(12) United States Patent
`Beaudoin et al.
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 6,800,299 B1
`Oct. 5, 2004
`
`US006800299B1
`
`(54) METHOD OF EXTRACTING LIPIDS FROM
`MARINE AND AQUATIC ANIMAL TISSUES
`
`(75) Inventors: Adrien Beaudoin, Rock Forest (CA);
`Geneviéve Martin, Sherbrooke (CA)
`
`(73) Assignee: Universite de Sherbrooke (CA)
`
`* N '
`ot1ce:
`
`(21) Appl. No.:
`(22) PCT Filed:
`
`s bj
`yd' 1 '
`h
`r h'
`u ect to an lsc a1mer, t e term 0 t is
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`09/830,146
`Oct. 21, 1999
`
`(86) PCT No.:
`
`PCT/CA99/00987
`
`§ 371 (9X1),
`Jul. 25, 2001
`(2), (4) Date:
`(87) PCT Pub. No.: WO00/23546
`
`PCT Pub. Date: Apr. 27, 2000
`Foreign Application Priority Data
`
`(30)
`
`Oct. 21, 1998
`
`(CA) ........................................... .. 2251265
`
`(51) Int. Cl.7 .............................................. .. A61K 35/56
`(52) US. Cl. ...................................... .. 424/522; 424/523
`(58) Field of Search ............................... .. 424/522, 523,
`424/94.1, 94.2
`
`(56)
`
`References Cited
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`6,055,936 A * 5/2000 Collin ....................... .. 11/215
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`Patent Abstracts of Japan, Jul. 2, 1976, JP 51 076467.
`Patent Abstracts of Japan V0. 009, No. 059, Mar. 15, 1985,
`JP 59196032.
`Thomas Carell, EdWard A. Wintner, A. Bashir—Hashemi,
`Julius Rebek, Jr. “A Novel Procedure for the Synthesis of
`Libraries Containing Small Organic Molecules”. AngeW.
`Chem. Int. Ed. Engl. 1994, 33, No. 20; p. 2059.
`Thomas Carell, EdWard A. Wintner, Julius Rebek, Jr. “A
`Solution—Phase Screening Procedure for the Isolation of
`Active Compounds from a Library of Molecules”. AngeW.
`Chem. Int. Ed. Engl. 1994, 33, No. 20; p. 2061.
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`Marie—Therése Chateau, Céline Ginestier—Verne, Jean
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`Takashi Kojima, Masao Yamamoto, Chihiro MochiZuki,
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`“Different Changes in Express and Function of Connexin 26
`and Connexin 32 During DNA Synthesis and Redifferenti
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`Kit S. Lam. “Application of conbinatorial library methods in
`cancer research and drug discovery”. Anti—Cancer Drug
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`Jose Prados, Consolacion MelguiZo, Juan Emilio Fernandez,
`Amelia Eva Aranega, Luis Alvarea and Antonia Arangea.
`“Actin, Tropomyosin and ol—Actinin as Markers of Differ
`entiation in Human Rhabdomyosarcoma Cell Lines Induced
`With Dimethyl Sulfoxide”. Cellular Molecular Biology.
`39(5) 525—536, 1993.
`HoWard M. Prentice, Stephen E. Moore, John G. Dickson,
`Patrick Doherty and Frank S. Walsh. “Nerve groWth fac
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`Stefan Sjolander and Csaba UrbanicZky, “Integrated Fluid
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`Analytical Chemistry, vol. 63, No. 29, Oct. 15, 1991.
`Alexander SZabo, Lesley StolZ and Russ GranZoW.. “Surface
`plasmon resonance and its use in biomolecular interaction
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`O. Trubiani, C. Peiri, M. Rapino, R. DiPrimo. “The c—myc
`gene regulats the polyamine pathWay in DMSO—induced
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`R.A. Houghten, J.R. Appel, S.E. Blondelle, J.H. Curevo,
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`Combinatorial Libraries for the Identi?cation of Bioactive
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`
`* cited by examiner
`
`Primary Examiner—Jean C. WitZ
`(74) Attorney, Agent, or Firm—Akerman Senter?tt
`
`(57)
`
`ABSTRACT
`
`Provided herein is a method for extracting lipid fractions
`from marine and aquatic animal material by acetone extrac
`tion. 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.
`
`39 Claims, 20 Drawing Sheets
`
`RIMFROST EXHIBIT 1016 page 0001
`
`

`

`U.S. Patent
`
`Oct. 5, 2004
`
`Sheet 1 0f 20
`
`US 6,800,299 B1
`
`
`
`‘m ' AHA. .. 20 30
`
`
`
`
`
`A 40
`
`
`
`k 50
`
`
`
`, 60
`
`70 [11111
`
`El
`
`1266
`1.455 — 12:0
`1.625
`1.612
`1.676
`2.056
`2.173
`2.331 — 1410
`2.505
`2.591 — 1411
`2.662
`2.802
`2.655
`3.078 -— Sid 1520
`8.309
`3.586
`6.610
`4.176 — 1610
`
`4.521 — 1621
`4.664
`4.891 — 161111"
`5.121
`5.426
`5.570
`6.037
`6.662
`6.871
`7.235
`7.925 — 18:0
`8.439 ~ 18:1
`8.640 - 18:11
`9.544
`9.801 — 18:2
`10.491
`10.625
`11.042
`
`11.687
`12.145 - 1633
`13.456
`15.626 .— 2030
`16.045
`16.462 — 2011
`17.017 — 2021((21811)
`19.344 — 2012
`22.606 — 2024(6,10,14,16)
`24.103
`26.247
`28.287
`31.295
`40.655
`49.721
`56.373
`62.225
`
`RIMFROST EXHIBIT 1016 page 0002
`
`

`

`U.S. Patent
`
`001. 5,2004
`
`Sheet 2 0f 20
`
`US 6,800,299 B1
`
`0A1
`1
`601
`66
`60
`45
`401
`35
`304
`261
`20%
`
`10
`
`20
`
`30
`
`mi“
`40
`60
`
`A
`
`60
`
`70 min
`
`E 11.7
`
`1.217
`1.264
`1.464 - 12.0
`1.624
`1.012
`1.876
`2.066
`2.171
`2.330 - 140
`2.606
`2.591 - 14.1
`2.680
`2.000
`2.064
`3.077 - $14 150
`3.306
`3.585
`
`3.806
`4.157 — 16:0
`4.616 — 16:1
`4.600
`4.091 — 16:1tr
`5.028
`5.109
`6.421
`6.662
`6.031
`6.642
`6.070
`7.230
`7.910 _ 1010
`0.419 ~ 1011
`8.622 - 10.111
`9.529
`
`9.786 — 18:2
`10.484
`10.013
`11.690
`12.136 - 1013
`13.447
`16.623 ~ 200
`16.026
`16.466 ~ 2011
`17.021 _ 20:1 (C1811)
`22.606 - 20:4 (6,10,14,10)
`24.100
`26.217
`20.241
`40.622
`66.417
`62.086
`
`RIMFROST EXHIBIT 1016 page 0003
`
`

`

`U.S. Patent
`
`08. 5,2004
`
`Sheet 3 0f 20
`
`US 6,800,299 B1
`
`L .hn n
`
`A) L.‘
`
`A 2‘
`
`j A *7
`
`20
`
`30
`
`40
`
`50
`
`60
`
`70 111111
`
`1.216
`1.262
`1.454 — 12:0
`1.624
`1.811
`1.875
`2.016
`2.054
`2.174
`2.330 — 14:0
`2.505
`2.589 — 14:]
`2.679
`2.799
`2.854
`2.981
`3.074 —- std 15:0
`3.304
`3.580
`3.804
`4.169 — 16:0
`4.296
`
`4.520 - 16:]
`4.683
`4.884 — 161111‘
`5.030
`5.111
`5.420
`5.581
`6.031
`6.642
`6.868
`7.226
`7.908 ~ 18:0
`8.444 — 18:1
`8.639 — 18:11!‘
`9.005
`9.536
`9.788 — 16:2
`10.267
`10.481
`10.807
`11.626
`12.140 * 18:3
`
`Eff}
`
`13.457
`13.943
`15.053
`15.572 4 20:0
`16.016
`16.486 — 20:1
`16.999 — 20:1(cis11)
`18.762
`19.303 — 2012
`20.474
`21.027 — 20:3
`22-575 — 20:4 (6,10,14,18)
`24.071
`26.215
`28.333
`31.180
`40.560
`46.595
`49.513
`56.292
`62.250
`
`RIMFROST EXHIBIT 1016 page 0004
`
`

`

`U.S. Patent
`
`061. 5,2004
`
`Sheet 4 0f 20
`
`US 6,800,299 B1
`
`20
`
`60
`
`40
`
`50
`
`60
`
`70
`
`mln'
`
`E4
`
`1.552 - 1210
`1749
`1.966
`2.095
`2.262
`2.485
`2.562 - 14.0
`2.764
`2666 - 1411
`» 6.004
`6.145
`6.476 - 8111 150
`6.720
`4.066
`4.625
`4.796 - 16:0
`5.196 - 16:1
`5.406
`
`5.675 - 16:1tr
`5.964
`6.264
`6.566
`6.655
`7.009
`7.159
`7.440
`7.674
`6.019
`6.462
`9.411 - 18:0
`10.000 ~ 18:1
`10.249 - 18:1tr
`10.716
`11.657
`11.647 - 18:2
`12.519
`
`2
`
`12.666.
`16.666
`14.017
`14.524 - 18:3
`16.107
`19.275 - 200
`20.112 - 20.1 (cisll)
`20.761 - 20:1
`27.556
`29.529
`62.161
`64.614
`69.240
`50.674
`61.692
`70.566
`77.694
`
`RIMFROST EXHIBIT 1016 page 0005
`
`

`

`U.S. Patent
`
`OCt. 5, 2004
`
`Sheet 5 0f 20
`
`US 6,800,299 B1
`
`PM 1
`
`60
`554
`50
`451
`40
`355
`30%
`
`251
`20
`
`=
`11.11 >
`
`f
`
`./\I
`
`10
`
`20
`
`30
`
`40
`
`60
`
`70
`
`min
`
`E 5
`
`1.556 - 12.0
`1.753
`1.972
`2.104
`2.304
`2.590 - 140
`2392 ~ 1411
`3.012
`3.153
`3.465 - S111 1510
`3.710
`4.096
`4.203
`4.333
`4.300 - 16:0
`5.206 - 16:!
`5.417
`5.711 - 16:1tr
`
`5.977
`6.294
`6.546
`7.020
`7.166
`7.309
`0.030
`3.473
`9.425 - 1310
`10.010 - 1311
`10.260 - 131111
`10.735
`11.394
`11.661 ~ 18:2
`12.540
`12.909
`13.402
`14.010
`
`14.540 — 1313
`16.133
`16.805
`13.131
`19.154 — 200
`19.375
`20.099 - 2021(Ci811)
`20.320 — 2011
`23.903
`27.533
`29.570
`32.195
`34.597
`39.334
`50.452
`70.660
`77.395 - 24.0
`
`RIMFROST EXHIBIT 1016 page 0006
`
`

`

`U.S. Patent
`
`08. 5,2004
`
`Sheet 6 0f 20
`
`US 6,800,299 B1
`
`4'0
`
`60
`
`0'0
`
`100
`
`1é0
`
`140
`
`100 min
`
`E5
`
`1.564 — 12:0
`1.762
`1977
`2.080
`2.273
`2.591 — 14:0
`2.887 —- 14:1
`3.008
`3.470 ~ std 15:0
`4.108
`4.341
`4.803 — 16:0
`5.210 — 16:11!‘
`5.683
`6.292
`6.514
`7.030
`7.810
`
`'
`
`34.677
`38.373 — 22:0
`40.163
`50.157
`57.532
`61.436
`70.271
`77.784 ~— 24:0
`110.694
`127.696
`
`8.025
`8.485
`9.360 — 18:0
`9.407
`10.147 — 18:11]“
`11.618 — 18:2
`12.858
`14.515 — 18:3
`16.162
`18.077
`19.355 — 20:1
`20.182 — 20:4 (01811)
`20.311
`23.205 — 20:2
`24.678
`27.411 — 20:4 (6,10,14,18)
`29.307
`31.990
`
`RIMFROST EXHIBIT 1016 page 0007
`
`

`

`U.S. Patent
`
`0a. 5,2004
`
`Sheet 7 0f 20
`
`US 6,800,299 B1
`
`Chaiesteml esters ...............
`
`Methyl esters|
`
`....
`
`..
`
`Triglycerides
`
`Free fatty acids
`Chaiesternl
`
`
`Diglycerides Mcnngfycerides Urigin
`
`RIMFROST EXHIBIT 1016 page 0008
`
`

`

`U.S. Patent
`
`0a. 5,2004
`
`Sheet 8 0f 20
`
`US 6,800,299 B1
`
`Cholesteml esters
`Methyl esters
`
`Triglycerides
`
`FI‘EE fati acids
`Che esters}
`Digiyceifides
`
`Monoglycerides ..... Gt‘igin
`
`RIMFROST EXHIBIT 1016 page 0009
`
`

`

`U.S. Patent
`
`Oct. 5, 2004
`
`Sheet 9 of 20
`
`US 6,800,299 B1
`
`
`
`RIMFROST EXHIBIT 1016 page 0010
`
`RIMFROST EXHIBIT 1016
`
`page 0010
`
`

`

`U.S. Patent
`
`0a. 5,2004
`
`Sheet 10 0f 20
`
`US 6,800,299 B1
`
`RIMFROST EXHIBIT 1016 page 0011
`
`

`

`U.S. Patent
`
`0a. 5,2004
`
`Sheet 11 0f 20
`
`US 6,800,299 B1
`
`RIMFROST EXHIBIT 1016 page 0012
`
`

`

`U.S. Patent
`
`0a. 5,2004
`
`Sheet 12 0f 20
`
`US 6,800,299 B1
`
`eutrl Hi5
`
`Lcithin
`Sphinm @
`3101311
`Lysnlsmiihi,
`rim
`
`RIMFROST EXHIBIT 1016 page 0013
`
`

`

`U.S. Patent
`
`0a. 5,2004
`
`Sheet 13 0f 20
`
`US 6,800,299 B1
`
`v01000001 lipids
`
`Cephalin
`
`Lecithin
`Sphingwmyelin
`Lysolegithin
`ngm
`
`m_l5
`
`RIMFROST EXHIBIT 1016 page 0014
`
`

`

`U.S. Patent
`
`0a. 5,2004
`
`Sheet 14 0f 20
`
`US 6,800,299 B1
`
`RIMFROST EXHIBIT 1016 page 0015
`
`

`

`U.S. Patent
`
`0a. 5,2004
`
`Sheet 15 0f 20
`
`US 6,800,299 B1
`
`RIMFROST EXHIBIT 1016 page 0016
`
`

`

`U.S. Patent
`
`0a. 5,2004
`
`Sheet 16 0f 20
`
`US 6,800,299 B1
`
`CHEF
`
`RIMFROST EXHIBIT 1016 page 0017
`
`

`

`U.S. Patent
`
`Oct. 5, 2004
`
`Sheet 17 0f 20
`
`US 6,800,299 B1
`
`N NE
`
`8 mm so 2 3 mm on S 8
`
`_ g _ _ _ _ _ _
`
`Q m 5:: $2 @5283 888:9: 5 2032523
`
`.3 N “O was 53235
`
`
`.35 266% 3 25:5
`
`2 ~
`
`2
`
`1mm
`'(sgseq m?gam qseu; e no '70) pp“
`
`RIMFROST EXHIBIT 1016 page 0018
`
`

`

`U.S. Patent
`
`0a. 5,2004
`
`Sheet 18 0f 20
`
`US 6,800,299 B1
`
`mm
`
`@N g mm ON 2 a ‘a
`
`“QM-E
`
`Q m $5 $2 5552 WBSZQE E. SOSMEHEBWQ
`
`
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`
`RIMFROST EXHIBIT 1016 page 0019
`
`

`

`U.S. Patent
`
`m.
`
`%
`
`m
`
`US 6,800,299 B1
`
`ENE
`
`m A
`
`w -m
`
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`
`M IN
`
`Q. m 52: £2 Eusmcgv WBSSE: E EOSQEEBEQ
`
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`
`RIMFROST EXHIBIT 1016 page 0020
`
`

`

`U.S. Patent
`
`Oct. 5, 2004
`
`Sheet 20 of 20
`
`US 6,800,299 B1
`
`OC=
`
`
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`RIMFROST EXHIBIT 1016 page 0021
`
`RIMFROST EXHIBIT 1016
`
`page 0021
`
`

`

`US 6,800,299 B1
`
`1
`METHOD OF EXTRACTING LIPIDS FROM
`MARINE AND AQUATIC ANIMALTISSUES
`
`FIELD OF THE INVENTION
`
`This invention relates to the extraction of lipid fractions
`from marine and aquatic animals such as knill, Calanus,fish
`and sea mammals. Morespecifically, this inventionrelates to
`an improved method of extracting lipid fractions by dehy-
`dration with solvents and recovering a solid residue rich in
`active enzymes.
`
`BACKGROUND OF THE INVENTION
`
`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
`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 ani-
`mal 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 marine and aquatic oils contain various
`antioxidants, which may have potential therapeutic proper-
`ties.
`
`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 developmentof 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.
`
`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.
`Various methods for extracting marine and aquatic animal
`oils are known. For example, it is knownto extract fish oil
`using organic solvents such as hexane and ethanol.It is also
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`known to measure the fat content in fish muscle tissue using
`solvents such as acetone.
`
`U.S. Pat. No. 4,331,695 describes a method using pres-
`surized solvents which are gaseous at room temperature,
`such as propane, butane or hexane. The extraction is per-
`formed at preferred temperatures of 15 to 80° C. on shredded
`vegetable or finely divided animal products. The extracted
`oils are then made to precipitate under high pressure and
`elevated temperatures of 50 to 200° C. However, hexaneis
`a poor extraction solvent for marine animals such askrill.
`Furthermore, the high temperatures used in the precipitation
`step negatively alters the lipids.
`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.
`U.S. Pat. No. 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 the aqueous phase and solid phase. The oil phase
`is then further treated with antioxidant to remove undesir-
`able odourortaste.
`
`Canadian Patent 1,098,900 describes a method for
`extracting oils from krill. The 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. biol.
`Chem. 226: 497-509 “A simple methodforthe isolation and
`purification of total 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 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 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.
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1. Gas-liquid chromatographyoffatty acids from dry
`krill (chloroform-methanol)
`FIG. 2. Gas-liquid chromatographyoffatty acids from dry
`krill (acetone)
`FIG. 3. Gas-liquid chromatography of fatty acids from
`frozen krill (acetone)
`FIG. 4. Gas-liquid chromatography of fatty acids from
`frozen krill (ethanol)
`FIG. 5. Gas-liquid chromatography of fatty acids from
`frozen krill (t-butanol)
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`US 6,800,299 B1
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`3
`FIG. 6. Gas-liquid chromatography of fatty acids from
`frozen krill (ethyl acetate)
`FIG. 7. Thin-layer chromatography of neutral lipids of
`Calanus sp. and M. norvegica
`FIG. 8. Thin-layer chromatographyof neutral lipids of EF.
`pacifica
`FIG. 9. Thin-layer chromatographyof neutral lipids of M.
`schmitti
`
`FIG. 10. Thin-layer chromatography of neutral lipids of
`G. galeus
`FIG. 11. Thin-layer chromatography of neutral lipids of
`Angel Shark
`FIG. 12. Thin-layer chromatography of phospholipids of
`Calanus sp. and M. norvegica
`FIG. 13. Thin-layer chromatography of phospholipids of
`E. pacifica
`FIG. 14. Thin-layer chromatography of phospholipids of
`M. schmitti
`
`FIG. 15. Thin-layer chromatography of phospholipids of
`G. galeus
`FIG. 16. Thin-layer chromatography of phospholipids of
`Angel Shark
`FIG. 17. Influence of the volume of acetone on lipid
`extraction (E. pacifica)
`FIG. 18. Influence of incubation time in acetone on lipid
`extraction (E. pacifica)
`FIG. 19. Influence of the volume of ethanol on lipid
`extraction (E. pacifica)
`FIG. 20. Influence of incubation time in ethanol on lipid
`extraction (I. 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 appli-
`cation 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 understoodthat 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 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.
`In a preferred embodiment, the extraction is carried out by
`successive acetone and alcohol treatments. Preferred alco-
`
`hols are isopropanol, and t-butanol. The alcohol mayalso 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 protein, including active enzymes.
`Recovery of total lipids is comparable to the Folch et al.
`(1957) procedure reported in the background of the inven-
`tion. It has been tested with krill, Calanus, fish and shark
`tissues.
`
`Surprisingly, it was found that successive extractiontreat-
`ments as proposed by the present invention hasa better yield
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`in lipid extraction that single solvent system extractions. The
`extraction using two successive solvents which starts with a
`ketone such as acetone is especially advantageoussince 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 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 besuffi-
`cient to allow a cost-effective recovery of lipid fractions and
`separate recovery 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 and preferably overnight. However
`extraction timeis notcritical to the yield of lipid extraction.
`To facilitate extraction, it is preferable to use particles of less
`than 5 mm in diameter. Extraction is preferably conducted
`under inert atmosphere and at a temperature in the order of
`about 5° C. or less.
`
`Preferably, the beginning of the extraction will be con-
`ducted underagitation for about 10 to 40 minutes, preferably
`20 minutes. Although extraction time is not critical, it 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 techniques including, for example,
`filtration, centrifugation or sedimentation. Filtration is pref-
`erably used.
`After separation by filtration on an organic solventresis-
`tant filter (metal, glass or paper) the residue is optionally
`washed with pure acetone, preferably two volumes(original
`volume of material) to recover yet more lipids. The com-
`bined 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.
`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). Thefiltrate is evapo-
`rated leaving a second fraction of lipids (identified as
`fraction II). Although the extraction periodis notcritical,it
`was found that an extraction time of about 30 minutes is
`
`sufficient at temperatures below about 5° C.
`Temperature of the organic solvents, except t-butanol, and
`temperature of the sample are notcritical parameters, but it
`is preferable to be as cold as possible. However, in the case
`of t-butanol whichis solid at room temperature, it is impor-
`tant 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 wasapplied to krill. This method is the reference
`for measuring efficiency of the extraction process. Another
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`US 6,800,299 B1
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`5
`comparison has been made with a technique using hexane as
`the extraction solvent. Lipid recovery by suspending lipid
`fractions in small volumes of their 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 translucent oil having appearance and properties
`more attractive than any oil obtained by the classical tech-
`nique of Folch et al. (1957).
`To analyze lipid composition, 780 ug of each extract was
`loaded on silica-gel plates and fractionated by thin layer
`chromatography, TLC (Bowyer et al. 1962) with the fol-
`lowing 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) including some modifications to the
`original technique: 2 h at 65° C. instead of 1 h 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.
`Fat was analyzed according to the American Oil Chem-
`ist’s Society (AOCS). The following criteria have been used
`to analyze the lipids extracted: saponification and Wys
`iodine indexes and moisture-volatile matter levels. Choles-
`
`terol content has also been determined by the method of
`Plummer 1987. The same 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, astaxanthin and canthaxan-
`tin contents. 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 showsthe results of lipid extraction from frozen
`Euphausia pacifica, a 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 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 separa-
`tions. These separations are influenced by the quantity of
`residual acetone in the water-oil solution after acetone
`
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`
`evaporation. This quantity of acetone varies from an experi-
`ment to 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 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, com-
`pared to acetone alone (1,86% extraction rate, and even
`greater extraction rates 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 (lipid fraction and solid
`protein-rich material). Indeed, samples of EF. pacifica incu-
`bated 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™ agar 1,5%
`(Difco Laboratories, Detroit, USA) then incubated at room
`temperature or 4° C. for 18 days. No significant bacterial
`growth was observedat 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
`(Goodman et al. 1980).
`Table 3 showsthe 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. Varia-
`tions can be attributable to diet and time (season) of
`collection, which are different for those two species.
`Table 4 showsthe 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 overthe classical method (4,46%
`versus 3,30%).
`It also shows that grinding may be an
`important factor when the species is large (4,46% versus
`3,53%).
`Table 5 reports on lipid extraction from Calanus. Consid-
`erable 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 dem-
`onstrated 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 more
`lipid than our method. Indeed small amounts oflipids 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 inven-
`tion. 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 of lipids for commercial
`uses (because of toxicity).
`In Tables 9 to 11, are shownresults of lipids extraction
`from shark liver tissues. There is no marked difference in
`
`results between techniques within a species.
`Tables 12 shows some characteristics features of fraction
`
`I (acetone) and fraction II (alcohol or ethyl acetate) for krill
`oil (e. pacifica). First, the saponification index offraction I
`(130,6) indicates that this fraction contains fatty acids with
`longer chains, compared to fraction II (185,7). The Wijs
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`US 6,800,299 B1
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`7
`iodine index of fraction I showsthat this fraction contains
`
`high levels of polyunsaturated fatty acids. As compared to
`olive oil which hasan 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 pointinferior 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 14 corroborates these
`iodine indexes: fraction I has a high percentage (30,24%) of
`polyunsaturated fatty acids (pentaenesthexaenes) and so
`fraction II (22,98%). Finally, Table 12 shows also that
`fraction I
`is comprised of 10,0% of volatile matter and
`humidity after evaporation of the solvent. For the sametest,
`the fraction II gives a value of 6,8%. To get rid of traces of
`solvents, it is important to briefly heat (to 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 12, 13, 14, 15, 16 and 17. It is noteworthy
`to mention that in Table 17, the carotenoids content was
`significantly high as measured in terms of two carotenoids
`namely astaxanthin and canthaxanthin. Indeed, duplicates
`analyzes revealed values of 92 to 124 ug/g of lipid fraction
`for astaxanthin and 262 to 734 ug/g for canthaxanthin. Thus,
`for the purpose of the present invention it may be said that
`the krill extract comprises astaxanthin at
`least 75 and
`preferably at least 90 ug/g of lipid fraction. In the case of
`canthaxanthin, at least 250 and preferably at least 270 ug/g
`of lipid fraction. Low values for peroxide and anisidine are
`advantageous and are due to the presence of high levels of
`natural antioxidants (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 migra-
`tion characteristics. Thus, krill extract is a good candidative
`for transdermal delivery of medicines.
`Table 18 shows the best mode of the method in accor-
`
`invention for lipid extraction of
`
`dance with the pres