`
`PCT/CA99/00075
`
`_42_
`
`50.
`
`Method as in claim 43 wherein said
`
`product is subject to digestion between approximately 0-70
`
`degrees Celsius.
`
`51.
`
`Method of producing a concentrated krill
`
`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.
`
`Mathod 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.
`
`54.
`
`Method as in claim 52 wherein the dry
`
`matter is selectted from the group of vegetable and/or
`
`vegetable and/or animal protein meals and by products.
`
`10
`
`15
`
`20
`
`RIMFROST EXHIBIT 1024 page 1351
`SUBSTITUTE SHEET fiiWéEfiT EXHIBIT 1024
`Page
`
`1351
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`FIG.IA
`
`RIMFROST EXHIBIT 1024 page 1352
`WT EXHIBIT 1024
`page 1352
`SUBSTITUTE SHEET 131%}:
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`2/l2
`
`
`
`FIG. IB
`
`RIMFROST EXHIBIT 1024 page 1353
`SUBSTITUTE SHEET MW?
`Page
`
`T EXHIBIT 1024
`
`1353
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`24
`
`3/2
`
`\
`
`
`
`
` E
`
`
`
`
`
`
`
`
`
`EUPHAUSIIDS OUT
`
`
`FIG. 2C
`
`RIMFROST EXHIBIT 1024 page 1354
`SUBSTITUTE SHEET M]???T EXHIBIT 1024
`
`page 1354
`
`
`
`
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`4/I2
`
`
`
`tangomm.:0m
`
`
`
`
`
`b.5050Huzoomn.lllll
`
`
`
`oz_hw_xmlil.’
`
`
`
`zozbmwm<m._u
`
`m.9“.
`
`
`
`WV
`
`
`
`m».«WW
`
`RIMFROST EXHIBIT 1024 page 1355
`SUBSTITUTE SHEEngflwT EXHIBIT 1024
`page 1355
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`
`
`-.II
`B.
`61
`>10
`
`
`OU"nn“‘-‘-=—-‘---‘-‘—
`
`
` .
`
`
`
`\\
`é
`
`‘I ~—
`
`1)
`'G
`
`
`FIG. 5
`
`RIMFROST EXHIBIT 1024 page 1356
`SUBSTITUTE SHEET fiMEEQ’ST EXHIBIT 1024
`page 1356
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`6/2
`
`mmmoomm
`
`22mm:
`
`VA
`
`
`
`RIMFROST EXHIBIT 1024 page 1357
`SUBSTITUTE SHEETM%
`Page
`
`T EXHIBIT 1024
`
`1357
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`7/l2
`
`2/0
`
`2/2
`
`2//
`
`KRILL
`HYDROLYSATE
`
`LIQUID IVIARINE
`PROTEIN -
`
`DRY
`
`CARRIER
`
`MIXING BLENDING PHASE
`
`GRINDING
`
`
`
`
`
`REACTOR CELL
`
`BALANCE TANK
`
`
`KIX OR OTHER
`DRYER SYSTEM
`
`COOLING
`
`DRIED KRILL PRODUCT
`
`
`
`FOR CARRIER
`
`DRIED KRILL
`
`PRODUCT
`
`MIXING BLENDING
`
`TEMPERATURE
`
`
`
`2/5
`
`2/7
`
`2/6
`
`220
`
`24/
`
`250
`
`PHASE
`
`SENSITIVE ENZYME
`
`
`
`242
`
`243
`
`FINAL PRODUCT
`
`ACTIVE PRODUCTS
`
`FIG. 7
`
`RIMFROST EXHIBIT 1024 page 1358
`SUBSTITUTE SHEET WES
`ST EXHIBIT 1024
`page 1358
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`8/I2
`
`220
`
`FIG. 8
`
`RIMFROST EXHIBIT 1024 page 1359
`SUBSTITUTE SHEET M%§T EXHIBIT 1024
`page 1359
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`9/!2
`
`~i<
`
`mmmmnmuw
`
`H<mr
` >ww
`
`omfifi_
`
`HUDQOmm
`
`m.07.
`
`MS%
`
`mQKDOm
`
`RIMFROST EXHIBIT 1024 page 1360
`SUBSTITUTE SHEETMERflfiT EXHIBIT 1024
`page 1360
`
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`lO/I2
`
`
`
`HYDROLYSED
`
`KRILL
`
`CENTRIFUGATION
`
`.
`
`CLARIFIED
`
`KRILL
`
`
`
`PRE—FILTRATION
`
`ENZYMES
`
`303
`
`305
`
`302
`
`
`
`
`
`FREEZE/FREEZE KRELL ENZYMES
` DRY/STORE BELOW 4°C
`
`
`
`
`
`CLARIFIED
`
`KRILL
`
`ENZYMES
`
`10,000 DA
`
`.A
`
`304
`
`CONCENTRATED
`
`3/3
`
`FIG.
`
`IO
`
`SUBSTITUTE SHEET
`
`RIMFROST EXHIBIT 1024 page 1361
`a e 1361
`u 33‘?
`p g
`
`T EXHIBIT 1024
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`ll/l2
`
`mODENFZmU
`
`
`
`mmmmm355m
`
`Emmmm
`
`‘55
`
`ITSm
`
`8mm
`
`TE25.2‘w.Fe
`
`5““ng
`
`vEA
`
`mwmmfiammmfiQMinm8352528m.H
`musswzw 2mm8up0%33mm9.83%jam__0—mM.
`
`
`
`RIMFROST EXHIBIT 1024 page 1362
`mmTEEHSET
`
`
`
`
`
`
`WO 99/39589
`
`PCT/CA99/00075
`
`IZ/IZ
`
`ACID STABILIZED
`SHELL WASTE
`
`NON ACID STABILIZED
`SHELL WASTE
`
`400
`
`40/
`
`GRINDING
`
`402
`
`4623 GRINDING
`
`DE-WATERING WATER
`
`GROUND SHELL
`
`404
`
`ADDITION WASTE
`
`ACID STABILIZED
`PROTEIN LIQUID
`
`4/0
`
`-
`
`
`KRILL ENZYME
`
`
`IN LIQUID OR
`CONCENTRATED
`
`
`
`
`
`FIRST DIGEST TO OPTIMISE PROTEIN QUALITY
`
`SECOND DIGEST TO REMOVE EXCESS PROTEIN
`
`DIGESTER FOR INCUBATION
`
`WASHING
`
`
`
`AND SECOND
`
`DIGEST
`
`
`CHITIN ASH
`
`DEWATERING IN
`
`
`
`
`PORTION
`
`PRESS OR CENTRIFUGE
`
`'
`
`4/3
`
`
`FURTHER TRADITIONAL
`TREATIVIENTS TO PURIFY
`
`
`
`
`
`
`
`
`PROTEIN LIQUID WITH ACTIVE ENZYMES
`FOR LOW TEMPERATURE DRYING, CO-DRYING,
`
`
`EVAPORATION, OR ACID STABILIZATION
`
`
`CHITIN
`
`4/4
`
`
`FIG. l2
`RIMFROST EXHIBIT 1024 page 1363
`IT 1 24
`u ewT EXHIB
`0
`page
`
`1363
`
`SUBSTITUTE SHEET
`
`
`
`INTERNATIONAL SEARCH REPORT
`lnte
`onal Application No
`
`
`CLASSIFICATION OF SUBJECT MATTER
`IPC 6
`A23K1/10
`A23K1/16
`C12N9/00
`
`PCT/CA 99/00075
`
`A23K1/18
`
`A23J1/04
`
`A23N17/00
`
`According to lntemational Patent Classification (IPC) or to both national classification and IPC
`B. FIELDS SEARCHED
`Minimum documentation searched (classification system followed by classification symbols)
`IPC 6
`A23K A23J
`C12N
`
`Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched
`
`Electronic data base consulted during the international search (name of data base and. where practical. search terms used)
`
`Relevant to claim No.
`
`1-54
`
`C. DOCUMENTS CONSIDERED TO BE RELEVANT
`
`Citation of document, with indication, where appropriate. of the relevant passages
`
`NO 98 34498 A (BIOZYME SYSTEMS INC ;SAXBY
`DAVID J (CA); SPENCE JOHN A (CA); ALOIS)
`13 August 1998
`see the whole document
`
`DATABASE HPI
`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
`
`NO 95 22893 A (SPECIALTY MARINE FEEDS INC)
`31 August 1995
`see page 15,
`line 19 — page 17,
`see claims 11—28,30—46
`
`line 19
`
`
`
`Further documents are listed in the continuation of box C.
`
`Patent family members are listed in annex.
`
`° SpeCIal categories of cited documents :
`
`"A" document defining the general state of the art which is not
`considered to be of particular relevance
`"E” eariier document but published on or after the international
`filing date
`"L" document which may throw doubts on priority claim(s) or
`which is cited to establish the publication date of another
`citation or other special reason (as specified)
`"0" document referring 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. PB. 5818 Patentlaan 2
`NL - 2280 HV Flijswijk
`Tel. (4-31-70) 340-2040. TX. 31 651 epo nl,
`Fax: (4-31.70) 340-3016
`
`Form PCT/ISNZI 0 (second sheet) (July 1992)
`
`"T" later document published after the international filing date
`or priority date and not in conflict with the application but
`cited to understand the principle or theory underlying the
`invention
`“X" document of particular relevance: the claimed invention
`cannot be considered novel or cannot be considered to
`involve an inventive step when the document is taken alone
`“Y" document of particular relevance; the claimed invention
`cannot be considered to involve an inventive step when the
`document is combined with one or more other such docu-
`ments, such combination being obvious to a person skilled
`in the art.
`"&" document member of the same patent family
`Date of mailing of the international search report
`
`29/06/1999
`Authorized officer
`
`Dekeirel, M
`
`RIMFROST EXHIBIT 1024 page 1364
`
`RIMFRQ§3g EXHIBIT 1024
`
`page 1364
`
`
`
`
`
`INTERNATIONAL SEARCH REPORT lnte
`,ional Application No
`PCT/CA 99/00075
`
`C.(Continuatlon) DOCUMENTS CONSIDERED TO BE RELEVANT
`
`Category °
`
`Citation of document. with indicationwhere appropriate, 0! the relevant passages
`
`Relevant to claim No.
`
`29-34,41
`
`NO 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
`
`NO 89 10960 A (PHARMACIA AB)
`16 November 1989
`
`last paragraph
`see page 8,
`see page 11, paragraph 3 - paragraph 5
`see page 14, paragraph 3
`see page 27, paragraph 4
`see claims 1,7,17
`
`NO 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
`
`DATABASE NPI
`
`Section Ch, Week 9602
`Derwent Publications Ltd., London, GB;
`Class D13, AN 96—018544
`XP002070860
`& RU 2 034 492 C (TROITSKII
`,
`10 May 1995
`see abstract
`
`B N)
`
`Form PCT/lSA/210 (continuation of second sheet) (July 1992)
`
`RIMFROST EXHIBIT 1024 page 1365
`
`RIMFRQfiE EXJHBIT 1024
`
`page 1365
`
`
`
`IPJTWEFUNIXTT()DLAIJSIQAJICHJ:RJEP()F¥T
`information on patent family members
`
`
`
`
`ink
`.ionai Application No
`
`
`
`
`Patent document
`cited in search report
`
`Publication
`date
`
`PCT/CA 99/00075
`Publication
`date
`
`Patent family
`member(s)
`
`
`
` 2197137
`
`NO 9834498
`13- 08- 1998
`07-08-1998
`29>
`
`5976698
`
`26-08-1998
`
`
`
` NO 9522893 A
` 31-08—1995
`CA
`2134515 A
`28-04-1996
`1802695 A
`11-09-1995
`
`
`
`
`1146709 A
`02-04-1997
`0758842 A
`26-02-1997
`
`
`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
`
`
`17-05-1990
`
`NO 9005026
`
`A .
`
`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
`
`
`
`
`Form PCT/ISAIQiO (patent family annax) (July 1992)
`
`RIMFROST EXHIBIT 1024
`
`RIMFROST EXHIBIT 1024 page 1366
`
`page 1366
`
`
`
`PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(51) International Patent Classification 7 :
`
`(1 1) International Publication Number:
`
`WO 00/23546
`
`A1
`C11B 1/10, C12N 9/64
`(43) International Publication Date:
`
`27 April 2000 (27.04.00)
`
`
`(21) International Application Number:
`
`PCT/CA99/00987
`
`
`
`(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, 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.
`
`(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 11K 2R1 (CA).
`
`(72) Inventors; and
`(7S) Inventors/Applicants (for US only): BEAUDOIN, Adrien
`[CA/CA]; 748, boulevard des Vétérans, Rock Forest, Que-
`bec J 1N 127 (CA). MARTIN, Genevieve [CA/CA]; 797,
`McManamy, Sherbrooke, Quebec J 1H 2N1 (CA).
`
`(74) Agents: DUBUC, Jean, H. et al.; Goudreau Gage Dubuc &
`Maltineau Walker, The Stock Exchange Tower, Suite 3400,
`PO. Box 242, 800 Place Victoria, Montreal, Quebec H4Z
`1E9 (CA).
`
`(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.
`
`
`
`RIMFROST EXHIBIT 1024
`
`RIMFROST EXHIBIT 1024 page 1367
`
`page 1367
`
`
`
`Zimbabwe '
`
`Albania
`Armenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Swit7erland
`Cote d’Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`ES
`FI
`
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`[L
`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 former Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`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.
`
`SI
`SK
`SN
`52
`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
`
`RIMFROST EXHIBIT 1024 page 1368
`RIMFROST EXHIBIT 1024‘
`page 1368
`
`
`
`WO 00/23546
`
`PCT/CA99/00987
`
`METHOD OF EXTRACTING LIPIDS FROM MARINE AND AQUATIC
`
`ANIMAL TISSUES
`
`1
`
`BACKGROUND OF THE INVENTION
`
`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.
`
`1O
`
`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, D and E and carotenoids.
`
`Cosmetics
`
`Various marine and aquatic animal oils are used for the production of moisturizing
`
`creams.
`
`RIMFROST EXHIBIT 1024
`
`RIMFROST EXHIBIT 1024 page 1369
`
`page 1369
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`Among the lipids found in krill, Ca/anus and fish, high concentrations of fatty acids
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`20:5 (eicosapentaenoic acid) and 22:6 (docosahexaenoic acid) are present. These
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`fatty acids are essential nutrients and are beneficial as fish feed. Furthermore, these
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`essential nutrients are carried over in human diet by eating the fish grown on such
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`diets.
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`Animal feed
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`Animal feed diets rich in omega-3 fatty acids may increase the level of unsaturated
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`fatty acids and decrease cholesterol levels of meat. This property is already exploited
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`in the poultry industry to improve the quality of eggs.
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`Various methods for extracting marine and aquatic animal oils are known. For
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`example, it is known to extract fish oil using organic solvents such as hexane and
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`ethanol.
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`It is also known to measure the 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
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`at room temperature, such as propane, butane or hexane. The extraction is
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`performed at preferred temperatures of 15 to 80°C on shredded vegetable or finely
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`divided animal products. The extracted oils are then made to precipitate under high
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`pressure and elevated temperatures of 50 to 200°C. However, hexane is a poor
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`extraction solvent for marine animals such as krill.
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`Furthermore,
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`the high
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`temperatures used in the precipitation step negatively alters the lipids.
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`Canadian Patent Application 2,115,571 describes a method for extracting oils from
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`various brown and read algae species. The method provides for example Soxhlet
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`extraction using nearly pure ethanol for 40 hours.
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`USP 5,006,281 describes a method for extracting oil from marine and aquatic
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`animals such as fish. The marine and aquatic animal
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`is first treated with an
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`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 defrosted krill in an aqueous medium. The oil
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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
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`simple method for the isolation and purification of total lipids from animal tissues”
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`proposes an extraction method using chloroform and methanol. This method is not
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`commercially feasible because of the toxicity of the solvents involved.
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`However, prior art processes are generally commercially unfeasible or provide low
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`quantitative yields. Thus,
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`it is an object of the present invention to provide an
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`improved marine and aquatic animal oil extraction method allowing recovery of a
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`valuable lipid fraction and separate recovery of a valuable protein rich solid residue
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`that comprises active enzymes.
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`Other objects and further scope of applicability of the present invention will become
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`apparent from the detailed description given hereinafter.
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`It should be understood,
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`however, that this detailed description, while indicating preferred embodiments of the
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`invention,
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`is given by way of
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`illustration only, since various changes and
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`modifications within the spirit and scope of the invention will become apparent to
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`those skilled in the art.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`Figure 1. Gas-liquid chromatography of fatty acids from dry krill
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`(chloroform-
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`methanol)
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`Figure 2. Gas-liquid chromatography of fatty acids from dry krill (acetone)
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`Figure 3. Gas-liquid chromatography of fatty acids from frozen krill (acetone)
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`Figure 4. Gas-liquid chromatography of fatty acids from frozen krill (ethanol)
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`Figure 5. Gas-liquid chromatography of fatty acids from frozen krill (t—butanol)
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`Figure 6. Gas—liquid chromatography of fatty acids from frozen krill (ethyl
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`acetate)
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`Figure 7. Thin-layer chromatography of neutral lipids of Calanus sp. and
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`M. norvegica
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`Figure 8. Thin-layer chromatography of neutral lipids of E. pacifica
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`Figure 9. Thin-layer chromatography of neutral lipids of M. schmitti
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`Figure 10. Thin—layer chromatography of neutral lipids of G. galeus
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`Figure 11. Thin-layer chromatography of neutral lipids of Angel Shark
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`Figure 12. Thin-layer chromatography of phospholipids of Calanus sp. and
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`M. norvegica
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`Figure 13. Thin-layer chromatography of phospholipids of E. pacifica
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`Figure 14. Thin-layer chromatography of phospholipids of M. schmitti
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`Figure 15. Thin-layer chromatography of phospholipids of G. ga/eus
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`Figure 16. Thin-layer chromatography of phospholipids of Angel Shark
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`Figure 17. Influence of the volume of acetone on lipid extraction (E. pacifica)
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`Figure 18. Influence of incubation time in acetone on lipid extraction
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`(E. pacifica)
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`Figure 19. Influence of the volume of ethanol on lipid extraction (E. pacifica)
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`Figure 20. Influence of incubation time in ethanol on lipid extraction
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`(T. raschii)
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`DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
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`Before describing the present invention in detail,
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`it is to be understood that the
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`invention is not limited in its application to the process details described herein. The
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`invention is capable of other embodiments and of being practised in various ways.
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`It is also to be understood that the phraseology or terminology used herein is for the
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`purpose of description and not limitation.
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`The method of the invention comprises suspending freshly collected marine and
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`aquatic material in acetone. Lipids are extracted with a ketone such as acetone.
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`This allows a rapid dehydration of animal tissue and a migration of the lipid fraction
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`to the solvent. The dry residue is a valuable product rich in active enzymes.
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`In a preferred embodiment, the extraction is carried out by successive acetone and
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`alcohol treatments. Preferred alcohols are isopropanol, and t—butanol. The alcohol
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`may also be substituted with an ester of acetic acid such as ethyl acetate. The
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`procedure produces two successive lipid fractions and a dry residue enriched in
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`protein, including active enzymes. Recovery of total lipids is comparable to the Folch
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`et al. (1957) procedure reported in the background of the invention. It has been
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`tested with krill, Calanus, fish and shark tissues.
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`Surprisingly, it was found that successive extraction treatments as proposed by the
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`present invention has a better yield in lipid extraction that single solvent system
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`extractions. The extraction using two successive solvents which starts with a ketone
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`such as acetone is especially advantageous since the acetone, in effect, dehydrates
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`the animal tissue. Having the animal tissue in dehydrated form greatly facilitates the
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`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 such as krill and Calanus and in the case of fish-filleting
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`by—products such as fish viscera, it is noted that extraction with acetone alone may
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`be sufficient to allow a cost-effective recovery of lipid fractions and separate recovery
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`of a dry solid product rich in proteins including active enzymes.
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`The general extraction method of the present invention will now be described. The
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`starting material consisting of freshly harvested and preferably finely divided marine
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`and aquatic animal material is subjected to acetone extraction, for at about two hours
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`and preferably overnight. However extraction time is not critical to the yield of lipid
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`extraction. To facilitate extraction, it is preferable to use particles of less than 5mm
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`in diameter. Extraction is preferably conducted under inert atmosphere and at a
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`temperature in the order of about 5°C or less.
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`Preferably, the beginning of the extraction will be conducted under agitation for about
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`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 material is best.
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`The solubilized lipid fractions are separated from the solid material by standard
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`techniques including, for example,
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`filtration, centrifugation or sedimentation.
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`Filtration is preferably used.
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`After separation by filtration on an organic solvent resistant filter (metal, glass or
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`paper) the residue is optionally washed with pure acetone, preferably two volumes
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`(original volume of material) to recover yet more lipids. The combined filtrates are
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`evaporated under reduced pressure. Optionally, flash evaporation or spray drying
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`may be used. The water residue obtained after evaporation is allowed to separate
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`from the oil phase (fraction l) at low temperature.
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`The solid residue collected on the filter is suspended and extracted with alcohol,
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`such as ethanol, isopropanol, t-butanol or alternatively with ethyl acetate, preferably
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`two volumes (original volume of material). The filtrate is evaporated leaving a second
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`fraction of lipids (identified as fraction II). Although the extraction period is not
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`critical,
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`it was found that an extraction time of about 30 minutes is sufficient at
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`temperatures below about 5°C.
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`Temperature of the organic solvents, except t—butanol, and temperature of the
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`sample are not critical parameters, but it is preferable to be as cold as possible.
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`However, in the case of t—butanol which is solid at room temperature, it is important
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`to warm it before using it and to perform the extraction at 25 °C immediately.
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`Comparative examples
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`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
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`reference for measuring efficiency of the extraction process. Another comparison has
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`been made with a technique using hexane as the extraction solvent. Lipid recovery
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`was estimated by suspending lipid fractions in small volumes of their original solvents
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`and measuring by gravimetry small aliquots after evaporation.
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`For all examples provided herein, the method of the present invention involving
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`acetone extraction followed by extraction with a second solvent (ethyl acetate, for
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`example) gave a translucent oil having appearance and properties more attractive
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`than any oil obtained by the classical technique of Folch et al. (1957).
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`To analyze lipid composition, 780 pg of each extract was loaded on silica-gel plates
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`1O
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`and fractionated by thin layer chromatography, TLC (Bowyer et al. 1962) with the
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`following solvents. Neutral lipids: hexane, ethyl ether, acetic acid (90:10:1, v/v) and
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`phospholipids: chloroform, methanol, water (80:25:2, v/v). Fatty acid composition of
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`E. pacifica was analyzed by gas liquid chromatography, GLC (Bowyer et al. 1962,
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`see bibliography) including some modifications to the original technique: 2h at 65°C
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`instead of 1h at 80°C, three washes with hexane instead of two and no wash with
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`water.
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`To get rid of traces of organic solvents, lipid fractions l and II are warmed to about
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`125°C for about 15 minutes under inert atmosphere.
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`Fat was analyzed according to the American Oil Chemist’s Society (AOCS). The
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`following criteria have been used to analyze the lipids extracted: saponification and
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`Wijs iodine indexes and moisture-volatile matter levels. Cholesterol content has also
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`been determined by the method of Plummer 1987 (see bibliography). The same
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`analyzes and others have been made by an independent laboratory under Professor
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`Robert Ackman’s supervision (Canadian Institute of Fisheries Technology, DalTech,
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`Dalhousie University, Halifax, Nova Scotia, Canada). This includes Wijs iodine index,
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`peroxide and anisidine values, lipid class composition, fatty acid composition, free
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`fatty acid FAME,
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`cholesterol,
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`tocopherol,
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`all-trans
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`retinol,
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`cholecalciferol,
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`asthaxanthin and canthaxantin contents.
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`Table 1 shows that higher levels of lipids are extracted from dry krill by acetone
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`followed by ethanol as compared to the classical procedure of Folch et al. (1957).
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`Table 2 shows the results of lipid extraction from frozen Euphausia pacifica, a
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`species of krill from Pacific Ocean. Assuming an eighty percent content of water, the
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`lipid content is comparable to dry krill as shown in Table 1. lsopropanol, t—butanol
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`and ethyl acetate, as solvent for the second extraction, give a yield less important
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`than ethanol, but are not necessarily less effective in lipid recovery since ethanol
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`carries more impurities than isopropanol, t—butanol or ethyl acetate. Then, they can
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`be used as second solvent after acetone as well. Variations between results from
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`acetone extractions are mainly due to the water—oil separations. These separations
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`are influenced by the quantity of residual acetone in the water-oil solution after
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`acetone evaporation. This quantity of acetone varies from an experiment to another,
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`because the evaporation system used at a small scale is less reproducible (at the
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`industrial scale, the evaporation step will be optimized). Single solvents have also
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`been tested to extract the totality of lipids from krill. This shows that ethyl acetate
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`(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
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`with an efficient acetone evaporation system).
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`One of the main advantages of the procedure is the removal of bacteria from extracts
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`(lipid fraction and solid protein-rich material).
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`Indeed, samples of E. pacifica
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`incubated in different ratios of acetone at 4°C for 112 days have been inoculated on
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`NA medium containing BactoTM beef extract 0,13%, BactoTM peptone 0.5% and BactoTM
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`agar 1,5% (Difco Laboratories, Detroit, USA) then incubated at room temperature or
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`4°C for 18 days. No significant bacterial growth was observed at a ratio of 1 volume
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`of acetone per gram of krill. At higher proportions of acetone (2 volumes and 5
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`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
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`(Goodman et al. 1980).
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`Table 3 shows the yield of lipids from M. norvegica. The percentage of lipids (3,67%)
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`is comparable to the one obtained with E. pacifica (3,11%) shown in Table 2.
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`Variations can be attributable to diet and time (season) of collection, which are
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`different for those two species.
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`Table 4 shows the influence of grinding on the efficiency of extraction of M.
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`non/egica lipids. These extractions were carried out under optimal conditions and
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`show the definite advantage of the procedure over the classical method (4,46 %
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`versus 3,30 %).
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`It also shows that grinding may be an important factor when the
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`species is large (4,46% versus 3,53 %).
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`Table 5 reports on lipid extraction from Calanus. Considerable quantities of lipids
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`were obtained. Some variations in Calanus species composition may explain the
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`variations between experiments 1 and 2 (8,22 % and 10,90 % of fresh weight).
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`Tables 6-8 report the total amount of lipids extracted from fish tissue. The method
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`of the present invention was demonstrated on mackerel, trout and herring. The
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`method was demonstrated on peripheral tissues (mainly muscles) and viscera.
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`Advantageously, the present method would permit the recovery of valuable lipid
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`fractions from parts of fish that are usually wasted after the withdrawal of fillets of the
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`fish. Those fish tissues not used after the transformation of the fish for human
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`consumption could be stored in acetone, and lipids extracted therefrom in
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`accordance with the present invention even if the method Folch [1957] recovers
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`more lipid than our method.
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`Indeed small amounts of lipids from mackerel (0.52%
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`from viscera and 1,45% from tissues) have been extracted by the method of Folch
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`after a first extraction with acetone and ethanol as described in the present invention.
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`Comparative extractions with the method described in the present invention carried
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`out in parallel with the method of Folch on trout and herring show superior recovery
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`with the latter. However, it is noteworthy that the Folch method can not be applied
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`30
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`for the recovery of lipids for commercial uses (because of toxicity).
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`In Tables 9 to 11, are shown results of lipids extraction from shark liver tissues.
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`There is no marked difference in results between techniques within a species.
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`Table 12 shows the fatty acid composition of krill oil (e. pacifica) follow