprising at least 65%
`
`(w/w) phospholipids.
`
`25
`
`In another aspect of the invention is a composition obtained from aquatic or marine sources,
`
`characterized by comprising 65% (w/w) phospholipids.
`
`In yet another aspect of the invention is a composition obtained from krill, characterized by
`
`comprising at least 65% (w/w) phospholipids.
`
`30
`
`In another aspect of the invention is a composition obtained from krill, characterized by
`
`comprising at least 65% (w/w) phospholipids and at least 39% omega-3 fatty acids (w/w).
`
`2
`
`RIMFROST EXHIBIT 1002 page 0005
`
`
`
`In yet another aspect of the invention is a composition obtained from krill, characterized by
`
`comprising at least 65% (w/w) phospholipids, at least 39% omega-3 fatty acids (w/w) and at
`
`least 580 mg/kg astaxanthin esters.
`
`5
`
`In another aspect of the invention is a composition obtained from krill, characterized by
`
`comprising at least 39% omega-3 fatty acids (w/w) and at least 580 mg/kg astaxanthin esters.
`
`In yet another aspect of the invention is a composition obtained from krill, characterized by
`
`comprising at least 65% (w/w) phospholipids and at least 580mg/kg astaxanthin esters.
`
`10
`
`In yet another aspect is a krill oil effective for reducing insulin resistance, improving blood
`
`lipid profile, reducing inflammation or reducing oxidative stress.
`
`In some embodiments, the present invention provides a composition comprising at least 65%
`
`15
`
`(w/w) of phospholipids, said phospholipids characterized in containing at least 35% omega-3
`
`fatty acid residues. In some preferred embodiments, the composition is derived from a marine
`
`or aquatic biomass. In some further preferred embodiments, the composition is derived from
`
`krill.
`
`In some embodiments, the composition comprises less than 2% free fatty acids. In
`
`some embodiments, composition comprises less than 10% triglycerides. In some preferred
`
`20
`
`embodiments, the phospholipids comprise greater than 50% phosphatidylcholine. In some
`
`embodiments, the composition comprises at least 500 mg/kg astaxanthin esters.
`
`In some
`
`embodiments, the composition comprises at least 500 mg/kg astaxanthin esters and at least
`
`36% (w/w) omega-3 fatty acids. In some embodiments, the composition comprises less than
`
`about 0.5g/1 OOg total cholesterol. In some embodiments, the composition comprises less than
`
`25
`
`about 0.45% arachidonic acid (w/w).
`
`In some embodiments, the present invention provides a krill lipid extract comprising at least
`
`500 mg/kg astaxanthin esters and at least 36% (w/w) omega-3 fatty acids.
`
`In further
`
`embodiments, the present invention provides a krill lipid extract comprising at least 100
`
`30 mg/kg astaxanthin esters, at least 20% (w/w) omega-3 fatty acids, and less than about 0.45%
`
`arachidonic acid (w/w).
`
`3
`
`RIMFROST EXHIBIT 1002 page 0006
`
`
`
`In some embodiments, the present invention provides methods comprising administering the
`
`foregoing compositions to a subject in an amount effective for reducing insulin resistance,
`
`reducing inflammation, improving blood lipid profile and reducing oxidative stress.
`
`5
`
`In some embodiments, the present invention provides a krill lipid extract comprising greater
`
`than about 80% triglycerides and greater than about 90 mg/kg astaxanthin esters. In some
`
`embodiments, the krill lipid extract is characterized in containing from about 5% to about
`
`15% omega-3 fatty acid residues.
`
`In some embodiments, the krill lipid extract is
`
`characterized in containing less than about 5% phospholipids. In some embodiments,, the
`
`10
`
`krill lipid extract ischaracterized in comprising from about 5% to about 10% cholesterol.
`
`In some embodiments, the present invention provides a krill meal composition comprising
`
`less than about 50g/kg total fat. In some embodiments, the krill meal composition comprises
`
`from about 5 to about 20 mg/kg astaxanthin esters.
`
`In some embodiments, the krill meal
`
`15
`
`composition comprises greater than about 65% protein. In some embodiments, the krill meal
`
`composition of comprises greater than about 70% protein. In some further embodiments, the
`
`present invention provides an animal feed comprising the krill meal of Claim 21.
`
`In some embodiments, the present invention provides methods of increasing flesh coloration
`
`20
`
`in an aquatic species comprising feeding said aquatic species a composition comprising the
`
`krill meal described above. In some embodiments, the present invention provides methods of
`
`increasing growth and overall survival rate of aquatic species by feeding the krill meal
`
`described above.
`
`25
`
`In some embodiments, the present invention provides methods of producing krill oil
`
`comprising: a) providing krill meal; and b) extracting oil from said krill meal. In some
`
`embodiments, the krill meal is produced by heat-treating krill. In some embodiments, the krill
`
`meal is stored prior to said extraction step.
`
`In some embodiments, the extracting step
`
`comprises extraction by supercritical fluid extraction. In some embodiments, the supercritical
`
`30
`
`fluid extraction is a two step process comprising an extraction with carbon dioxide and an
`
`extraction including ethanol as an entrainer.
`
`In some embodiments, the present invention
`
`provides an oil produced by the foregoing method.
`
`4
`
`RIMFROST EXHIBIT 1002 page 0007
`
`
`
`In some embodiments, the present invention provides methods of production of krill oil
`
`comprising: a) providing fresh krill; b) treating said fresh krill to denature lipases and
`
`phospholipases in said fresh krill to provide a denatured krill product; and c )extracting oil
`
`from said denatured krill product.
`
`In some embodiments, the denaturation step comprises
`
`5
`
`heating of said fresh krill. In some embodiments, the denaturation step comprises heating said
`
`fresh krill after grinding. In some embodiments, the methods further comprise storing said
`
`denatured krill product at room temperature or below between the denaturation step and the
`
`extraction step.
`
`In some embodiments, the enzyme denaturation step is achieved by
`
`application of heat. In some embodiments, the extraction step comprises use of supercritical
`
`10
`
`carbon dioxide, with or without use of a polar modifier. In some embodiments, the extraction
`
`step comprises use of ethanol. In some embodiments, the extraction step is comprises ethanol
`
`extraction followed by acetone to precipitation of phospholipids. In some embodiments, the
`
`denatured krill product is a meal. In some embodiments, the present invention provides an oil
`
`produced by the foregoing method.
`
`15
`
`In some embodiments, the present invention provides a composition compnsmg an oil
`
`extracted from krill having a phosphatidylcholine content of greater then about 50% (w/w). In
`
`some embodiments, the oil has a phosphatidylcholine content of greater then about 70%
`
`(w/w). In some embodiments, the oil has a phosphatidylcholine content of greater then about
`
`20
`
`80% (w/w). In some embodiments, the composition comprises less than 2% free fatty acids.
`
`In some embodiments, the composition comprises less than 10% triglycerides.
`
`In some
`
`embodiments, the composition comprises at least 500 mg/kg astaxanthin esters.
`
`In some
`
`embodiments, the composition comprises less than about 0.45% arachidonic acid (w/w).
`
`25
`
`In some embodiments, the present invention provides composition comprising odorless krill
`
`oil. In some embodiments, the odorless krill oil comprises less than about 10 mg/kg (w/w)
`
`trimethylamine.
`
`In some further embodiments, the present invention provides an odorless
`
`krill oil produced by the method comprising: extracting a neutral krill oil from a krill oil
`
`containing material by supercritical fluid extraction to provide a deodorized krill material,
`
`30 wherein said neutral krill oil contains odor causing compounds and extracting a polar krill oil
`
`from said deodorized krill material by supercritical fluid extraction with a polar entrainer to
`
`provide an essentially odorless krill oil.
`
`5
`
`RIMFROST EXHIBIT 1002 page 0008
`
`
`
`In some embodiments, the present invention provides a composition compnsmg krill oil
`
`containing less than about 70 micrograms/kilogram (w/w) astaxanthin esters.
`
`In some
`
`embodiments, the compositions comprise less than about 50 micrograms/kilogram (w/w)
`
`astaxanthin esters.
`
`In some embodiments, the compositions comprise less than about 20
`
`5 micrograms/kilogram (w/w) astaxanthin esters.
`
`In some embodiments, the compositions
`
`comprise less than about 5 micrograms/kilogram (w/w) astaxanthin esters.
`
`In some embodiments, the present invention provides a krill oil produced by the process
`
`comprising: pumping fresh krill from a trawl onto a ship, heating the krill to provide a krill
`
`10 material, and extracting oil from the krill material.
`
`In further embodiments, the present invention provides a blended krill oil composition
`
`comprising: from about 45% to 55% w/w phospholipids; from about 35% to 45% w/w
`
`triglycerides; and from about 400 to about 1500 ppm astaxanthin. In some embodiments, the
`
`15
`
`blended krill oil product comprises a blend of lipid fractions obtained from Euphausia
`
`superba. In some embodiments, the composition comprises from about 25% to 30% omega-3
`
`fatty acids as a percentage of total fatty acids and wherein from about 80% to 90% of said
`
`omega-3 fatty acids are attached to said phospholipids.
`
`20
`
`In still other embodiments, the present invention provides a Euphausia superba krill oil
`
`composition comprising: from about 30% to 60% w/w phospholipids; from about 30% to 50%
`
`triglycerides; from about 400 to about 1500 ppm astaxanthin; and from about 20% to 35%
`
`omega-3 fatty acids as a percentage of total fatty acids in said composition, wherein from
`
`about 70% to 95% of said omega-3 fatty acids are attached to said phospholipids.
`
`25
`
`In still further embodiments, the present invention provides a dietary supplement comprising
`
`encapsulated Euphausia superba krill oil comprising from about 30% to 60% w/w
`
`phospholipids; from about 30% to 50% triglycerides; from about 400 to about 1500 ppm
`
`astaxanthin; and from about 20% to 35% omega-3 fatty acids as a percentage of total fatty
`
`30
`
`acids in said composition, wherein from about 70% to 95% of said omega-3 fatty acids are
`
`attached to said phospholipids.
`
`In some embodiments, the present invention provides methods of making a Euphausia
`
`superba krill oil composition comprising: contacting Euphausia superba with a polar solvent
`
`6
`
`RIMFROST EXHIBIT 1002 page 0009
`
`
`
`to provide an polar extract comprising phospholipids; contacting Equphasia superba with a
`
`neutral solvent to provide a neutral extract comprising triglycerides and astaxanthin;
`
`combining said polar extract and said neutral extract to provide Euphausia superba krill oil
`
`comprising from about 30% to 60% w/w phospholipids; from about 30% to 50%
`
`5
`
`triglycerides; from about 400 to about 1500 ppm astaxanthin; and from about 20% to 35%
`
`omega-3 fatty acids as a percentage of total fatty acids in said composition, wherein from
`
`about 70% to 95% of said omega-3 fatty acids are attached to said phospholipids. In some
`
`embodiments, the methods further comprise the step of encapsulating the Euphausia superba
`
`krill oil. In some embodiments, the present invention provides a Euphausia superba krill oil
`
`10
`
`produced by the methods described above.
`
`In some embodiments, the present invention provides methods of producing a dietary
`
`supplement comprising; contacting Euphausia superba with a polar solvent to provide an polar
`
`extract comprising phospholipids; contacting Equphasia superba with a neutral solvent to
`
`15
`
`provide a neutral extract comprising triglycerides and astaxanthin; combining said polar
`
`extract and said neutral extract to provide Euphausia superba krill oil comprising from about
`
`30% to 60% w/w phospholipids; from about 30% to 50% triglycerides; from about 400 to
`
`about 1500 ppm astaxanthin; and from about 20% to 35% omega-3 fatty acids as a percentage
`
`of total fatty acids in said composition, wherein from about 70% to 95% of said omega-3 fatty
`
`20
`
`acids are attached to said phospholipids; and encapsulating said Euphausia superba krill oil.
`
`DESCRIPTION OF THE FIGURES
`
`Figure 1. 31P NMR of Total Lipid of Residue powder from lab scale 3-stage extraction
`
`25
`
`(313 K, 300 bar and up to 24% ethanol).
`
`Figure 2. Cumulative extraction yields for pilot scale trials on krill meal B where: all neutral
`
`lipids are extracted (full N extn); 1h the neutral lipids are extracted and ethanol is recycled
`
`from the previous run (1/2 N extn); 1h the neutral lipids are extracted and ethanol is recycled
`
`30
`
`for a second time (R2); 1h the neutral lipids are extracted using fresh ethanol (1/2 N, Fr); 1h the
`
`neutral lipids are extracted and the ethanol recycled from this and the previous run.
`
`7
`
`RIMFROST EXHIBIT 1002 page 0010
`
`
`
`Figure 3. FLipid profile for krill meal feed (type-A) and extract samples. Sl/1 =neutral lipid
`
`main product, S 1/2 = phospholipid main product, S2/1 = neutral lipid minor product, S2/2
`
`etoh = high ethanol fraction dissolved lipids, S2/2 etoh insol = precipitated lipids.
`
`Figure 4. Lipid profile for krill meal feed (type-B) and extract samples. Sl/1 =neutral lipid
`
`5 main product, S 1/2 = phospholipid main product, S2/1 = neutral lipid minor product, S2/2 - 4
`
`= high ethanol second separator fractions at increasing extraction times.
`
`Figure 5. Lipid profile for krill meal feed (type-B) and extract samples. Sl/1 =neutral lipid
`
`main product, S 1/2 = phospholipid main product, S2/1 - 7 = second separator fractions at
`
`10
`
`increasing extraction times. Run performed with partial extraction of neutral lipids using C02
`+ 5 % ethanol, then in-situ blending/extraction with C02 + ~ 20 % ethanol.
`
`Figure 6. Second separator product composition and masses as a function of C02 usage, solid
`
`symbols LH axis, lines RH axis.
`
`15
`
`Figure 7. Summary of 500 bar extraction runs. Legend key, SI =first separator, 0 or 5 =zero
`
`or 5 % ethanol for first stage of extraction, 0.5 = ~ 1/2 neutral lipid extracted, final figure is
`
`average ethanol at high ethanol flow rate, R = recycle.
`
`20
`
`Figure 8. Phospholipid content in main product as a function of the percentage of neutral
`
`lipids extracted in the minor product.
`
`DEFINITIONS
`
`25 As used herein, "phospholipid" refers to an organic compound having the following general
`
`structure:
`
`8
`
`RIMFROST EXHIBIT 1002 page 0011
`
`
`
`0
`
`II
`o--c--R1
`0
`
`11
`2
`- -O - -C - -R
`
`0
`
`II
`0-1-_ O-R
`
`3
`
`wherein RI is a fatty acid residue, R2 is a fatty acid residue or -OH, and R3 is a -H or
`
`nitrogen
`
`containing
`
`compound
`
`choline
`
`(HOCH2CH2N+(CH3 ) 30H-),
`
`ethanolamine
`
`5
`
`(HOCH2CH2NH2), inositol or serine. RI and R2 cannot simultaneously be OH. When R3 is
`
`an -OH, the compound is a diacylglycerophosphate, while when R3 is a nitrogen-containing
`
`compound, the compound is a phosphatide such as lecithin, cephalin, phosphatidyl serine or
`
`plasmalogen.
`
`10 An "ether phospholipid" as used herein refers to a phospholipid having an ether bond at
`
`position I the glycerol backbone. Examples of ether phospholipids include, but are not
`
`limited
`
`to,
`
`alkylacylphosphatidylcholine
`
`(AAPC),
`
`lyso-alkylacylphosphatidylcholine
`
`(LAAPC), and alkylacylphosphatidylethanolamine (AAPE). A "non-ether phospholipid" is a
`
`phospholipid that does not have an ether bond at position I of the glycerol backbone.
`
`15
`
`As used herein, the term omega-3 fatty acid refers to polyunsaturated fatty acids that have the
`
`final double bond in the hydrocarbon chain between the third and fourth carbon atoms from
`
`the methyl end of the molecule. Non-limiting examples of omega-3 fatty acids include,
`
`5,8,I I,I4,I 7-eicosapentaenoic acid (EPA), 4,7,10,13,I6,I9-docosahexanoic acid (DHA) and
`
`20
`
`7,10,13,I6,I9-docosapentanoic acid (DPA).
`
`As used herein, astaxanthin refers to the following chemical structure:
`
`9
`
`RIMFROST EXHIBIT 1002 page 0012
`
`
`
`OH
`
`HO
`
`0
`
`As used herein, astaxanthin esters refer to the fatty acids esterified to OH group in the
`
`astaxanthin molecule.
`
`5 As used herein, the term w/w (weight/weight) refers to the amount of a given substance in a
`
`composition on weight basis. For example, a composition comprising 50% w/w
`
`phospholipids means that the mass of the phospholipids is 50% of the total mass of the
`
`composition (i.e., 50 grams of phospholipids in 100 grams of the composition, such as an oil).
`
`10 DETAILED DESCRIPTION OF THE INVENTION
`
`This invention discloses novel krill oil compositions characterized by containing high levels
`
`of astaxanthin, phospholipids and omega-3 fatty acids. The krill oils compositions are
`
`extracted from krill meal using supercritical fluid extraction (SFE) with a solvent modifier.
`
`The krill meal has been processed on board a ship in Antarctica using live krill as starting
`
`15 material in order to ensure the highest possible quality of the krill meal. The krill oils are
`
`extracted from the krill meal in two stages, in step 1 the neutral fraction is extracted using neat
`
`supercritical C02 . The neutral fraction consisted mostly of triglycerides and cholesterol. In
`
`stage 2, the polar lipids (phospholipids) are extracted by adding 20% ethanol to the
`
`supercritical C02 extraction medium. The polar krill oil extracted where found to contain 94%
`
`20
`
`phospholipids (w/w), 46% omega-3 fatty acids (w/w) and 580 mg/kg astaxanthin esters.
`
`The present invention provides methods to avoid decomposition of glycerides and
`
`phospholipids in krill oil and compositions produced by those methods. The product obtained
`
`by these new methods is virtually free of enzymatically decomposed oil constituents. The
`
`25
`
`solution to the problem is to incorporate a protein denaturation step on fresh krill prior to use
`
`of any extraction technology. Denaturation can be achieved by thermal stress or by other
`
`means. After denaturation, the oil can be extracted by an optional selection of nonpolar and
`
`polar solvents including use of supercritical carbon dioxide. Krill is adapted to a very efficient
`
`10
`
`RIMFROST EXHIBIT 1002 page 0013
`
`
`
`nutrient digestion at very low temperatures. Therefore the enzymes are sensitive to heat and
`
`the step of applying thermal denaturation of lipases and phospholipases does not imply use of
`
`very high temperatures. Suprisingly, it has been found that the use of mild denaturation
`
`conditions can greatly enhance the quality of krill oil.
`
`5
`
`Additionally, a major obstacle of several processes of extraction is the cost of removing water.
`
`This is particularly true for methods feasible for extraction of highly unsaturated lipids where
`
`freeze drying has been regarded as the method of choice to avoid oxidative breakdmvn of
`
`lipids. However, the lipids in krill are surprisingly stable against oxidative deterioration.
`
`10
`
`Therefore, a process including moderate use of heat in the water removing process is feasible
`
`provided that the enzymes have been inactivated.
`
`A.
`
`Krill Processing
`
`15
`
`The present invention provides methods for processing freshly caught krill at the site of
`
`capture and preferably on board a ship. After processing on board, the krill can be further
`
`subjected to extraction processes on board the ship or at a remote location away from the ship.
`
`The processing steps described herein also also allow for the storage of krill material,
`
`preferably a krill meal for from about 1,2, 3, 4, 5, 6, 8, 9, 10, 11, or 12 months to about 24 to
`
`20
`
`36 months prior to processing.
`
`In some preferred embodiments, freshly caught is first subjected to a protein denaturation
`
`step. The present invention is not limited to any particular method of protein dentatuation. In
`
`some embodiments, the denaturation is accomplished by application of chemicals, heat, or
`
`25
`
`combinations thereof. In some embodiments, freshly caught krill is wet pressed to obtain oil
`
`and meal. In some embodiments, the meal is then heated to a temperature of about 50 degrees
`
`Celsius to about 100 degrees Celsius for about 20 minutes to about an hour, preferably about
`
`40 minutes to coagulate the proteins. In some embodiments, this material is then pressed to
`
`yield a press cake. When this method is used on krill, only a small amount of oil is released.
`
`30 Most of the oil is still present in the denaturated meal. In some embodiments, antioxidants
`
`such as ethoxyquin or Vitamin E are added to the meal. However, as shown in the examples,
`
`the resulting meal is surprisingly stable. The stability can only partly be explained by addition
`
`of an antioxidant to the meal. This antioxidant can after extraction of the oil from denaturated
`
`meal be removed by further processing steps. Alternatively the oil can be extracted rather
`
`11
`
`RIMFROST EXHIBIT 1002 page 0014
`
`
`
`shortly after production of the meal without any addition of antioxidant in the process.
`
`Further, storage conditions at a low to very low temperature can be applied if addition of
`
`antioxidant is not desired.
`
`5
`
`Krill oil extracted from denaturated krill meal by supercritical fluid extraction even 19 months
`
`after the production of the meal con