`
`PCT/CA99/00075
`
`~ 42 -
`
`50.
`Method as in claim 43 wherein said
`product is subject to digestion between approximately 0-70
`degrees Celsius.
`
`Method of producing a concentrated krill
`51.
`hydrolysate comprising the steps of harvesting, digesting
`and evaporating the krill hydrolysate to provide a partial
`hydrolysis for a predetermined time and temperature so as to
`enhance the nutrient characteristics of said krill.
`
`10
`
`15
`
`20
`
`Method of producting a dry krill premix
`52.
`or feedstuff comprising the steps of producing a
`predetermined amount of concentrated krill hydrolysate,
`producing a predetermined amount of dry matter and mixing
`said concentrated krill hydrolysate and said dry carrier
`matter and co-drying said mixture.
`
`Method as in claim 52 wherein the dry
`54.
`matter is selectted from the group of vegetable and/or
`vegetable and/or animal protein meals and by products.
`
`RIMFROST EXHIBIT 1024 page 1351
`SUBSTITUTE SHEET (WieSQ)1 EXHIBIT 1024
`page
`
`1351
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`FIG.1A
`
`RIMFROST EXHIBIT 1024 page 1352
`RQST EXHIBIT 1024SUBSTITUTE SHEET Rule
`
`page 1352
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`C/\2
`
`
`
`FIG. 1B
`
`RIMFROST EXHIBIT 1024 page 1353
`SUBSTITUTE SHEET (Rute80}
`page
`
`T EXHIBIT 1024
`
`1353
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`
`EUPHAUSTIDS OUT
`
`
`FIG. 2C
`RIMFROST EXHIBIT 1024 page 1354
`SUBSTITUTE SHEET RRR3T EXHIBIT 1024
`
`page 1354
`
`
`
`WO 99/39589
`
`4/\2
`
`
`rdaaOTzg-|=|FE|beDN|f3]
`
`fo—_a—e=N
`
`fo
`
`Londoyd---——=NOILO3SYv370T\SLind4id
`
`
`
`
`
`
`
`
`YTN0g~-4-pgpS-—7faONILSIX3——-—EPXfc=LINDYID
`
`
`
`PCT/CA99/00075
`
`
`
`nM%.Sp¢‘Ol5QoQ.
`
`nN
`
`OSx
`
`RIMFROST EXHIBIT 1024 page 1355
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`reiH—‘
`>2|VY
`
`ȣ @
`
`——
`
`H50 OUT ~—
`
`D
`
`64
`
`
` 6/
`
`dfWeOFSSOLEee
`
`
`
`
`
`Meiy|Foal!ANS
`
`
`
`{|ft
`
`
`
`3 —t—— HO IN
`
`
`
`
`
`
`
`—— PRODUCT
`
`FIG. 5
`
`RIMFROST EXHIBIT 1024 page 1356
`SUBSTITUTE SHEET RMEBBPT EXHIBIT 1024
`page 1356
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`WnIdSw
`
`6/l2
`
`$$3904d|DK 9SIs
`
`
`
`T EXHIBIT 1024page1357
`
`RIMFROST EXHIBIT 1024 page 1357
`SUBSTITUTE SHEET(Rule50)
`page
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`C/\2
`
`2/0
`
`LIZ
`
`2//
`
`KRILL
`HYDROLYSATE
`
`LIQUID MARINE
`PROTEIN.
`
`DRY
`CARRIER
`
`MIXING BLENDING PHASE
`
`GRINDING
`
`
`
`
`
`
`TEMPERATURE
`SENSITIVE ENZYME
`
`ACTIVE PRODUCTS
`242
`
`215
`
`2/7
`
`ZIG
`
`220
`
`eH
`
`250—)
`
`243—|
`
`REACTOR CELL
`BALANCE TANK
`
`KIX OR OTHER
`
`COOLING
`
`DRIED KRILL
`PRODUCT
`
`
`
`
`DRIED KRILL PRODUCT
`
`FOR CARRIER
`
`MIXING BLENDING
`DUASE
`
`FINAL PRODUCT
`
`FIG. 7
`
`RIMFROST EXHIBIT 1024 page 1358
`SUBSTITUTE SHEET[ie8R}
`page
`
`T EXHIBIT 1024
`
`1358
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`8/12
`
`220
`
`FIG. 8
`
`RIMFROST EXHIBIT 1024 page 1359
`SUBSTITUTE SHEET(Rute$8}> EXHIBIT 1024
`page 1559
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`9/12
`
`Q3|ud
`
`LOndosd
`
`6Sls
`
`YAEENYOS
`
`YIV
`
`RIMFROST EXHIBIT 1024 page 1360
`SUBSTITUTE SHEETRATERQSEXHIBIT 1024
`page 1360
`
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`lO/l2
`
`3O/
`
`.|CENTRIFUGATION
`
`HYDROLYSED
`|
`CLARIFIED
`
` KRILL
`
`KRILL
`
`PRE-FILTRATION
`
`ENZYMES
`
`303
`
`JOD
`
`CLARIFIED
`
`KRILL
`
`502
`
`ENZYMES
`
`
`
`
`
`C2
`a
`JOF
`
`10,000 DA
`
`CONCENTRATED
`
`FREEZE/FREEZE
`
`KRILL ENZYMES DRY/STORE BELOW 4°C
`
`
`BIZ
`
`FIG.
`
`lO
`
`RIMFROST EXHIBIT 1024 page 1361
`SUBSTITUTE SHEET(ule0)
`pag
`
`T EXHIBIT 1024
`
`page 1361
`
`
`
`ALVaWNad=Vd00001\7seC)
` SAWAZNA Naa=.Q,OshMOTETYOLS/AUG“TINYllOls:AZATMAAZATWAoIME=GHLVULNADNODpa=rad
`
`
`
`SHINAZNG2=PnTINeCanivo7;
`
`fo=e=ea
`
`RIMFROST EXHIBIT 1024 page 1362
`
`WO 99/39589
`
`PCT/CA99/00075
`
`II/ 12
`
`FONARLLNAD
`
`
`
`dINoOITHSad
`
`(SAWAZNA
`
`TINO
`
`rr
`
`fal
`
`
`
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`ACID STABILIZED
`SHELL WASTE
`
`ii4O
`
`le/l2
`
`NON ACID STABILIZED
`SHELL WASTE
`
`40/
`
`GRINDING
`
`4O2
`
`405—~ GRINDING
`
`DE-WATERING|WATER GROUND SHELL
`
`ADDITION |WASTE
`
`FOF
`ACID STABILIZED
`PROTEIN LIQUID
`
`FI
`O
`
`
`KRILL ENZYME
`
`
`IN LIQUID OR
`CONCENTRATED
`
`
`
`
`DIGESTER FOR INCUBATION
`SECOND DIGEST TO REMOVE EXCESS PROTEIN
`
`FIRST DIGEST TO OPTIMISE PROTEIN QUALITY
`
`
`DEWATERING IN
`PRESS OR CENTRIFUGE
`
`
`WASHING
`
`AND SECOND
`DIGEST
`CHITIN ASH
`PORTION
`FID
`
`
`4/3 FURTHER TRADITIONAL
`
`TREATMENTS TO PURIFY
`CHITIN
`Ald
`
`
`
`PROTEIN LIQUID WITH ACTIVE ENZYMES
`FOR LOW TEMPERATURE DRYING, CO-DRYING,
`
`
`EVAPORATION, OR ACID STABILIZATION
`
`
`FIG. l2
`RIMFROST EXHIBIT 1024 page 1363
`Vi RST EXHIB
`0
`page
`
`IT 1024
`
`1363
`
`SUBSTITUTE SHEET
`
`
`
`INTERNATIONAL SEARCH REPORT
`
`inte
`onal Application No
`PCT/CA 99/00075
`
`CLASSIFICATION OF SUBJECT MATTER
`pe 6
`A23K1/10
`A23K1/16
`C€12N9/00
`
`A23K1/18
`
`A23J1/04
`
`A23N17/00
`
`According to International Patent Classification (IPC) or to both nationalclassification and IPC
`B. FIELDS SEARCHED
`Minimum documentation searched (classification system followed by classification symbols)
`IPC 6
`A@3K A23J
`C12N
`
`Documentation searched other than minimum documentation to the extent that such documentsare inciuded in the fields searched
`
`Electronic data base consulted during the international search (name of data base and, where practical, search terms used)
`
`C. DOCUMENTS CONSIDEREDTO BE RELEVANT
`
`Citation of document, with indication, where appropriate, of the relevant passages
`
`Relevant to claim No.
`
`PX
`
`WO 98 34498 A (BIOZYME SYSTEMS INC ;SAXBY
`DAVID J (CA); SPENCE JOHN A (CA); ALOIS)
`13 August 1998
`see the whole document
`
`DATABASE WPI
`Section Ch, Week 8447
`Derwent Publications Ltd., London, GB;
`Class C03, AN 84-293719
`XP002070859
`& SU 1 084 005 A (N BASSIN FISHING IND)
`,
`7 April 1984
`see abstract
`
`WO 95 22893 A (SPECIALTY MARINE
`31 August 1995
`see page 15,
`line 19 - page 17,
`see claims 11-28,30-46
`
`FEEDS INC)
`
`line 19
`
`1-54
`
`1,20,52
`
`Further documentsare listed in the continuation of box C.
`
`Patent family members arelisted in annex.
`
`Dekeirel, M
`
`"T" later document publishedafter the internationalfiling date
`or priority date and notin conflict with the application but
`cited to understand theprinciple or theory underlying the
`invention
`“X" documentof particular relevance: the claimed invention
`cannot be considered novel or cannot be considered to
`involve an inventive step when the documentis taken alone
`"Y" document of particular relevance; the claimed invention
`cannot be considered to involve an inventive step when the
`documentis combined with one or more other such docu-
`ments, such combination being obvious to a person skilled
`in the art.
`"&" document memberof the samepatent family
`Date of mailing of the international search report
`
`° Special catagories ot cited documents :
`
`"&" documentdefining the general state of the art which is not
`considered to be of particular relevance
`"“E" earlier document but published on or after the international
`filing date
`"L* document which may throw doubts on priority claim(s) or
`whichis cited to establish the publication date of ancther
`citation or other special reason (as specified)
`"©" documentreferring to an oral disclosure, use, exhibition or
`other means
`“P” document published prior to the international
`later than the priority date claimed
`Date of the actual completion of the international search
`
`filing date but
`
`9 June 1999
`
`Name and mailing address of the ISA
`European Patent Office, P.B. 5818 Patentlaan 2
`NL - 2280 HV Rijswijk
`Tal. (431-70) 340-2040, Tx. 31 651 epo ni,
`Fax: (+31-70) 340-3016
`
`Fom PCT/ISA/210 (second sheet) (July 1992)
`
`29/06/1999
`Authorized officer
`
`RIMFROST EXHIBIT 1024 page 1364
`RIMFRQSE EXSHIBIT 1024
`page 1364
`
`
`
`C.(Continuation) DOCUMENTS CONSIDEREDTO BE RELEVANT
`
`
`
`Category *|Citation of document, with indication,where appropriate, of the relevant passages Relevantto claim No.
`
`jonal Application No
`
`PCT/CA 99/00075
`
`WO 89 01031 A (PHARMACIA AB)
`9 February 1989
`see page 5, paragraph 2
`see page 7, paragraph 1
`see examples 1-3
`see claim 1
`
`WO 89 10960 A (PHARMACIA AB)
`16 November 1989
`see page 8,
`last paragraph
`see page 11, paragraph 3 - paragraph 5
`see page 14, paragraph 3
`see page 27, paragraph 4
`see claims 1,7,17
`
`WO 90 05026 A (AKT CONSULTANTS)
`17 May 1990
`see figure 1
`
`PATENT ABSTRACTS OF JAPAN
`vol. 017, no. 315 (C-1071), 16 June 1993
`& JP 05 030923 A
`(RIKEN VITAMIN CO LTD),
`9 February 1993
`see abstract
`
`29-34 ,41
`
`1,20,52
`
`INTERNATIONAL SEARCH REPORT Inte
`
`DATABASE WPI
`Section Ch, Week 9602
`Derwent Publications Ltd., London, GB;
`Class D13, AN 96-018544
`XP002070860
`& RU 2 034 492 C (TROITSKII BN)
`,
`10 May 1995
`see abstract
`
`
`
`Form PCT/ISA/21 0 (continuation of secand sheet) (July 1992)
`
`RIMFROST EXHIBIT 1024 page 1365
`RIMFROSE EXHIBIT 1024
`page 1365
`
`
`
`
`
` INTERNATIONAL SEARCH REPORT
`
` Inte donal Application No
`information on patent family members
`
`PCT/CA 99/00075
`
`Patent document
`cited in search report
`
`Publication
`date
`
`WO 9834498
`
`13-08-1998
`
`A
`
`u
`
`Patent family
`member(s)
`
`2197137 A
`
`5976698 A
`
`Publication
`date
`
`07-08-1998
`
`26-08-1998
`
`
`
`
`
`WO 9522893
`
`A
`
`31-08-1995
`
`28-04-1996
`2134515 A
`CA
`
`
`11-09-1995
`1802695 A
`AU
`
`
`02-04-1997
`1146709 A
`CN
`
`
`26-02-1997
`0758842 A
`EP
`FI
`963343 A
`27-08-1996
`
`
`06-01-1998
`10500004 T
`
`
`
`
`124085 T
`15-07-1995
`
`
`AU
`2259788 A
`01-03-1989
`
`DE
`3854050 D
`27-07-1995
`
`
`EP
`0393035 A
`24-10-1990
`
`2504465 T
`
`20-12-1990
`
`
`
`WO 9005026
`
`17-05-1990
`
`28-05-1990
`4518389 A
`AU
`03-05-1990
`2002193 A
`CA
`
`
`22-08-1990
`158990 A
`DK
`27-12-1990
`0403608 A
`EP
`
`
`
`
`31-12-1990
`89100729 A
`GR
`PT
`92192 A,B
`31-05-1990
`
`5105560 A
`
`21-04-1992 Fom PCT/ISA/210 (patent family annex) (July 1992)
`
`RIMFROST EXHIBIT 1024
`
`page 1366
`
`RIMFROST EXHIBIT 1024 page 1366
`
`
`
`PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(11) International Publication Number:
`
`WO 00/23546
`
`(43) International Publication Date:
`
`27 April 2000 (27.04.00)
`
`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[CA/CA]; University Boulevard, Sher-
`brooke, Quebec JIK 2R1 (CA).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): BEAUDOIN, Adrien
`[CA/CA]; 748, boulevard des Vétérans, Rock Forest, Que-
`bec JIN 1Z7 (CA). MARTIN, Genevieve [CA/CA]; 797,
`McManamy, Sherbrooke, Quebec J1H 2N1 (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
`1E9 (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, JS, 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, TT, 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, Fl, 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.
`
`(51) International Patent Classification 7;
`C11B 1/10, CL2N 9/64
`
`
`(21) International Application Number:
`
`
`
`
`
`(54) Titles 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 butanolor 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 amountof active enzymes. Also provided herein is a krill extract.
`
`RIMFROST EXHIBIT 1024
`
`page 1367
`
`RIMFROST EXHIBIT 1024 page 1367
`
`
`
`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.
`
`Zimbabwe -
`
`Albania
`Armenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Cate dIvoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`SI
`SK
`SN
`SZ
`TD
`TG
`TJ
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`
`™T
`
`R
`TT
`UA
`UG
`US
`UZ
`VN
`YU
`ZW
`
`ES
`FI
`
`GA
`GB
`GE
`GH
`GN
`GR
`AU
`TE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`Lc
`LI
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Treland
`Israel
`Iceland
`Ttaly
`Japan
`Kenya
`Kyrgyzstan
`Democratic People’s
`Republic of Korea
`Republic of Korca
`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 former Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`RIMFROST EXHIBIT 1024
`RIMFROST EXHIBIT 1024 page 1368
`
`page 1368
`
`
`
`WO 00/23546
`
`PCT/CA99/00987
`
`METHOD OF EXTRACTING LIPIDS FROM MARINE AND AQUATIC
`
`ANIMAL TISSUES
`
`4
`
`
`BACKGROUND OFTHE INVENTION
`
`This invention relates to the extraction oflipid 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.
`
`Lipid fractions obtained from marine and aquatic animals such as krill, Ca/anus, fish
`and sea mammals havevarious 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 animaloils have
`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 animaloils are reported as suppressing the development ofcertain forms of
`lupus and renal diseases. As a further example,krill may be used as a source of
`enzymesfor debridement of ulcers and woundsorto facilitate food digestion. Also
`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
`essential for proper development of the brain and the eye. Marine and aquatic
`animal oils are also rich in liposoluble vitamins A, D and E and carotenoids.
`
`Various marine and aquatic animaloils are used for the production of moisturizing
`
`Cosmetics
`
`creams.
`
`10
`
`15
`
`20
`
`25
`
`RIMFROST EXHIBIT 1024=page 1369
`
`RIMFROST EXHIBIT 1024 page 1369
`
`
`
`WO 00/23546
`
`,
`
`PCT/CA99/00987
`a
`
`Fish farming
`Amongthelipids foundin krill, Ca/anus 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 humandiet 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.
`
`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
`ethanol.
`It is also known to measurethe fat content in fish muscle tissue using
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`solvents such as acetone.
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`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 orfinely
`divided animal products. The extracted oils are then madeto 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 usedin the precipitation step negatively alters thelipids.
`
`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
`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
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`the aqueous phase and solid phase. The oil phase is then further treated with
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`antioxidant to remove undesirable odour or taste.
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`Canadian Patent 1,098,900 describes a method for extracting oils from krill. The
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`method involves emulsifying fresh or defrostedkrill in an aqueous medium. Theoil
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`fraction is recovered by centrifugation.
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`Folch in the article published in the year 1957 in J. biol. Chem. 226: 497-509 “A
`simple methodfor the isolation and purification oftotal lipids from animal tissues”
`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
`improved marine and aquatic animaloil extraction method allowing recovery of a
`valuable lipid fraction and separate recovery of a valuable protein rich solid residue
`
`that comprises active enzymes.
`
`Otherobjects and further scope of applicability of the present invention will become
`apparentfrom 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 ofillustration only, since various changes and
`modifications within the spirit and scope of the invention will become apparent to
`
`those skilled in the art.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`Figure 1. Gas-liquid chromatography of fatty acids from dry krill
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`(chloroform-
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`methanol)
`Figure 2. Gas-liquid chromatographyof fatty acids from dry krill (acetone)
`Figure 3. Gas-liquid chromatographyof fatty acids from frozenkrill (acetone)
`Figure 4. Gas-liquid chromatographyof fatty acids from frozen krill (ethanol)
`Figure 5. Gas-liquid chromatographyof fatty acids from frozenkrill (-butanol)
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`Figure 6. Gas-liquid chromatographyof fatty acids from frozen krill (ethyl
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`acetate)
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`Figure 7. Thin-layer chromatography of neutrallipids of Calanus sp. and
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`M. norvegica
`
`Figure 10.
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`Figure 8. Thin-layer chromatography of neutrallipids of E. pacifica
`Figure 9. Thin-layer chromatography of neutrallipids of M. schmitt
`Thin-layer chromatography of neutral lipids of G. galeus
`Thin-layer chromatography of neutrallipids of Angel Shark
`Thin-layer chromatography of phospholipids of Ca/anus sp. and
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`Figure 11.
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`Figure 12.
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`M. norvegica
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`Figure 13.
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`Figure 14.
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`Figure 15.
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`Figure 16.
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`Figure 17.
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`Figure 18.
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`Figure 19.
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`Figure 20.
`
`Thin-layer chromatography of phospholipids of E. pacifica
`Thin-layer chromatography of phospholipids of M. schmitti
`Thin-layer chromatography of phospholipids of G. galeus
`Thin-layer chromatography of phospholipids of Angel Shark
`influence of the volume of acetone on lipid extraction (E. pacifica)
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`Influence of incubation time in acetone on lipid extraction
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`(E. pacifica)
`Influence of the volume of ethanolonlipid extraction (E. pacifica)
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`Influence of incubation time in ethanol on lipid extraction
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`(T. raschii)
`
`DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
`
`it is to be understood that the
`Before describing the present invention in detail,
`invention is not limited in its application to the process details described herein. The
`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 hereinis for the
`
`purpose of description and notlimitation.
`
`The method of the invention comprises suspending freshly collected marine and
`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 productrich in active enzymes.
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`In a preferred embodiment, the extraction is carried out by successive acetone and
`alcohol treatments. Preferred alcohols are isopropanol, and ¢-butanol. The alcohol
`may also be substituted with an ester of acetic acid such as ethyl acetate. The
`procedure produces two successivelipid fractions and a dry residue enriched in
`protein, including active enzymes. Recovery oftotallipids is comparable to the Foich
`et al. (1957) procedure reported in the background of the invention. It has been
`tested with krill, Calanus, fish and shark tissues.
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`Surprisingly, it was found that successive extraction treatments as proposed by the
`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 acetoneis especially advantageoussince the acetone,in effect, dehydrates
`the animal tissue. Having the animaltissue in dehydrated form greatly facilitates the
`extraction process with the second solvent, alcohol or an ester of acetic acid such
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`as ethyl acetate.
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`In the case of zooplancton suchaskrill and Ca/anus andin the caseoffish-filleting
`by-products suchasfish viscera, it is noted that extraction with acetone alone may
`be sufficient to allow a cost-effective recovery oflipid fractions and separate recovery
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`of a dry solid productrich 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
`and preferably overnight. Howeverextraction 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 underinert atmosphere and at a
`temperature in the order of about 5°C orless.
`
`Preferably, the beginning ofthe extraction will be conducted underagitation for about
`10 to 40 minutes, preferably 20 minutes. Although extraction time is not critical, it
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`was found that a 2 hour extraction with 6:1 volume ratio of acetone to marine and
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`aquatic animal materialis best.
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`The solubilized lipid fractions are separated from the solid material by standard
`techniques including, for example,
`filtration, centrifugation or sedimentation.
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`Filtration is preferably used.
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`After separation byfiltration on an organic solventresistantfilter (metal, glass or
`paper) the residue is optionally washed with pure acetone, preferably two volumes
`(original volume of material) to recover yet morelipids. The combinedfiltrates 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 |) at low temperature.
`
`The solid residue collected on the filter is suspended and extracted with alcohol,
`such as ethanol, isopropanol, f-butanolor alternatively with ethyl acetate, preferably
`two volumes(original volume of material). Thefiltrate is evaporated leaving a second
`fraction oflipids (identified as fraction Il). Although the extraction period is not
`critical,
`it was found that an extraction time of about 30 minutesis sufficient at
`temperatures below about 5°C.
`
`Temperature of the organic solvents, except t-butanol, and temperature of the
`sample are notcritical parameters, butit is preferable to be as cold as possible.
`However, in the case of tbutanol whichis solid at room temperature, it is important
`to warm it before using it and to perform the extraction at 25 °C immediately.
`
`Comparative examples
`To comparetheefficiency of the extraction process, a classical technique (Folch et
`al. 1957) using chloroform and methanol was applied to krill. This method is the
`reference for measuring efficiency of the extraction process. Another comparison has
`been madewith a technique using hexane as the extraction solvent. Lipid recovery
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`was estimated by suspendinglipid fractions in small volumesoftheir original solvents
`and measuring by gravimetry small aliquots after evaporation.
`
`For all examples provided herein, the method of the present invention involving
`acetone extraction followed by extraction with a second solvent (ethyl acetate, for
`example) gave a translucentoil having appearance and properties more attractive
`than any oil obtained by the classical technique of Folchet al. (1957).
`
`To analyzelipid composition, 780 yg of each extract was loadedonsilica-gel plates
`and fractionated by thin layer chromatography, TLC (Bowyeret al. 1962) with the
`following solvents. Neutrallipids: 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 gasliquid chromatography, GLC (Bowyeret al. 1962,
`see bibliography) including some modifications to the original technique: 2h at 65°C
`instead of 1h at 80°C, three washes with hexaneinstead of two and no washwith
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`water.
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`To getrid of traces of organic solvents,lipid fractions | and Il are warmed to about
`425°C for about 15 minutes under inert atmosphere.
`
`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 matterlevels. Cholesterol content has also
`been determined by the method of Plummer 1987 (see bibliography). The same
`analyzes and others have been madebyan independentlaboratory under Professor
`Robert Ackman’s supervision (CanadianInstitute 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,
`asthaxanthin and canthaxantin contents.
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`Table 1 showsthat higherlevels of lipids are extracted from dry krill by acetone
`followed by ethanol as comparedtothe classical procedure of Folch et al. (1957).
`
`Table 2 showsthe results of lipid extraction from frozen Euphausia pacifica, a
`speciesofkrill from Pacific Ocean. Assuming an eighty percent content of water, the
`lipid content is comparable to dry krill as shown in Table 1. Isopropanol, 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, butanolor ethyl acetate. Then, they can
`be used as second solventafter 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 experimentto another,
`because the evaporation system used at a small scale is less reproducible (at the
`industrial scale, the evaporation step will be optimized). Single solvents have also
`been tested to extract thetotality 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).
`
`One of the main advantagesof the procedureis the removal of bacteria from extracts
`(lipid fraction and solid protein-rich material).
`Indeed, samples of E. pacifica
`incubatedin different ratios of acetone at 4°C for 112 days have beeninoculated on
`NA medium containing Bacto™ beef extract 0,3%, Bacto™ peptone 0,5% and Bacto™
`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 ofkrill. 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
`(Goodmanet al. 1980).
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`Table 3 showstheyield oflipids from M. norvegica. The percentage oflipids (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.
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`Table 4 shows the influence of grinding on the efficiency of extraction of M.
`norvegicalipids. These extractions were carried out under optimal conditions and
`show the definite advantage of the procedure overthe classical method (4,46 %
`versus 3,30 %).
`It also showsthat grinding may be an important factor when the
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`speciesis large (4,46% versus 3,53 %).
`
`Table 5 reports onlipid extraction from Ca/anus. Considerable quantities of lipids
`were obtained. Some variations in Calanus species composition may explain the
`
`variations between experiments 1 and 2 (8,22 % and 10,90 % of fresh weight).
`
`Tables 6-8 report the total amountoflipids 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
`fractions from parts of fish that are usually wasted after the withdrawaloffillets 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
`morelipid than our method.
`Indeed small amountsoflipids from mackerel (0.52%
`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
`for the recovery oflipids for commercial uses (becauseoftoxicity).
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`In Tables 9 to 11, are shown results of lipids extraction from sharkliver tissues.
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`There is no markeddifference in results between techniques within a species.
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`Table 12 showsthe fatty acid composition ofkrill oil (e. pacifica) following extraction
`
`in various solvents.
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`Tables 13 shows some characteristics features of fraction | (acetone) and fraction
`Il (alcohol or ethyl acetate)forkrill oil (e. pacifica). First, the saponification index of
`fraction | (130,6) indicates that this fraction contains fatty acids with longer chains,
`comparedto fraction II (185,7). The Wi