peT
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`(51) International Patent Classification 7 :
`CUB 1/10, C12N 9/64
`
`A1
`
`(11) International Publication Number:
`
`WO 00/23546
`
`(43) International Publication Date:
`
`27 April 2000 (27.04.00)
`
`(21) International Application Number:
`
`PCT/CA99/00987
`
`(22) International Filing Date:
`
`21 October 1999 (21.10.99)
`
`(30) Priority Data:
`2,251,265
`
`21 October 1998 (21.10.98)
`
`CA
`
`(71) Applicant (for all designated States except US): UNIVERSITE
`DE SHERBROOKE rCA/CAl; University Boulevard, Sher(cid:173)
`brooke, Quebec JlK 2RI (CA).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for us only): BEAUDOIN, Adrien
`rCA/CAl; 748, boulevard des Veterans, Rock Forest, Que(cid:173)
`bec JlN IZ7 (CA). MARTIN, Genevieve rCA/CAl; 797,
`McManamy, Sherbrooke, Quebec JlH 2NI (CA).
`
`(74) Agents: DUBUC, Jean, H. et al.; Goudreau Gage Dubuc &
`Martineau Walker, The Stock Exchange Tower, Suite 3400,
`P.O. Box 242, 800 Place Victoria, Montreal, Quebec H4Z
`IE9 (CA).
`
`(81) Designated States: AE, AL, AM, AT, AU, AZ, BA, BB, BG,
`BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE,
`ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP,
`KE,KG,KP, KR,KZ,LC, LK,LR,LS, LT,LU, LV, MA,
`MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU,
`SD, SE, SG, SI, SK, SL, TJ, TM, TR, IT, TZ, UA, UG,
`US, UZ, VN, YU, ZA, ZW, ARIPO patent (GH, GM, KE,
`LS, MW, SD, SL, SZ, TZ, UG, ZW), Eurasian patent (AM,
`AZ, BY, KG, KZ, MD, RU, TJ, TM), European patent (AT,
`BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU,
`MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM,
`GA, GN, GW, ML, MR, NE, SN, TD, TG).
`
`Published
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`(54) Title: METHOD OF EXTRACTING LIPIDS FROM MARINE AND AQUATIC ANIMAL TISSUES
`
`(57) Abstract
`
`Provided herein is a method for extracting lipid fractions from marine and aquatic animal material by acetone extraction. The
`resulting non-soluble and particulate fraction is preferably subjected to an additional solvent extraction with an alcohol, preferably ethanol,
`isopropanol or t-butanol or an ester of acetic acid, preferably ethyl acetate to achieve extraction of the remaining soluble lipid fraction from
`the marine and aquatic animal material. The remaining non-soluble particulate contents is also recovered since it is enriched in proteins
`and contains a useful amount of active enzymes. Also provided herein is a krill extract.
`
`000001
`
`

`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`AL
`AM
`AT
`AU
`AZ
`BA
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`CI
`CM
`CN
`CU
`CZ
`DE
`DK
`EE
`
`Albania
`Annenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Cote d'Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Gennany
`Denmark
`Estonia
`
`ES
`FI
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People's
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`Lesotho
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`The fonner Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`SI
`SK
`SN
`SZ
`TD
`TG
`TJ
`TM
`TR
`TT
`UA
`UG
`US
`UZ
`VN
`YU
`ZW
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`Zimbabwe
`
`000002
`
`

`
`WO 00/23546
`
`PCT/CA99/00987
`
`1
`METHOD OF EXTRACTING LIPIDS FROM MARINE AND AQUATIC
`
`ANIMAL TISSUES
`
`BACKGROUND OF THE INVENTION
`
`5
`
`This invention relates to the extraction of lipid fractions from marine and aquatic
`
`animals such as krill, Calanus, fish and sea mammals. More specifically,
`
`this
`
`invention relates to an improved method of extracting lipid fractions by dehydration
`
`with solvents and recovering a solid residue rich in active enzymes.
`
`10
`
`Lipid fractions obtained from marine and aquatic animals such as krill, Calanus, fish
`
`and sea mammals have various applications:
`
`Medical applications
`
`Marine and aquatic animal oils and fractions thereof contain various therapeutic
`
`agents. For example, it is reported that various marine and aquatic animal oils have
`
`15
`
`anti-inflammatory properties. Marine and aquatic animal oils are also reported as
`
`helpful in reducing the incidence of cardiovascular disease. Also, some marine and
`
`aquatic animal oils are reported as suppressing the development of certain forms of
`
`lupus and renal diseases. As a further example, krill may be used as a source of
`
`enzymes for debridement of ulcers and wounds or to facilitate food digestion. Also
`
`20
`
`marine and aquatic oils contain various antioxidants, which may have potential
`
`therapeutic properties.
`
`Nutraceuticals
`
`Considering the beneficial effects of omega-3 fatty acids, oils from krill, Calanus and
`
`fish could be used as dietary supplements to human diet. These fatty acids are
`
`25
`
`essential for proper development of the brain and the eye. Marine and aquatic
`
`animal oils are also rich in liposoluble vitamins A, 0 and E and carotenoids.
`Cosmetics
`
`Various marine and aquatic animal oils are used for the production of moisturizing
`
`creams.
`
`000003
`
`

`
`WO 00/23546
`
`PCT/CA99/00987
`
`2
`Fish farming
`
`Among the lipids found in krill, Calanus and fish, high concentrations of fatty acids
`
`20:5 (eicosapentaenoic acid) and 22:6 (docosahexaenoic acid) are present. These
`
`fatty acids are essential nutrients and are beneficial as fish feed. Furthermore, these
`
`essential nutrients are carried over in human diet by eating the fish grown on such
`diets.
`
`Animal feed
`
`Animal feed diets rich in omega-3 fatty acids may increase the level of unsaturated
`fatty acids and decrease cholesterol levels of meat. This property is already exploited
`in the poultry industry to improve the quality of eggs.
`
`5
`
`10
`
`Various methods for extracting marine and aquatic animal oils are known. For
`
`example, it is known to extract fish oil using organic solvents such as hexane and
`It is also known to measure the fat content in fish muscle tissue using
`ethanol.
`
`15
`
`solvents such as acetone.
`
`USP 4,331,695 describes a method using pressurized solvents which are gaseous
`
`at room temperature, such as propane, butane or hexane. The extraction is
`
`performed at preferred temperatures of 15 to 80°C on shredded vegetable or finely
`
`20
`
`divided animal products. The extracted oils are then made to precipitate under high
`
`pressure and elevated temperatures of 50 to 200°C. However, hexane is a poor
`
`extraction solvent
`
`for marine animals such as krill.
`
`Furthermore,
`
`the high
`
`temperatures used in the precipitation step negatively alters the lipids.
`
`25
`
`Canadian Patent Application 2,115,571 describes a method for extracting oils from
`
`various brown and read algae species. The method provides for example Soxhlet
`
`extraction using nearly pure ethanol for 40 hours.
`
`USP 5,006,281 describes a method for extracting oil from marine and aquatic
`
`30
`
`animals such as fish. The marine and aquatic animal
`
`is first treated with an
`
`antioxidant compound, finely divided and centrifuged to separate the oil phase from
`
`000004
`
`

`
`WO 00/23546
`
`PCT/CA99/00987
`
`3
`the aqueous phase and solid phase. The oil phase is then further treated with
`
`antioxidant to remove undesirable odour or taste.
`
`Canadian Patent 1,098,900 describes a method for extracting oils from krill. The
`
`5
`
`method involves emulsifying fresh or defrosted krill in an aqueous medium. The oil
`
`fraction is recovered by centrifugation.
`
`Folch in the article published in the year 1957 in J. bioI. Chem. 226: 497-509 t~
`simple method for the isolation and purification of total lipids from animal tissues"
`
`10
`
`proposes an extraction method using chloroform and methanol. This method is not
`
`commercially feasible because of the toxicity of the solvents involved.
`
`However, prior art processes are generally commercially unfeasible or provide low
`
`quantitative yields. Thus,
`
`it is an object of the present invention to provide an
`
`15
`
`improved marine and aquatic animal oil extraction method allowing recovery of a
`
`valuable lipid fraction and separate recovery of a valuable protein rich solid residue
`
`that comprises active enzymes.
`
`Other objects and further scope of applicability of the present invention will become
`
`20
`
`apparent from the detailed description given hereinafter.
`
`It should be understood,
`
`however, that this detailed description, while indicating preferred embodiments of the
`
`invention,
`
`is given by way of
`
`illustration only, since various changes and
`
`modifications within the spirit and scope of the invention will become apparent to
`
`those skilled in the art.
`
`25
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`Figure 1. Gas-liquid chromatography of fatty acids from dry krill
`
`(chloroform(cid:173)
`
`methanol)
`
`Figure 2. Gas-liquid chromatography of fatty acids from dry krill (acetone)
`
`30
`
`Figure 3. Gas-liquid chromatography of fatty acids from frozen krill (acetone)
`
`Figure 4. Gas-liquid chromatography of fatty acids from frozen krill (ethanol)
`
`Figure 5. Gas-liquid chromatography of fatty acids from frozen krill (t-butanol)
`
`000005
`
`

`
`WO 00/23546
`
`PCT/CA99/00987
`
`4
`Figure 6. Gas-liquid chromatography of fatty acids from frozen krill (ethyl
`
`acetate)
`
`Figure 7. Thin-layer chromatography of neutral lipids of Calanus sp. and
`
`M. norvegica
`
`5
`
`Figure 8. Thin-layer chromatography of neutral lipids of E. pacifica
`
`Figure 9. Thin-layer chromatography of neutral lipids of M. schmitti
`
`Figure 10. Thin-layer chromatography of neutral lipids of G. galeus
`
`Figure 11. Thin-layer chromatography of neutral lipids of Angel Shark
`
`Figure 12. Thin-layer chromatography of phospholipids of Calanus sp. and
`
`10M . norvegica
`
`Figure 13. Thin-layer chromatography of phospholipids of E. pacifica
`
`Figure 14. Thin-layer chromatography of phospholipids of M. schmitti
`
`Figure 15. Thin-layer chromatography of phospholipids of G. galeus
`
`Figure 16. Thin-layer chromatography of phospholipids of Angel Shark
`
`15
`
`Figure 17. Influence of the volume of acetone on lipid extraction (E. pacifica)
`
`Figure 18. Influence of incubation time in acetone on lipid extraction
`
`(E. pacifica)
`
`Figure 19. Influence of the volume of ethanol on lipid extraction (E. pacifica)
`
`Figure 20. Influence of incubation time in ethanol on lipid extraction
`
`20
`
`(T. raschii)
`
`DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
`
`Before describing the present invention in detail, it is to be understood that the
`
`invention is not limited in its application to the process details described herein. The
`
`25
`
`invention is capable of other embodiments and of being practised in various ways.
`
`It is also to be understood that the phraseology or terminology used herein is for the
`
`purpose of description and not limitation.
`
`The method of the invention comprises suspending freshly collected marine and
`
`30
`
`aquatic material in acetone. Lipids are extracted with a ketone such as acetone.
`
`This allows a rapid dehydration of animal tissue and a migration of the lipid fraction
`
`to the solvent. The dry residue is a valuable product rich in active enzymes.
`
`000006
`
`

`
`WO 00/23546
`
`PCT/CA99/00987
`
`5
`In a preferred embodiment, the extraction is carried out by successive acetone and
`
`alcohol treatments. Preferred alcohols are isopropanol, and t-butanol. The alcohol
`
`may also be substituted with an ester of acetic acid such as ethyl acetate. The
`
`procedure produces two successive lipid fractions and a dry residue enriched in
`
`5
`
`protein, including active enzymes. Recovery of total lipids is comparable to the Folch
`
`et al. (1957) procedure reported in the background of the invention. It has been
`
`tested with krill, Calanus, fish and shark tissues.
`
`Surprisingly, it was found that successive extraction treatments as proposed by the
`
`10
`
`present invention has a better yield in lipid extraction that single solvent system
`
`extractions. The extraction using two successive solvents which starts with a ketone
`
`such as acetone is especially advantageous since the acetone, in effect, dehydrates
`
`the animal tissue. Having the animal tissue in dehydrated form greatly facilitates the
`
`extraction process with the second solvent, alcohol or an ester of acetic acid such
`
`15
`
`as ethyl acetate.
`
`In the case of zooplancton such as krill and Calanus and in the case of fish-filleting
`
`by-products such as fish viscera, it is noted that extraction with acetone alone may
`
`be sufficient to allow a cost-effective recovery of lipid fractions and separate recovery
`
`20
`
`of a dry solid product rich in proteins including active enzymes.
`
`The general extraction method of the present invention will now be described. The
`
`starting material consisting of freshly harvested and preferably finely divided marine
`
`and aquatic animal material is subjected to acetone extraction, for at about two hours
`
`25
`
`and preferably overnight. However extraction time is not critical to the yield of lipid
`
`extraction. To facilitate extraction, it is preferable to use particles of less than 5mm
`
`in diameter. Extraction is preferably conducted under inert atmosphere and at a
`
`temperature in the order of about 5°C or less.
`
`30
`
`Preferably, the beginning of the extraction will be conducted under agitation for about
`
`10 to 40 minutes, preferably 20 minutes. Although extraction time is not critical, it
`
`000007
`
`

`
`WO 00/23546
`
`PCT/CA99/00987
`
`6
`was found that a 2 hour extraction with 6: 1 volume ratio of acetone to marine and
`aquatic animal material is best.
`
`The solubilized lipid fractions are separated from the solid material by standard
`
`5
`
`techniques including,
`
`for example,
`
`filtration, centrifugation or sedimentation.
`
`Filtration is preferably used.
`
`After separation by filtration on an organic solvent resistant filter (metal, glass or
`paper) the residue is optionally washed with pure acetone, preferably two volumes
`
`10
`
`(original volume of material) to recover yet more lipids. The combined filtrates are
`
`evaporated under reduced pressure. Optionally, flash evaporation or spray drying
`
`may be used. The water residue obtained after evaporation is allowed to separate
`
`from the oil phase (fraction I) at low temperature.
`
`15
`
`The solid residue collected on the filter is suspended and extracted with alcohol,
`
`such as ethanol, isopropanol, t-butanol or alternatively with ethyl acetate, preferably
`
`two volumes (original volume of material). The filtrate is evaporated leaving a second
`
`fraction of lipids (identified as fraction II). Although the extraction period is not
`
`critical, it was found that an extraction time of about 30 minutes is sufficient at
`
`20
`
`temperatures below about 5°C.
`
`Temperature of the organic solvents, except t-butanol, and temperature of the
`
`sample are not critical parameters, but it is preferable to be as cold as possible.
`
`However, in the case of t-butanol which is solid at room temperature, it is important
`
`25
`
`to warm it before using it and to perform the extraction at 25°C immediately.
`
`Comparative examples
`To compare the efficiency of the extraction process, a classical technique (Folch et
`
`al. 1957) using chloroform and methanol was applied to krill. This method is the
`
`30
`
`reference for measuring efficiency of the extraction process. Another comparison has
`been made with a technique using hexane as the extraction solvent. Lipid recovery
`
`000008
`
`

`
`WO 00/23546
`
`PCT/CA99/00987
`
`was estimated by suspending lipid fractions in small volumes of their original solvents
`
`and measuring by gravimetry small aliquots after evaporation.
`
`7
`
`For all examples provided herein, the method of the present invention involving
`
`5
`
`acetone extraction followed by extraction with a second solvent (ethyl acetate, for
`
`example) gave a translucent oil having appearance and properties more attractive
`
`than any oil obtained by the classical technique of Folch et al. (1957).
`
`To analyze lipid composition, 780 I-Ig of each extract was loaded on silica-gel plates
`
`10
`
`and fractionated by thin layer chromatography, TLC (Bowyer et al. 1962) with the
`
`following solvents. Neutral lipids: hexane, ethyl ether, acetic acid (90: 10: 1, v/v) and
`
`phospholipids: chloroform, methanol, water (80:25:2, v/v). Fatty acid composition of
`
`E. pacifica was analyzed by gas liquid chromatography, GLC (Bowyer et al. 1962,
`
`see bibliography) including some modifications to the original technique: 2h at 65°C
`
`15
`
`instead of 1h at 80°C, three washes with hexane instead of two and no wash with
`
`water.
`
`To get rid of traces of organic solvents, lipid fractions I and II are warmed to about
`
`125°C for about 15 minutes under inert atmosphere.
`
`20
`
`Fat was analyzed according to the American Oil Chemist's Society (AOCS). The
`
`following criteria have been used to analyze the lipids extracted: saponification and
`
`Wijs iodine indexes and moisture-volatile matter levels. Cholesterol content has also
`
`been determined by the method of Plummer 1987 (see bibliography). The same
`
`25
`
`analyzes and others have been made by an independent laboratory under Professor
`
`Robert Ackman's supervision (Canadian Institute of Fisheries Technology, DalTech,
`
`Dalhousie University, Halifax, Nova Scotia, Canada). This includes Wijs iodine index,
`
`peroxide and anisidine values, lipid class composition, fatty acid composition, free
`
`fatty acid FAME,
`
`cholesterol,
`
`tocopherol, all-trans retinol,
`
`cholecalciferol,
`
`30
`
`asthaxanthin and canthaxantin contents.
`
`000009
`
`

`
`WO 00/23546
`
`PCT/CA99/00987
`
`8
`Table 1 shows that higher levels of lipids are extracted from dry krill by acetone
`
`followed by ethanol as compared to the classical procedure of Folch et al. (1957).
`
`Table 2 shows the results of lipid extraction from frozen Euphausia pacifica, a
`
`5
`
`species of krill from Pacific Ocean. Assuming an eighty percent content of water, the
`
`lipid content is comparable to dry krill as shown in Table 1. Isopropanol, t-butanol
`
`and ethyl acetate, as solvent for the second extraction, give a yield less important
`
`than ethanol, but are not necessarily less effective in lipid recovery since ethanol
`
`carries more impurities than isopropanol, t-butanol or ethyl acetate. Then, they can
`
`10
`
`be used as second solvent after acetone as well. Variations between results from
`
`acetone extractions are mainly due to the water-oil separations. These separations
`
`are influenced by the quantity of residual acetone in the water-oil solution after
`
`acetone evaporation. This quantity of acetone varies from an experiment to another,
`
`because the evaporation system used at a small scale is less reproducible (at the
`
`15
`
`industrial scale, the evaporation step will be optimized). Single solvents have also
`
`been tested to extract the totality of lipids from krill. This shows that ethyl acetate
`
`(1,37% extraction rate), as hexane (0,23% extraction rate) are not good solvents,
`
`compared to acetone alone (1,86% extraction rate, and even greater extraction rates
`
`with an efficient acetone evaporation system).
`
`20
`
`One of the main advantages of the procedure is the removal of bacteria from extracts
`Indeed, samples of E. pacifica
`
`(lipid fraction and solid protein-rich material).
`
`incubated in different ratios of acetone at 4°C for 112 days have been inoculated on
`
`NA medium containing Bacto™ beef extract 0,3%, Bacto™ peptone 0,5% and Bacto™
`
`25
`
`agar 1,5% (Difco Laboratories, Detroit, USA) then incubated at room temperature or
`
`4°C for 18 days. No significant bacterial growth was observed at a ratio of 1 volume
`
`of acetone per gram of krill. At higher proportions of acetone (2 volumes and 5
`
`volumes), there was no bacterial growth at all, which means that acetone preserves
`
`krill samples. Acetone is known as an efficient bactericidal and viricidal agent
`
`30
`
`(Goodman et al. 1980).
`
`0000010
`
`

`
`WO 00123546
`
`PCTICA99/00987
`
`9
`Table 3 shows the yield of lipids from M. norvegica. The percentage of lipids (3,67%)
`is comparable to the one obtained with E. pacifica (3,11 %) shown in Table 2.
`Variations can be attributable to diet and time (season) of collection, which are
`different for those two species.
`
`5
`
`Table 4 shows the influence of grinding on the efficiency of extraction of M.
`
`norvegica lipids. These extractions were carried out under optimal conditions and
`
`show the definite advantage of the procedure over the classical method (4,46 %
`
`versus 3,30 %). It also shows that grinding may be an important factor when the
`
`10
`
`species is large (4,46% versus 3,53 %).
`
`Table 5 reports on lipid extraction from Ca/anus. Considerable quantities of lipids
`
`were obtained. Some variations in Ca/anus species composition may explain the
`
`variations between experiments 1 and 2 (8,22 % and 10,90 % of fresh weight).
`
`15
`
`Tables 6-8 report the total amount of lipids extracted from fish tissue. The method
`
`of the present invention was demonstrated on mackerel, trout and herring. The
`
`method was demonstrated on peripheral tissues (mainly muscles) and viscera.
`Advantageously, the present method would permit the recovery of valuable lipid
`
`20
`
`fractions from parts of fish that are usually wasted after the withdrawal of fillets of the
`
`fish. Those fish tissues not used after the transformation of the fish for human
`
`consumption could be stored in acetone, and lipids extracted therefrom in
`
`accordance with the present invention even if the method Folch [1957] recovers
`
`more lipid than our method.
`
`Indeed small amounts of lipids from mackerel (0.52%
`
`25
`
`from viscera and 1,45% from tissues) have been extracted by the method of Folch
`
`after a first extraction with acetone and ethanol as described in the present invention.
`
`Comparative extractions with the method described in the present invention carried
`
`out in parallel with the method of Folch on trout and herring show superior recovery
`
`with the latter. However, it is noteworthy that the Folch method can not be applied
`
`30
`
`for the recovery of lipids for commercial uses (because of toxicity).
`
`0000011
`
`

`
`WO 00/23546
`
`PCT/CA99/00987
`
`10
`In Tables 9 to 11, are shown results of lipids extraction from shark liver tissues.
`
`There is no marked difference in results between techniques within a species.
`Table 12 shows the fatty acid composition of krill oil (e. pacifica) following extraction
`in various solvents.
`
`5
`
`Tables 13 shows some characteristics features of fraction I (acetone) and fraction
`
`II (alcohol or ethyl acetate) for krill oil (e. pacifica). First, the saponification index of
`
`fraction I (130,6) indicates that this fraction contains fatty acids with longer chains,
`compared to fraction II (185,7). The Wijs iodine index of fraction I shows that this
`
`10
`
`fraction contains high levels of polyunsaturated fatty acids. As compared to olive oil
`
`which has an index of 81.1.
`
`It explains why fraction I is liquid at room temperature.
`
`It is well known that unsaturated fatty acids have a fusion point inferior to the one
`
`of their saturated homologues. The same observations are made for fraction II which
`
`has a iodine index of 127,2. The fatty acid composition shown in Table 12
`
`15
`
`corroborates these iodine indexes: fraction I has a high percentage (30,24%) of
`
`polyunsaturated fatty acids (pentaenes+hexaenes) and so fraction II (22,98%).
`Finally, Table 13 shows also that fraction I is comprised of 10,0% of volatile matter
`
`and humidity after evaporation of the solvent. For the same test, the fraction II gives
`
`a value of 6,8%. To get rid of traces of solvents, it is important to briefly heat (to
`
`20
`
`about 125°C, for about 15 min) the oil under nitrogen.
`
`Results on krill oils obtained in accordance with the method of the present invention
`
`(fraction I extracted with acetone and fraction II extracted with ethyl acetate) are
`
`provided in Tables 13, 14, 15, 16, 17 and 18.
`
`It is noteworthy to mention that in
`
`25
`
`Table 18, the carotenoids content was significantly high as measured in terms of two
`
`carotenoids namely asthaxanthin and canthaxanthin. Indeed, duplicates analyzes
`
`revealed values of 92 to 124 IJg/g of lipid fraction for asthaxanthin and 262 to 734
`
`IJg/g for canthaxanthin. Thus, for the purpose of the present invention it may be said
`
`that the krill extract comprises asthaxanthin at least 75 and preferably at least 90
`
`30
`
`ug/g of lipid fraction.
`
`In the case of canthaxanthin, at least 250 and preferably at
`
`least 270 IJg/g of
`
`lipid fraction.
`
`Low values for peroxide and anisidine are
`
`advantageous and are due to the presence of high levels of natural antioxidants
`
`0000012
`
`

`
`WO 00/23546
`
`PCT/CA99/00987
`
`11
`
`(astaxanthin and canthaxanthin). These compounds are indicative of favourable
`pharmaceutical or cosmetological properties of the krill extract whereby high levels
`
`of carotenoids indicate excellent transdermal migration characteristics. Thus, krill
`
`extract is a good candidative for transdermal delivery of medicines.
`
`5
`
`Table 19 shows the best mode of the method in accordance with the present
`
`invention for lipid extraction of aquatic animal tissues.
`
`Table 20 shows that the enzyme activity of the solid fraction is maintained following
`
`10
`
`the method of the present invention.
`
`Indeed, the demonstration was completed for
`
`solid krill residue obtained after successive acetone and ethyl acetate extraction.
`
`Proteolytic activities were measure by the liberation of amino groups by
`
`spectrophotometric assay using o-pthaldialdehyde as
`
`reagent.
`
`Protein
`
`concentrations were measured by the Bradford method. Soluble proteins were
`extracted with water and added to a 10% lactoserum protein concentrate obtained
`
`by ultrafiltration. At the end of incubation at 37°C in 50mM potassium phosphate
`buffer, trichloroacetic acid was added and the amount of NH3 group was measured
`in the supernatant according to the method of Church et al. [1983, J Dairy Sci 66:
`
`1219-1227].
`
`Figures 1 to 6 show chromatograms of fatty acid composition of E. pacifica lipids.
`
`On each of them, high proportions of 20:5 and 22:6 fatty acids (characteristic of
`
`marine and aquatic oils) are noticeable and represented by two distinct peaks. Data
`
`are shown in Table 12.
`
`15
`
`20
`
`25
`
`Variations in lipid patterns of neutral
`
`lipids (from Figure 7 to Figure 11) from one
`
`species to another are attributable to the differences in food sources. Within a
`
`species (E. pacifica, for example) there is no marked variation between lipid patterns
`
`obtained from different techniques of lipid extraction. Concerning phospholipids
`
`30
`
`(Figure 12 to Figure 16), the opposite is observed: variations are explained by the
`
`different extraction processes of lipids since the same species do not lead to the
`same lipid pattern. Lipids from shark species (extracted by the mentioned methods)
`
`0000013
`
`

`
`WO 00/23546
`
`PCT/CA99/00987
`
`12
`
`and commercial cod-liver oil (sample available from Uniprix drugstores, Province of
`
`Quebec, Canada) are mainly composed of neutral
`
`lipids as opposed to
`
`phospholipids.
`
`5
`
`The influence of the volume of solvent and incubation time on the efficiency of the
`
`acetone to extract
`
`lipids from E. pacifica is illustrated in Figures 17 and 18,
`
`respectively. A ratio of 1:6 (w/v) produced optimal yield with near complete extraction
`
`after 2h. The second extraction step has been experimented with ethanol. The
`
`volume of this solvent does not appear to be critical since the same yield was
`
`10
`
`obtained with different volumes of ethanol (Figure 19), but
`
`incubations time in
`
`ethanol should be at least 30 minutes as indicated by the results on Figure 20.
`
`15
`
`20
`
`25
`
`One of the inventors, Dr. Adrien Beaudoin, has ingested the different lipid fractions
`
`of krill. No side effect profile was observed.
`
`Although the invention has been described above with respect with one specific form,
`
`it will be evident to a person skilled in the art that it may be modified and refined in
`
`various ways. It is therefore wished to have it understood that the present invention
`
`should not be limited in scope, except by the terms of the following claims.
`
`Demonstration that krill residue, obtained after acetone and ethyl acetate extraction,
`
`contains enzyme proteolytic activities. Proteolytic activities were measured by the
`
`liberation of amino groups by spectrophotometric assay using o-phthaldialdehyde
`
`as reagent. Protein concentrations were measured by the Bradford method.
`
`The enzyme source was the residue obtained after acetone and ethyl acetate
`
`extractions of lipids. Soluble proteins were extracted with water and added to a 10%
`
`lactoserum protein concentrate obtained by ultrafiltration.
`
`30
`
`At
`
`the end of
`
`incubation at 37°C in 50 mM potassium phosphate buffer,
`
`trichloroacetic acid was added and the amount of NH3 groups were measured in the
`supernatant according to Church and al. 1983.
`
`0000014
`
`

`
`WO 00123546
`
`PCTICA99/00987
`
`13
`
`BIBLIOGRAPHY
`
`Bowyer, D.E., Leat, W.M.F., Howard, A.N. and Gresham, G.A. 1962. The
`
`determination of the fatty acid composition of serum lipids separated by thin-layer
`
`5
`
`chromatography; and a comparison with column chromatography. BBA. 70: 423-431.
`
`10
`
`Chandrasekar, B., Troyer, D.A., Venkatraman, J.T. and Fernandes, G. 1996. Tissue
`
`specific regulation of transforming growth factor beta by omega-3 lipid-rich krill oil in
`
`autoimmune murine lupus. Nutr Res. 16(3): 489-503.
`
`Christensen, M.S., Hoy, C-E. and Redgrave, T.G. 1994. Lymphatic absorption of n-3
`
`polyunsaturated fatty acids from marine oils with different intramolecular fatty acid
`
`distributions. BBA. 1215: 198-204.
`
`15
`
`Church, F.C., Swaisgood, H.E., Porter, D.H. and Catignani, G.L. 1983.
`
`Spectrophotometric assay using 0- Phthaldialdehyde for determination of proteolysis
`
`in milk and isolated milk proteins. J Dairy Sci. 66: 1219-1227.
`
`20
`
`25
`
`30
`
`Difco laboratories. 1984. Difco Manual Dehydrated Culture Media and Reagents for
`Microbiology. 10th ed. Detroit.
`
`Folch, J., Lees, M. and Sloane-Stanley, G.H. 1957. A simple method for the isolation
`
`and purification of total lipids from animal tissues. J. bioI. Chem. 226: 497-509.
`
`Goodman Gilman, A., Goodman, L.L. and Gilman, A. 1980. The Pharmacological
`Basis of Therapeutics. 6th ed. Collier Macmillan Canada ltd, Toronto.
`
`Harwood, H.J. and Geyer, RP. 1964. Biology Data Book. The Federation of
`
`American Societies for Experimental Biology, Washington.
`
`Hellgren, L., Karlstam, B., Mohr, V. and Vincent, J. 1991. Krill enzymes. A new
`
`concept for efficient debridement of necrotic ulcers. Int J Dermatol. 30(2): 102-103
`
`0000015
`
`

`
`WO 00/23546
`
`PCT/CA99/00987
`
`14
`Plummer, D.T. 1987. An introduction to practical biochemistry. 3th ed. McGraw-Hili
`Book Company, London.
`
`Rawn, J.D. 1990. Traite de biochimie. De Boeck-Wesmael, Bruxelles.
`
`Runge, J.A. and Joly, P. 1994. Rapport sur I'etat des invertebres en 1994: 7:0
`
`Zooplancton (Euphausiaces et Calanus) de l'Estuaire et du Golfe du Saint-Laurent.
`
`Sargent, J.R. 1997. Fish oils and human diet. Br J Nutr.78 Suppl1: S5-S13.
`
`5
`
`10
`
`0000016
`
`

`
`WO 00/23546
`
`PCT/CA99/00987
`
`15
`TABLE 1. EXTRACTION OF DRY KRILL LIPIDS (E. pacifica)
`
`Exp. No.
`
`Technique
`
`Yield (%)
`
`Total (%)
`
`Mean (%) + s.d.
`
`5
`
`1-
`
`acetone a)
`ethanol b)
`
`2-
`
`3-
`
`4-
`
`5-
`
`6-
`
`1/
`
`1/
`
`1/
`
`chlor : MeOH c)
`
`1/
`
`8,00
`7,60
`
`19,70
`6,90
`
`8,15
`11,20
`
`6,80
`13,60
`
`15,60
`
`26,60
`
`19,35
`
`20,40
`
`15,50
`
`14,90
`
`20,49±3,95
`
`15,20±0,30
`
`10
`
`15
`
`20
`
`25
`
`Determinations in triplicates (variation < 5 %).
`a) :Extraction made with a sample-solvent ratio of 1:9 (w/v) , no incubation.
`b) :Extraction made with a sample-solvent ratio of 1:4 (w/v) , incubated 1 night at 4°C, following
`a first extraction with acetone.
`c) :Folch et al. 1957.
`
`30
`
`TABLE 2. EXTRACTION OF FROZEN KRILL LIPIDS (E. pacifica)
`
`Exp. No.
`
`35
`
`1-
`
`2-
`
`40
`
`3-
`
`4-
`
`5-
`
`6-
`
`45
`
`50
`
`Technique
`
`acetone a)
`ethanol b)
`
`1/
`
`1/
`
`acetone a)
`isopropanol b)
`
`1/
`
`1/
`
`Yield (%)
`
`Total (%)
`
`Mean (%) + s.d.
`
`1,17
`1,23
`
`3,05
`1,09
`
`1,53
`1,26
`
`2,45
`0,70
`
`1,80
`0,80
`
`1,60
`0,80
`
`2,40
`
`4,14
`
`2,79
`
`3,15
`
`2,60
`
`2,40
`
`3,11±0,91
`
`2,72±0,39
`
`0000017
`
`

`
`WO 00/23546
`
`PCT/CA99/00987
`
`16
`
`TABLE 2 (continued). EXTRACTION OF FROZEN KRILL LIPIDS (E. pacifica)
`
`5
`
`Exp. No.
`
`7-
`
`10
`
`8-
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`9-
`
`10-
`
`11-
`
`12-
`
`13-
`
`14-
`
`15-
`
`16-
`
`17-
`
`18-
`
`19-
`
`20-
`
`21-
`
`22-
`
`Technique
`
`acetone a)
`t-butanol c)
`
`"
`
`"
`
`acetone a)
`ethyl acetate b)
`
`"
`
`"
`
`co