O P I C
`
`OFFICE DE LA PROPRIETE
`
`..
`
`‘-
`
`.
`
`. .. . -‘
`
`(12)(19) (CA) Demande-Application
`C I P 0
`CANADIAN INTELLECTUAL
`
`INTELLECTUELLE DU CANADA
`
`‘.\
`
`PROPERTY OFFICE
`
`(21) (A1)
`(22)
`(43)
`
`1998/10/21
`2000/04/21
`
`(72) BEAUDOIN, Adrien, CA
`(72) MARTIN, Genevieve, CA
`(71) UNIVERSITE DE SHERBROOKE, CA
`
`(51) Int.Cl.6 Cl lB 1/10, A2311/04, A23D 9/02
`(54) PROCEDE D’EXTRACTION DES LIPIDES DE TISSUS
`
`D’ANIMAUX AQUATIQUES PRODUISANT UN RESIDU
`DESHYDRATE
`
`(54) PROCESS FOR LIPID EXTRACTION OF AQUATIC ANHVIAL
`TISSUES PRODUCDIG A DEHYDRATED RESIDUE
`
`(57) The procedure includes the suspension of freshly collected material in an equal Volume of acetone under inert gas
`atmosphere. Lipids are extracted by successive acetone and ethanol treatments. The procedure produces two lipid
`fractions and a dry residue enriched in protein and other material insoluble in organic solvents. Recovery of total lipids
`is comparable or superior to the Folch et al. (1957) procedure. It has been tested with krill, Calanus and fish tissues.
`
`Idt'C d
`n us rue
`ana a
`
`|dtC d
`n usry ana a
`
`000001
`
`AKBM 1003
`
`000001
`
`

`
`CA 02251265 1998-10-21
`
`TITLE QF THE INVENTION
`
`PROCESS FOR LIPID EXTRACTION OF AQUATIC ANIMAL TISSUES
`
`PRODUCING A DEHYDRATED RESIDUE
`
`FIELD QF THE INVENTION
`
`The present invention relates to a method for lipid extraction of
`
`animal tissues and to the lipid and dry residue fractions obtained therefrom.
`
`More particularly, the present invention relates to a lipid extraction method
`
`using krill, Calanus and fish tissues as starting material.
`
`000002
`
`000002
`
`

`
`CA 02251265 1998-10-21
`
` flIDfl
`
`Extraction process
`Fresh (or frozen) material (Euphausia pacifica and other species) is
`suspended in cold acetone for a given period of time at low temperature (5°C or
`lower). A ratio of krill-acetone 1:6 (wlv) and an incubation time of 2 h in acetone
`were found to be optimal. Alternatively the material can be kept in an equal
`volume of acetone at low temperature for long periods of time (months) under
`inert atmosphere. The size of the material
`is an important factor for
`the
`penetration of acetone. Indeed, it is preferable to grind material with dimensions
`superior to 5 mm before getting it in contact with acetone. ‘lire suspension is
`swirled for a short period of time (about 20 min) after acetone addition. After
`filtration on an organic solvent resistant filter (metal, glass or paper) the residue
`is washed with two volumes of pure acetone. The combined filtrates are
`evaporated under
`reduced pressure. 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 two volumes (original volume of frozen material) of 100% ethanol. The
`ethanol filtrate is evaporated leaving a second fraction of lipids (identified as
`fraction II).
`'
`
`Variations of the process
`Variable volumes of acetone relative to the levels of sample can be used.
`. It is also applicable to the volume of acetone used to wash and to the volume of
`ethanol used to extract Incubation times in solvents may vary. Particle size effect
`the recovery of lipids and the material could be ground in various sizes of
`particles. depending on the grinder used. Temperature of the organic solvents
`and temperature of the sample are not critical parameters, but it is preferable to
`. be as cold as possible.
`
`000003
`
`000003
`
`

`
`CA 02251265 1998-10-21
`
`.
`Methods
`To compare the efficiency of the extraction process. a classical technique
`(Folch et al. 1957) implying chloroform and methanol was applied to krill. This is
`the standard of reference for the efficiency of the extraction process. Lipid
`recovery was estimated by suspending lipid fractions in small volumes of their
`original solvents and measuring by gravimetry small aliquots after evaporation.
`To analyze lipid composition, small aliquots of the various extracts were
`loaded on silica-gel plates and fractionated by thin layer chromatography. TLC
`(Bowyer et al. 1962) with the following solvents. Neutral lipids: hexane. ethyl
`ether, acetic acid (90:10:1 vlv) and phospholipids: chloroform. methanol. water
`(80:25:2 vlv). Fatty acid composition of E. pacifica was analyzed by gas liquid
`chromatography. GLC (Bowyer et al. 1962) including some modifications to the
`original technique: 1h at 65°C instead of 2h at 80°C. three washes with hexane
`instead of two and no wash with water.
`The dry residue is wetted with ethanol
`rehydratation of the proteins.
`To get rid of traces of organic solvents. lipid fraction l and ll are wanned
`(60°C for fraction I and 70°C for fraction ll) for 5 min under inert atmosphere.
`
`to facilitate a progressive
`
`000004
`
`000004
`
`

`
`CA 02251265 1998-10-21
`
`Applications
`The different fractions (oil. proteins, and others) of aquatic animal biomass
`extracted by the current procedure could be used in many fields:
`
`1 -Aquaculture
`As mentioned in results, fatty acids 20:5 (eicosapentaenoic acid) and 22:6
`(docosahexaenoic acid) are found in high concentrations in krill, Calanus, and
`fish. Farming fish on high quality marine oils rich in docosahexaenoic and
`eicosapentaenoic (EPA) acids is an efficient means of delivering these essential
`nutrients in human diets and also efficiently exploiting a strictly limited marine
`bioresource (Sargent 1997). Krill may be used as food supplement for fish and
`shrimp (Sargent 1997) because of its capacity to improve growth and survival
`capacity against diseases
`(Runge 1994), as pigmentation enhancer
`for
`ornamental fish species and as starter diet for marine and fresh water species
`(Prawn Hatchery Food 1997).
`
`2-Nutraceuticals
`
`Considering the beneficial effects of omega-3 fatty acids, the marine oils
`from krill, Calanus and fish could be used as dietary supplements to human dlet.
`22:6 n-3 fatty acid is essential for proper development of the brain and the eye
`(Sargent 1997).The beneficial effects of n-3 polyunsaturated fatty acids in
`‘reducing the
`incidence of cardiovascular disease by
`lowering plasma
`triacylglycerol level and altering platelet function towards a more anti-atherogenic
`state has been reviewed (Christensen 1994). Also, dietary krill oil, like fish oil.
`can suppress the development of autoimmune murine lupus: EPA substitutes for
`arachidonic acid, a substrate for cycloxygenase thereby reducing the production
`of prostaglandins (Chandrasekar 1996). The effects of dietary supplementation
`with w-3 lipid-rich krill oil includes decreased expression of TGF, in kidneys and
`of the oncogene-c-ras in splenocytes (Chandrasekar 1996). Krill oil has
`beneficial effects on life span and amelioration of renal disease similar to those
`previously described in studies with fish oil (Chandrasekar 1996).
`
`3—AnimaI food
`
`Feeding the animals with omega-3 fatty acids may increase the level of
`unsaturated fatty acids and decrease cholesterol levels of meat. This property is
`exploited in the poultry industry to improve the quality of eggs. Calanus.
`in
`particular, is a full of promise ingredient of domestic animal's food (Runge 1994).
`
`4-Cosmetic industry
`Calanus is used for the production of moisturizing creams (Runge 1994).
`
`5-Medical applications
`Krill may be used as a source of enzymes for medical application like the
`debridement of ulcers and wounds (Hellgren 1991) or to facilitate food digestion.
`
`000005
`
`000005
`
`

`
`CA 02251265 1998-10-21
`
`Finally, these marine products are also rich in liposoluble
`
`vitamins A, D, E and K and carotenoids that are extracted with lipids. The
`
`chitin of krill and Calanus could be exploited to protect plants against fungi.
`
`Also, marine oils contain unidentified antioxidants which may have potential
`
`therapeutic properties.
`
`Other objects, advantages and features of the present
`
`invention will become more apparent upon reading of the following non-
`
`restrictive description of preferred embodiments with reference to the
`
`accompanying drawing which is exemplary and should not be interpreted as
`
`limiting the scope of the present invention.
`
`000006
`
`000006
`
`

`
`CA 02251265 1998-10-21
`
` EM
`
`Results
`
`Note on experimental conditions
`
`is practicable under
`then ethanol
`The lipid extraction with acetone.
`different experimental conditions. as mentioned on page 1 of this document
`(variation of the process). Moreover, the majority of data shown in this document
`are from experiments made with sample-acetone ratio of 1:9 (wlv) incubated
`overnight at 4°C and with sample-ethanol ratio of 1:4 (wlv) incubated 1h at 4°C.
`In addition. no material has been ground in most experiments. Only later. tests
`have been made to standardize the method for extraction of lipids with acetone.
`then ethanol. As shown in Figure 9 and 11.
`it appears that optimal ratios of
`sample-solvent are 1:6 (wlv) for acetone and 1:2 (wlv) for ethanol. Figure 10 and
`Figure 12 show that optimal incubation times are 2 h for the first solvent and 30
`min for the second. Grinding has been experimented and it is clear that solvents
`have a better impact on ground material. as shown in Table 5. Then,
`experimental conditions are specified for each experiment
`Diagram 1
`illustrates the procedure of lipid extraction from frozen krill
`whiohisthesameusedwithdryknuarldotherfreshspedesascalanus.
`mackerel. trout and hening.
`
`Interpretation of results
`
`Table1showsthathigherlevelsofIipidsareextractedbyacetone
`followed byethanolascomparedtothedassical procedureofFolch etal. (1957).
`Thesameinformationisfoundinhblesconceminganotherknllspecies
`(Megayctiphanes norvegica). Back to Table 1, one can see that the combination
`ofacetoneandeflianolasasinglestepdidnotimproveflieezdracfionprocess
`Table 2 shows the results of lipid extraction from frozen Euphausie
`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.
`Samples of E. pacifica incubated in different ratios of acetone at 4°C for 112 days
`have been inoculated on NA medium containing Bacto beefextract 0.3%. Bacto
`peptone 0.5% and Bactc agar 1.5% (Difco 1984) then incubated at room
`temperature or 4°C for 18 days. No significant bacterial growth was observed at
`a ratio of 1 volume of acetone per gram of krill. At higher proportions of acetone
`(2 volumes and 5 volumes). there was no bacterial growth at all. whidr means
`that acetone preserves krill samples. Acetone is known as an efficient
`bactericidal and viricidal agent (Goodman et al. 1980).
`Table 3 shows the yield of lipids from M. norvegica. The percentage of
`lipids is lower (3.67 %) than for E. padfica (4.04 96) shown in Table 2. These
`variations can be attributable to the season of catch.
`
`Table 4 shows the krill composition obtained from experiments 3 and 4
`with frozen E. pecifica (‘I'able 2). One finds about 83% of water, 4% of lipids and
`12% of dry residue.
`
`000007
`
`,__,_.___
`
`,,.,
`
`,, _-._ ir—r >1
`
`000007
`
`

`
`CA 02251265 1998-10-21
`
`Table 5 shows the influence of grinding on the efliciency of extraction of
`M. norvegica lipids. These extractions were carried out under optimal conditions
`and show the definite advantage of the procedure over the classical method
`(4.46 % versus 3,30 %). It also shows that grinding may be an important factor
`when the species is large (4,46% versus 3.53 %).
`Considerable quantity of lipids were obtained from Calanus (Table-6).
`Some variations in Calanus species composition may explain the variations
`between experiments 1 and 2 (8.22 % and 10,90 % of fresh weight).
`When the technique was applied to fish (mackerel) peripheral tissues
`(mainly muscles) or viscera. an amount of lipids was extracted (Table 7) but it
`appeared less efficient than the classical method since extractions of the residue
`with the latter technique allowed us to recover less lipid. Overall, our technique
`would allow us to exploit parts of fish that are usually wasted atter the withdrawal
`of fillets of the fish or lipid extracts from fishes not used for human consumption.
`Those fish tissues not used alter the transformation of the fish for human
`
`consumption could be stored in acetone, then lipids could be extracted with our
`process. Extraction of lipids from trout and herring were carried out in parallel
`with the classical method. Results appear in Table 3 and 9. The yield is not
`significantly different for the viscera whereas with peripheral tissues (muscles)
`the classical technique is superior (14.93 % versus 6.70 %). Technique using
`acetone followed by ethanol for trout and herring (and maybe for other species)
`seems applicable as well as for mackerel. Table 11 shows the suggested
`procedure for lipid extraction of aquatic animal tissues.
`of E.
`Figures 1
`to 4 show chromatograms of fatty acid composition
`pecifica lipids. On each of them, high proportions of 20:5 and 22:6 fatty acids
`(characteristic of marine oils) are noticeable and represented by two distinct
`peaks. The concentration of the sample on Figure 4 was lower than the others,
`so the peaks don't have the same amplitude. With retention times and amounts
`gave by the chromatograph, identification and compilation of the majority of the
`fatty acids have been done (see Table 10).
`Figures 5 to 8 (TLC) show a higher proportion of neutral
`compared to phospholipids in marine oils.
`The influence of incubation time on the efficiency of the acetone to extract
`lipids from E. pacifica is illustrated in Figure 9. Extraction is already completed at
`2 h. With this time. we proceeded to determine the influence of the sample-
`acetone ratio (Figure 10). Results show that a ratio of 1:6 (wlv) produce the best
`yield. The second lipid extraction is carried out with ethanol. The incubation time
`in this solvent should be at least 30 min as indicated by the results of Figure 11.
`The volume of ethanol does not appear to be critical since the same yield was
`obtained with different volumes of ethanol.
`
`lipids as
`
`7
`
`000008
`
`000008
`
`

`
`CA 02251265 1998-10-21
`
`One of the inventors, Mr Adn'en Beaudoiri, has tested the different lipid
`fractions. No side effect was observed. The fraction I has the taste of the cod
`liver oil and the insoluble material tastes like salty shrimps.
`
`000009
`
`000009
`
`

`
`CA 02251265 1998-10-21
`
`DIAGRAM 1. KRILL LIPID EXTRACTION PROCESS
`
`
`
`0 Starting matenal
`
`- Acetone extraction
`(overnight)
`
`000
`
`- fmsh {Q}?
`
`6 000 L
`
`o Filtration and washing with acetone
`(vacuum)
`
`1 000 L-2 000 L
`
`recycling
`
`o Evaporation
`
`o Ethanol extraction
`
`2000L
`
`o Filtration
`
`0 Evaporation
`
`"« tofkn‘=loi1:Ok CECE
`
`recycling
`
`0000010
`
`0000010
`
`

`
`CA 02251265 1998-10-21
`
`TABLE 1. EXTRACTION OF DRY KRILL LIPIDS (E. paciflca)
`
`
`
`ggg. No.
`
`Technigue
`
`Yield (%)
`
`Total (%)
`
`1-
`
`2-
`
`3-
`
`4-
`
`6-
`
`7-
`
`8,00
`7,60
`
`19,70
`Ei90
`
`8,15
`1 1,20
`
`6.80
`13.60
`
`acetone "
`ethanol "’
`
`"
`
`"
`
`"
`
`Chiorz MeOH °’
`
`"
`
`Combined acetone-ethanol “’
`
`15,30
`
`25.50
`
`19,35
`
`20, -‘.0
`
`i=20,49
`o-- 3.95
`
`15,50
`
`14,90
`
`7=15,20
`
`a- 0.30
`
`14.30
`
`
`
`Determinations in triplicates (variation < 5 96).
`" :Extraction made with a sample-acetone ratio of 1:9 (wlv). no incubation.
`"’ :Extraction made with a sample-ethanol ratio of 1:4 (wlv),
`incubated 1 night at 4°C.
`°’ :Folch et al. 1957
`" :Extraction made with a sample-acetone-ethanol ratio of 1:5:5. no incubation.
`
`‘O
`
`0000011
`
`0000011
`
`

`
`CA 02251265 1998-10-21
`
`TABLE 2. EXTRACTION OF FROZEN KRILL LIPIDS (E. pacifica)
`
`
`Exp. No.
`
`Technigue
`
`Weld %
`
`Total (°/o)
`
`'
`
`1-
`
`2-
`
`3-
`
`4-
`
`5-
`
`6—
`
`7-
`
`8-
`
`9-
`
`acetone "
`ethanol "’
`
`"
`
`"
`
`"
`
`"
`
`"
`
`"
`
`"
`
`”
`
`2,26
`2.14
`
`2,25
`1,13
`
`2,71
`1,80
`
`2.94
`1,45
`
`2.44
`1,43
`
`2,54
`1,23
`
`2.53
`1,46
`
`2,43
`1.39
`
`2,46
`1,72
`
`4,40
`
`3,33
`
`4,50 °’
`
`4,39 ‘’
`
`3,87
`
`3,77
`
`4,04
`
`3.97
`
`4,13
`
`‘i=4.o4
`a=0,34
`
`
`
`Deteminations in triplicates (variation < 5 Va).
`" :Ex'traction made with a sample-acetone ratio of 1:9 (wlv).
`incubated 1 night at 4°C.
`"’ :Extraction made with a sampie-ethanol ratio of 1:4 (wlv). incubated 1h at 4°C.
`°’ :See Table 4 for total composition.
`
`N
`
`0000012
`
`0000012
`
`

`
`CA 02251265 1998-10-21
`
`TABLE 3. EXTRACTION OF FROZEN KRILL LIPIDS
`(M. norveglca)
`
`
`
`Exp. No.
`
`Technigue
`
`Weld (%)
`
`Total g%)
`
`1-
`
`2-
`
`3-
`
`acetone "
`ethanol "’
`
`"
`
`"
`
`1,82
`1,82
`
`1,15
`2,35
`
`1,68
`2,19
`
`3,8;
`
`3, so
`
`3.37
`
`i'=3,67
`a=0,15
`
`
`
`‘ Detenninations in triplicates (variation < 5 %).
`" :Extraction made with a sample-acetone ratio of 1:9 (wlv),
`incubated 1 night at 4°C.
`"’ :Extraction made with a sample-ethanol ratio of 1:4 (wlv), incubated 1 h at 4°C.
`
`TL
`
`0000013
`
`0000013
`
`

`
`CA 02251265 1998-10-21
`
`TABLE 4. FROZEN KRILL COMPOSITION (E. pacifica)
`on a fresh weight basis
`
`Exg. N9.
`
`gig
`
`In
`
`I
`
`I m
`
`ri
`
`I
`
`flag;
`
`3-
`
`4-
`
`4,50
`
`4,39
`
`12,50
`
`11,50
`
`83,00
`
`84,11
`
`‘i=83,55
`i=12,oo
`:?=4,44
`(F 0,55
`:38 0.50
`o=0,05
`
`
`Determinations in triplicates (variation < 5 %).
`Experience numbers refer to Table 2.
`
`15
`
`0000014
`
`0000014
`
`

`
`CA 02251265 1998-10-21
`
`TABLE 5. INFLUENCE OF GRINDING ON EXTRACTION OF FROZEN
`KRILL LIPIDS (M. norveglca)
`
`
`
`Exg. No.
`
`Tggnigue
`
`Krill ground before 1“extragign
`
`weld (34.1
`
`Total g%)
`
`1-
`
`2-
`
`3-
`
`4-
`
`5-
`
`acetone "
`ethanol "’
`
`"
`
`~
`
`Chlor: MeOH °’
`
`"
`
`yes
`
`no
`
`yes
`
`yes
`
`yes
`
`3,10
`1,07
`
`2,14
`1,39
`
`3,32
`1,14
`
`4,17
`
`3,53
`
`4,45
`
`3,30
`
`3.26
`
`.__________.__..__._____..__..______..___._________._______.
`
`Determinations in triplicates (variation < 5 96).
`" :Extraction made with a sample-acetone ratio of 1:6, incubated 2 h at 4°C
`"’ :Extraction made with a sample-ethanol ratio of 1:2, incubated 30 min at 4°C.
`°’ :Folch et al. 1957.
`
`\‘|
`
`0000015
`
`0000015
`
`

`
`CA 02251265 1998-10-21
`
`TABLE 6. EXTRACTION OF FROZEN Calanus LIPIDS
`(calanus sp.)
`
`
`Exp. N9.
`
`Igagigge
`
`Yiglg (°[;)
`
`Tmgl (Z9)
`
`1-
`
`2-
`
`acetone "
`ethanol "’
`
`"
`
`6,18
`2,04
`
`8.64
`2.26
`
`8,22
`
`10.90
`
`'i'=9,56
`o=1,34
`
`
`
`Detenninations in triplicates (variation < 5 %).
`"’ :Extraction made with a sample-acetone ratio of 1:9 (wlv),
`incubated 1 night at 4°C.
`"’ :Extraction made with a sample-ethanol ratio of 1:4 (wlv).
`incubated 1 h at 4°C.
`
`\5
`
`0000016
`
`0000016
`
`

`
`CA 02251265 1998-10-21
`
`TABLE 7. EXTRACTION OF FRESH F|SH.L|P|DS (Mackerel)
`
`Egg. No. Weld %
`
`Totgl (%)
`
`1-viscera
`fish 1
`
`2-tissues
`fish 1
`
`3-viscera
`fish 2
`
`4-tissues
`fish 2
`
`5-viscera
`fish 3
`
`6-tissues
`fish 3
`
`-
`
`7-viscera
`fish 4
`
`8-tissues
`fish 4
`
`9-viscera
`fish 1
`
`acetone "
`ethanol °’
`
`"
`
`"
`
`”
`
`"
`
`"
`
`"
`
`"
`
`cmocMeoH °’
`
`6,11
`0,59
`
`3.78
`0.91
`
`10,46
`0.57
`
`6.65
`1.41
`
`8.39
`0.66
`
`5.27
`0.97
`
`8.47
`0,69
`
`8.40
`1.02
`
`10-tissues
`fish 1
`
`"
`
`6,70
`
`4,69
`
`11,03
`
`8,06
`
`9.05
`
`6,24
`
`9,16
`
`9.4:
`
`0,52
`
`1,45
`
`
`
`" :FJctraction made with a sample-acetone ratio of 1:9 (wlv),
`incubation time:
`
`-fish 1 viscera: 4h, fish 1 tissues: 23h
`-fish 2 viscera: 23h45. fish 2 tissues: 45h3O
`-fish 3 viscera: 8 days 2h20, fish 3 tissues: 8 days 22h3O
`-fish 4 viscera: 17 days 23h, fish 4 tissues: 18 days 2h25
`'0 :Extraction made with a sample-ethanol ratio of 1:4 (wlv), incubated 1n at 4°C.
`°’ :Fo|ch et al. 1957.
`
`\6
`
`0000017
`
`0000017
`
`

`
`CA 02251265 1998-10-21
`
`TABLE 8. EXTRACTION OF FRESH FISH LIPIDS (Trout)
`
`
`Exg. No.
`
`Technigue
`
`Yield (%)
`
`Tgtal (%)
`
`1 -viscera
`
`acetone "
`ethanol ">
`
`2-tissues
`
`"
`
`3-viscera
`
`Ch|onMeOH °’
`
`4-tissues
`
`"
`
`34,70
`2,13
`
`5,53
`1,17
`
`36,88
`
`6,70
`
`39.31
`
`14,93
`
`
`
`Determinations in triplicates (variation < 5 %).
`" :Extraction made with a sample-acetone ratio of 1:9 (wlv),
`incubated 1 night at 4°C.
`"’ :Extraction made with a sample-ethanol ratio of 1:4 (wlv), incubated 1 h at 4°C.
`°’ :Foich et al. 1957.
`
`\7
`
`0000018
`
`0000018
`
`

`
`CA 02251265 1998-10-21
`
`TABLE 9. EXTRACTION OF FRESH FISH LIPIDS (Herring)
`
`__E_)_<.Q;fl>_.
`
`Techngue
`
`Ylgld 1°/9)
`
`Total 1%}
`
`1-tissues and
`viscera
`
`acetone "
`ethanol °’
`
`2,09
`0,68
`
`2-tissues and
`viscera
`
`Chlor:MeOH °’
`
`2.77
`
`5.95‘
`
`Determination in triplicates (variation < 5 % ).
`" :Extraction made with a samp|e—acetone ratio of 1:9 (wlv),
`incubated 1 night at 4°.
`*” :Extraction made with a sample—ethanol ratio of 1:4 (wlv), incubated 1 h at 4°C.
`°’ :Fo|ch et al. 1957.
`
`\‘8
`
`0000019
`
`0000019
`
`

`
`CA
`
`02251265 1998-10-21
`
`Table 10: Fatty acid composition E. pacifica
`Unsaturated
`Solvent
`Saturated
`Mono
`
`Di
`
`chlo-math
`acetone
`acetone
`ethanol
`
`26,18
`21 ,4
`19,09
`45,93
`45,96
`
`22,54
`22,18
`22,11
`22,96
`22,98
`
`1,91
`1,75
`2,03
`1,23
`1.24
`
`Unidentified
`
`Poly
`4,31
`4,67
`4,79
`2,72
`2.48
`
`H-Poly
`26,34
`24,52
`30,24
`11,11
`11,18
`
`18,72
`25,49
`21,72
`16.05 (500 pglmL)
`16,15 (200 pgImL)
`
`Data expressed in percentage of total fatty acids (96).
`
`\3
`
`0000020
`
`0000020
`
`

`
`CA 02251265 1998-10-21
`
`TABLE 11. OPTIMAL CONDITIONS FOR LIPID EXTRACTION OF
`AQUATIC ANIMAL TISSUES (suggested procedure)
`
`
`STEP
`
`C NDITIONS
`
`Grinding (if particles > 5mm)
`
`4°C
`
`Lipid extraction
`
`Filtration
`
`Washing
`
`Filtration
`
`Evaporation
`
`Oil-water separation
`
`Lipid extraction
`
`Filtration
`
`Evaporation
`
`sample—acetone ratio of 1:6 (wlv)
`2h (including swirling 20 min)
`4°C
`
`organic solvent resistant filter
`under reduced pressure
`
`sampIe—acetone ratio of 1:2 (wlv)
`pure and cold acetone
`
`organic solvent resistant filter
`under reduced pressure
`
`under reduced pressure
`
`4°C
`
`sample-ethanol ratio of 1:2 (wlv)
`pure ethanol
`30 min
`
`4°C
`
`organic solvent resistant filter
`under reduced pressure
`
`,
`
`under reduced pressure
`
`21>
`
`0000021
`
`0000021
`
`

`
`CA 02251265 1998-10-21
`
`Bibliography
`
`Bowyer. D.E., Leaf, W.M.F., Howard, AN. and Gresham. GA 1962. The
`determination of the fatty acid composition of serum lipids separated by
`thin-layer chromatography; and a comparison with column chromatogra-
`phy. BBA 70: 423-431
`
`Chandrasekar. B., Troyer, DA, Venkatraman, J.T. and Fernandes, G. 1996.
`Tissue specific regulation of transforming growth factor beta by omega-3
`lipid—rich krill oil in autoimmune murine lupus. Nutr Res. 16(3): 489-503
`
`Christensen, M.S., I-toy, C-E. and Redgrave, T.G. 1994. Lymphatic absorption
`of n-3 polyunsaturated fatty acids from marine oils with different intramole-
`cular fatty acid distributions. BBA 1215: 198-204
`
`Difco laboratories. 1984. Difco Manual Dehydrated Culture Media and
`Reagents for Microbiology. 10"‘ ed. Detroit.
`
`Foich, J., Lees, M. and Sloane-Stanley, G.H. 1957. A simple method for the
`isolation and purification of total lipids from animal tissues. J. biol. Chem.
`226: 497-509
`
`Goodman Gilman, A, Goodman, L.L. and Gilman, A 1980. The Phannacological
`Basis of Therapeutics. 6"’ ed. Collier Macmillan Canada ltd. Toronto.
`
`Hellgren, L., Karlstam, B., Mohr, V. and Vincent, J. 1991. Krill enzymes. A
`new concept for efficient debridement of necrotic ulcers. Int J Derrnatol.
`30(2): 102-103
`
`Prawn Hatchery Food. 1997. ht_tp://www.kk-teclLcom/kIill.html
`
`Runge, JA and Joly, P. 1994. Rapport sur l'état des invertébrés en 1994:
`7:0 Zooplancton (Euphausiacés et Calanus) de |'Estuaire et du Golfe
`du Saint-Laurent.
`
`Sargent, J.R. 1997. Fish oils and human diet. Br J Nutr.78 Suppl 1: S5-S13
`
`2|
`
`0000022
`
`0000022
`
`

`
`CA 02251265 1998-10-21
`
`Although the present invention has been described herein
`
`above by way of preferred embodiments thereof, it can be modified, without
`
`departing from the spirit and nature of the subject invention as defined in the
`
`appended claims.
`
`0000023
`
`0000023
`
`

`
`CA 02251265 1998-10-21
`
`WHAT IS CLAIMED IS:
`
`1.
`
`A method for extracting lipids from an aquatic animal tissue
`
`comprising the steps of:
`
`a) suspending said animal aquatic tissue in an organic solvent;
`
`b) extracting lipids by successive organic solvent treatment;
`
`and
`
`c)
`
`collecting said lipids in a first fraction and an organic
`
`insoluble fraction.
`
`2.
`
`The method of claim 1, wherein said organic solvent of a) is
`
`acetone.
`
`3.
`
`The method of claim 1 or 2, wherein said organic solvent of b)
`
`is selected from at least one of acetone and alcohol.
`
`4.
`
`The method of claim 1, 2 or 3, wherein said organic insoluble
`
`fraction comprises a dry residue fraction which is enriched in protein.
`
`5.
`
`The method of claim 1, 2, 3 or 4, wherein said aquatic animal
`
`tissue is at least one tissue selected from the group consisting of krill tissue,
`
`Calanus tissue and fish tissue.
`
`6.
`
`A lipid extract obtained by the method of claim 2, 3, 4 or 5.
`
`22
`
`0000024
`
`0000024
`
`

`
`CA 02251265 1998-10-21
`
`7.
`
`8.
`
`A protein rich fraction obtained by the method of claim 4 or 5.
`
`A lipid extract having the properties in accordance with the
`
`present invention.
`
`23
`
`0000025
`
`0000025
`
`

`
`CA 02251265 1998-10-21
`
`CUNFIIJENTIEL
`: 98-03-24 20:09:39
`:
`7
`»
`: Chantal Beaudoin
`
`injecéion bate
`Sample Name
`Acq. Operator
`
`—
`seq. Line :
`1
`Vial
`:
`1
`Inj
`:
`Inj Volume : Manually
`
`: C:\HPCHEM\1\METHODS\ALAIN2.M
`Method
`: 98-03-24 19:56:07
`by Chantal Beaudoin
`Last changed
`(modified after loading)
`'
`Méthode corrigée lors de l'installation de la nouvelle colonne 12 septembre
`1997. Température du four 170 degré C et purge flow = 150 ml/min. Flux dans
`la colonne : 4,0 ml/min. Augmentation de la temperature a 175 degré C et
`le
`pur e flow est descendu a 140 ml/min,
`le 13 mars 1998.
`FID1 A, of GENOOOOZD
`
`pA:;:-
`‘_'
`
`_
`
`:1
`
`5.570
`
`28.287
`
`
`
`
`
`»-22.00020:4(41.10.1415)MAW
`
`‘.19:fifi$[g%hmn)
`
`wan2&2
`
`v25.247
`
`Mzu
`
`>4o5s5
`
`49721
`
`)55373
`
`
`
`__LIZ:::——52225
`
`Figure 1: Gas-liquid chromatography of fatty acids from dry krill (chloroform-
`methanol).
`
`CONFIDFNl‘IEI.
`
`0000026
`
`I ;
`
`I
`
`C
`
`i I i a
`
`0000026
`
`

`
`,'
`
`,
`
`Injection Date
`Sample Name
`Acq. Operator
`
`CA 02251265 1- 8110-21
`WM‘ M 111111:
`_
`: 98-03-25 20:00:46 -
`: 11
`: Chantal Beaudoin
`
`_-----_-_
`—
`Seq. Line :
`1
`Vial
`:
`1
`Inj
`:
`Inj Volume : Manually
`
`Method
`
`: C:\HPCHEM\1\METHODS\ALAIN2.M
`
`Last changed
`
`by Chantal Beaudoin
`: 98-03-25 18:55:58
`(modified after loading)
`Méthode corrigée lors de l'installation de la nouvelle colonne 12 septembre
`1997. Temperature du four 170 degré C et purge flow = 150 ml/min. Flux dans
`la colonne : 4,0 ml/min. Augmentation de la temperature a 175 degré C et
`le
`purge flow est descendu a 140 ml/min,
`le 13 mars 1998.
`FlD1A. otGENOOO05.D
`
`:1?
`
` >22565-20:4
`(6.10,14,16)
`
`Figure 2: Gas-liquid chromatography of fatty acids from dry krill (acetone)
`
`CONFIDENTIEL
`
`
`
`0000027
`
`0000027
`
`

`
`'
`
`CA 02251265 1998-10-21
`
`CONFIDENTIEL
`
`Injection Date
`Sample Name
`Acq. Operator
`
`: 98-04-01 18:48:05
`: 26
`: Chantal Beaudoin
`
`—
`Seq. Line :
`1
`Vial
`:
`1
`Inj
`:
`Inj Volume : Manually
`
`Method
`Last changed
`
`: C:\HPCHEM\1\METHODS\ALAIN2.M
`: 98-04-01 18:45:50
`by Chantal Beaudoin
`(modified after loading)
`Méthode corrigée lors de l'insta11ation de la nouvelle colonne 12 septembre
`1997. Temperature du four 170 degré C et purge flow = 150 ml/min. Flux dans
`la colonne : 4,0 ml/min. Augmentation de la temperature a 175 degré C et
`le
`ourge flow est descendu a 140 ml/min,
`le 13 mars 1998.
`FID1 A ofGENOO0O8.D
`
`
`
`1
`55%
`1
`1
`50%
`3
`453'
`o
`403
`_
`v‘
`t
`7-.3;
`._
`3
`3
`:
`=
`0
`2
`351
`”
`3.
`1
`h§¢_-an;
`9"‘:
`3:
`§ 28-?
`3°}
`..
`:
`"
`.a
`.2
`mm ::
`1.
`251 1
`'-."*0<9 ‘° d
`
`130-
`‘V N
`i
`~
`.
`"' ' A
`1
`20-‘
`
`I
`
`
`
`'
`
`s
`3
`i
`x
`§
`3
`I
`1
`f
`I
`5
`:
`3
`:
`!
`-'
`E
`I
`
`3
`N
`g
`IA‘;
`Z
`f‘
`1
`
`I
`
`'
`
`
`
`Figure 3: Gas—liquid chromatography of fatty acids from frozen krill (acetone).
`
`CONFIDENHEI.
`
`
`
`0000028
`
`0000028
`
`

`
`(
`
`‘
`
`"
`
`‘
`
`CA 02251265 1998-10-21
`
`UUNIWENTIEL
`
`Injection Date
`Sample Name
`Acq. Operator
`
`: 98~04—O2 17:35:45
`: 28
`: Chantal Beaudoin
`
`-
`Seq. Line :
`1
`Vial
`:
`1
`Inj
`:
`Inj Volume : Manually
`
`Method
`Last changed
`
`: C: \HPCHEM\l\METHODS\AI_.AIN2.M
`: 98~04—O2 17:28:39
`by Chantal Beaudoin
`(modified after loading)
`Méthode corrigée lors de l'installation de la nouvelle colonne 12 septembre
`1997. Temperature du four 170 degré C et purge flow = 150 ml/min. Flux dans
`la colonne : 4,0 ml/min. Augmentation de la temperature a 175 degré C et
`le
`.purge flow est descendu a 140 ml/min,
`le 13 mars 1998.
`Fm1A:uasmmwan
`.
`
`
`
`
`
` _21.523-20:4(s.1o,14,1a) 2L300-204
`
`32::-25.096
`26457
`
`
`Figure 4: Gas-liquid chromatography of fatty acids from frozen kn'l| (ethanol).
`
`CONFIDENTIEL
`
`
`
`0000029
`
`0000029
`
`

`
`
`
`
`
`$157.4!...".(.....#:..<.
`
`PA
`
`.n22.0
`
`1
`
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`
`.2.
`
`56
`
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`
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`
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`
`.2.»>5.:.A....T
`
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`
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`j of neutqal I
`ipid$__ pg Qalan
`£9810
`_ as g;
`(ethlfiol
`) sample of other
`9'esi7é‘r‘6I
`interést, ch
`e.22;ri
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`ice
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`
`0000030
`
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`
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`
`0000031
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`
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`
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`
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`:2, vlv)
`
`(acetone); -7-
`
`0000032
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`vi.)rh..~uh.r.V.rlr|rL.lKKo.r|tP~or>.F..»r.v«‘LE
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`
`CA 02251265 1998-10-21
`
`In
`
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`
`no
`
`‘(us-«I :13!“ um: I no ‘-/.) ppm
`
`0000034
`
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`
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`
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`
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`
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`
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`
`on9.2.2onRonm_
`
`CA 02251265 1998-10-21
`
`N
`
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`
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`
`m-o
`
`‘(tyne uman I19-IJ 3 no ‘-/.) ppm
`
`0000035
`
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`
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`
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`
`
`
`
`
`
`
`CA 02251265 1998- 10-21
`
`2.
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`
`8.82S2.on2
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`
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`
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`
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`
`0000036
`
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`
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`
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`
`
`
`
`
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`
`0000036
`
`

`
`3.
`
`2:2.on3
`
`CA 02251255 1993-10-21
`
`T'
`
`
`
`Aaofioam.5ZO=.U<~—.—.Nn.—.=.:._Z0aoz<E.mE0m:5._o>HEBmomuzn5amz_.§....=S._:...
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`
`0000037
`
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