CA2346979C - Method of extracting lipids from marine and aquatic animal tissues - Google Patents
Method of extracting lipids from marine and aquatic animal tissues Download PDFInfo
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- CA2346979C CA2346979C CA002346979A CA2346979A CA2346979C CA 2346979 C CA2346979 C CA 2346979C CA 002346979 A CA002346979 A CA 002346979A CA 2346979 A CA2346979 A CA 2346979A CA 2346979 C CA2346979 C CA 2346979C
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
- C11B1/10—Production of fats or fatty oils from raw materials by extracting
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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.
Description
TlTLE OF THE INVENTION
METHOD OF EXTRACTING LIPIDS FROM MARINE AND AQUATIC
ANlMAL TISSUES
FIELD OF THE INVENTION
This invention relates to the extraction of lipid fracdons from marine and aquatic animals such as krill, Calanus, fish and sea mammals. More specificaliy, this invention relates to an improved method of extracting lipid fracpons by dehydration with solvents and recovering a solid residue rich In active enzymes.
BACKGROUND OF THE INVENTION
Lipid fractions obtained from marine and aquafic animals such as kriif, Ca/anus, fish and sea mammals have various appiications:
Medical applications Marine and aquatic animal oiis 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 aquabc animal oils are reported as suppressing the deveiopment of certain forms of lupus and renal diseases. As a further example, krili may be used as a source of enzymes for debridement of ulcers and wounds or to facditate fooq digestion.
Also marine and aquatic oils contain various antioxidants, which may have potential therapeutic properties.
Nutraceuticais Considering the benefiaai efterts of omega-3 fatty acids, oils fnxn krfU, Calanus and fish could be used as dietary supplements to human diet. These fatty acids are essential for proper development of the brain and the eye. Marine and aquatic animal oils are also rich in liposoluble vitamins A, D and E and carotenoids.
Cosmetics AMENDED SHEET
Lw_ ; Prir~~~d 1~O.~r2f1Q1 ~
CA 02346979 2001-04-12~
la Various marine and aquatic animat oils are used for itus proquction of moisturizing creams.
.E..~ .
~~ ~ N AMENDED SHEET
METHOD OF EXTRACTING LIPIDS FROM MARINE AND AQUATIC
ANlMAL TISSUES
FIELD OF THE INVENTION
This invention relates to the extraction of lipid fracdons from marine and aquatic animals such as krill, Calanus, fish and sea mammals. More specificaliy, this invention relates to an improved method of extracting lipid fracpons by dehydration with solvents and recovering a solid residue rich In active enzymes.
BACKGROUND OF THE INVENTION
Lipid fractions obtained from marine and aquafic animals such as kriif, Ca/anus, fish and sea mammals have various appiications:
Medical applications Marine and aquatic animal oiis 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 aquabc animal oils are reported as suppressing the deveiopment of certain forms of lupus and renal diseases. As a further example, krili may be used as a source of enzymes for debridement of ulcers and wounds or to facditate fooq digestion.
Also marine and aquatic oils contain various antioxidants, which may have potential therapeutic properties.
Nutraceuticais Considering the benefiaai efterts of omega-3 fatty acids, oils fnxn krfU, Calanus and fish could be used as dietary supplements to human diet. These fatty acids are essential for proper development of the brain and the eye. Marine and aquatic animal oils are also rich in liposoluble vitamins A, D and E and carotenoids.
Cosmetics AMENDED SHEET
Lw_ ; Prir~~~d 1~O.~r2f1Q1 ~
CA 02346979 2001-04-12~
la Various marine and aquatic animat oils are used for itus proquction of moisturizing creams.
.E..~ .
~~ ~ N AMENDED SHEET
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 ornega-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 known to extract fish oil using organic solvents such as hexane and ethanol. It is also known to measure the fat content in fish muscle tissue using '! 5 solvents such as acetone.
USP 4,331,695 describes a method using pressurized solvents which are gaseous at room temperature, such as propane, butane or hexane. The extraction is performed at preferred temperatures of 15 to 80 C on shredded vegetable or finely ;20 divided animal products. The extracted oils are then made to precipitate under high pressure and elevated ternperatures of 50 to 200 C. However, hexane is a poor extraction solvent for marine animals such as krill. Furthermore, the high temperatures used in the precipitation step negatively alters the lipids.
:25 Canadian Patent Application 2,115,571 describes a method for extracting oils from various brown and read algae species. The method provides for example Soxhlet extraction using nearly pure ethanol for 40 hours.
USP 5,006,281 describes a method for extracting oil from marine and aquatic 30 animals such as fish. The marine and aquatic animal is first treated with an antioxidant compound, finely divided and centrifuged to separate the oil phase from the aqueous phase and solid phase. The oil phase is then further treated with antioxidant to remove undesirable odour or taste.
Canadian Patent 1,098,900 describes a method for extracting oils from krill.
The 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 method for the 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 andi 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
Figure 1. Gas-liquid chiroma-tography of fatty acids from dry krill (chloroform-methanol) Figure 2. Gas-liquid chromatography of fatty acids from dry krill (acetone) Figure 3. Gas-liquid chromatography of fatty acids from frozen krill (acetone) Figure 4. Gas-liquid chromatography of fatty acids from frozen krill (ethanol) Figure 5. Gas-liquid chromatography of fatty acids from frozen krill (t-butanol) Figure 6. Gas-liquid chromatography of fatty acids from frozen krill (ethyl acetate) Figure 7. Thin-layer chromatography of neutral lipids of Calanus sp. and M. norvegica Figure 8. Thin-layer chromatography of neutral lipids of E. pacifica Figure 9. Thin-layer chromatography of neutral lipids of M. schmitti Figure 10. Thin-layer chrornatography of neutral lipids of G. galeus Figure 11. Thin-layer chrornatography of neutral lipids of Angel Shark Figure 12. Thin-layer chrornatography of phospholipids of Calanus sp. and M. norvegica Figure 13. Thin-layer chrornatography of phospholipids of E. pacifica Figure 14. Thin-layer chrornatography of phospholipids of M. schmitti Figure 15. Thin-layer chrornatography of phospholipids of G. ga/eus Figure 16. Thin-layer chrornatography of phospholipids of Angel Shark Figure 17. Influence of the volume of acetone on lipid extraction (E.
pacifica) Figure 18. Influence of incubation time in acetone on lipid extraction (E. pacifica) Figure 19. Influence of the volume of ethanol on lipid extraction (E.
pacifica) Figure 20. Influence of incubation time in ethanol on lipid extraction (T. raschii) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Before describing the present invention in detail, it is to be understood that the invention is not limited in its application to the process details described herein. The invention is capable of other embodiments and of being practised in various ways.
It is also to be understood that the phraseology or terminology used herein is for the purpose of description anci 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 alcohols are isopropanol, and t-butanol. The alcohol may also be substituted with an ester of acetic acid such as ethyl acetate.
The procedure produces two successive lipid fractions and a dry residue enriched in 5 protein, including active enzymes. Recovery of total lipids is comparable to the Folch et al. (1957) procedure reported in the background of the invention. It has been tested with krill, Ca/anus, fish and shark tissues.
Surprisingly, it was found that successive extraction treatments as proposed by the present invention has a better yield in lipid extraction that single solvent system extractions. The extraction using two successive solvents which starts with a ketone such as acetone is especially advantageous since the acetone, in effect, dehydrates the animal tissue. Having the animal tissue in dehydrated form greatly facilitates the extraction process with the second solvent, alcohol or an ester of acetic acid such as ethyl acetate.
In the case of zooplankton such as krill and Calanus and in the case of fish-filleting by-products such as fish viscera, it is noted that extraction with acetone alone may be sufficient to allow a cost-effective recovery of lipid fractions and separate recovery 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 time is not critical to the yield of lipid extraction. To facilitate extraction, it is preferable to use particles of less than 5mm in diameter. Extraction is preferably conducted under inert atmosphere and at a temperature in the order of about 5 C or less.
Preferably, the beginning of the extraction will be conducted under agitation for about 10 to 40 minutes, preferably 20 minutes. Although extraction time is not critical, it 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 preferably useci.
After separation by filtration on an organic solvent resistant filter (metal, glass or paper) the residue is optionally washed with pure acetone, preferably two volumes (original volume of material) to recover yet more lipids. The combined filtrates are evaporated under reduced pressure. Optionally, flash evaporation or spray drying may be used. The water residue obtained after evaporation is allowed to separate from the oil phase (fraction I) at low temperature.
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 volurrie of material). The filtrate is evaporated leaving a second fraction of lipids (identifiecl as fraction II). Although the extraction period is not critical, it was found that an extraction time of about 30 minutes is sufficient at temperatures below about 5 C.
Temperature of the orgariic solvents, except t-butanol, and temperature of the sample are not critical parameters, but it is preferable to be as cold as possible.
However, in the case of f-butanol which is solid at room temperature, it is important to warm it before using it and to perform the extraction at 25 C immediately.
Comparative examples To compare the efficiency of the extraction process, a classical technique (Foich et al. 1957) using chloroforrri and methanol was applied to krill. This method is the reference for measuring efficiency of the extraction process. Another 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 pnosent invention invoiving acetone extraction followed by extracbon with a second solvent (ethyl acetate, for example) gave a translucent oil havinp appearance and properties more attractive than any oil obtained by the classical tecnnique of Folch et al. (1957).
To analyze lipid aamposition. 780 Ng of each extract was loaded on afl'ica-gel pMates and fractionated by thin layer chromatography, TLC (Bowyer et al. 1962) with the following solvents. Neutral lipids: hsxane, ethyl ether. aosticc acid (90:10:1, v/v) and phospholipids: chlorofonn, methand, water (80:25:2, v/v). Fatty acid camposiUon of E. pacifica was analyzed by gas liquid chromatography, GLC (Bowyer et al.
1962) including some modifications to the origlnal technique: 2h at 65 C instead of 1h at 80 C, three washes with hexane instead of two and no wash with water_ To get rid of traees of organic solvent$, lipid fraictions I and !I are warmed to about 125 C for about 15 minutes under inert atmosphere.
Fat was analyzed according to the American Oil ChemiBt's Society (AOCS). The following criteria have been used to analyze the lipids extracted:
saponification and Wijs iodine indexes and moisture-volatile matter levels. Cholesterol corltent has also been determined by the method of Plummer 1987. The same anayyzes and others have been made by an independent laboratory under Professor Robert Ackman's supervision (Canadian Institute of Fisheries Technology, palTech, Dalhousie University. Halifax, Nova Scotia, Canada). This includes Wijs iodine index, peroxide and anisidine values, lipid class c:omposition, fatty acid composition, free fatty acid FAME, cholesterol, tocopherol, aN-trans retinol. cholecalcderol, astaxanthin and canthaxantin contents.
r~ ~~ x ~ AMENDED SHEET
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 ornega-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 known to extract fish oil using organic solvents such as hexane and ethanol. It is also known to measure the fat content in fish muscle tissue using '! 5 solvents such as acetone.
USP 4,331,695 describes a method using pressurized solvents which are gaseous at room temperature, such as propane, butane or hexane. The extraction is performed at preferred temperatures of 15 to 80 C on shredded vegetable or finely ;20 divided animal products. The extracted oils are then made to precipitate under high pressure and elevated ternperatures of 50 to 200 C. However, hexane is a poor extraction solvent for marine animals such as krill. Furthermore, the high temperatures used in the precipitation step negatively alters the lipids.
:25 Canadian Patent Application 2,115,571 describes a method for extracting oils from various brown and read algae species. The method provides for example Soxhlet extraction using nearly pure ethanol for 40 hours.
USP 5,006,281 describes a method for extracting oil from marine and aquatic 30 animals such as fish. The marine and aquatic animal is first treated with an antioxidant compound, finely divided and centrifuged to separate the oil phase from the aqueous phase and solid phase. The oil phase is then further treated with antioxidant to remove undesirable odour or taste.
Canadian Patent 1,098,900 describes a method for extracting oils from krill.
The 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 method for the 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 andi 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
Figure 1. Gas-liquid chiroma-tography of fatty acids from dry krill (chloroform-methanol) Figure 2. Gas-liquid chromatography of fatty acids from dry krill (acetone) Figure 3. Gas-liquid chromatography of fatty acids from frozen krill (acetone) Figure 4. Gas-liquid chromatography of fatty acids from frozen krill (ethanol) Figure 5. Gas-liquid chromatography of fatty acids from frozen krill (t-butanol) Figure 6. Gas-liquid chromatography of fatty acids from frozen krill (ethyl acetate) Figure 7. Thin-layer chromatography of neutral lipids of Calanus sp. and M. norvegica Figure 8. Thin-layer chromatography of neutral lipids of E. pacifica Figure 9. Thin-layer chromatography of neutral lipids of M. schmitti Figure 10. Thin-layer chrornatography of neutral lipids of G. galeus Figure 11. Thin-layer chrornatography of neutral lipids of Angel Shark Figure 12. Thin-layer chrornatography of phospholipids of Calanus sp. and M. norvegica Figure 13. Thin-layer chrornatography of phospholipids of E. pacifica Figure 14. Thin-layer chrornatography of phospholipids of M. schmitti Figure 15. Thin-layer chrornatography of phospholipids of G. ga/eus Figure 16. Thin-layer chrornatography of phospholipids of Angel Shark Figure 17. Influence of the volume of acetone on lipid extraction (E.
pacifica) Figure 18. Influence of incubation time in acetone on lipid extraction (E. pacifica) Figure 19. Influence of the volume of ethanol on lipid extraction (E.
pacifica) Figure 20. Influence of incubation time in ethanol on lipid extraction (T. raschii) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
Before describing the present invention in detail, it is to be understood that the invention is not limited in its application to the process details described herein. The invention is capable of other embodiments and of being practised in various ways.
It is also to be understood that the phraseology or terminology used herein is for the purpose of description anci 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 alcohols are isopropanol, and t-butanol. The alcohol may also be substituted with an ester of acetic acid such as ethyl acetate.
The procedure produces two successive lipid fractions and a dry residue enriched in 5 protein, including active enzymes. Recovery of total lipids is comparable to the Folch et al. (1957) procedure reported in the background of the invention. It has been tested with krill, Ca/anus, fish and shark tissues.
Surprisingly, it was found that successive extraction treatments as proposed by the present invention has a better yield in lipid extraction that single solvent system extractions. The extraction using two successive solvents which starts with a ketone such as acetone is especially advantageous since the acetone, in effect, dehydrates the animal tissue. Having the animal tissue in dehydrated form greatly facilitates the extraction process with the second solvent, alcohol or an ester of acetic acid such as ethyl acetate.
In the case of zooplankton such as krill and Calanus and in the case of fish-filleting by-products such as fish viscera, it is noted that extraction with acetone alone may be sufficient to allow a cost-effective recovery of lipid fractions and separate recovery 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 time is not critical to the yield of lipid extraction. To facilitate extraction, it is preferable to use particles of less than 5mm in diameter. Extraction is preferably conducted under inert atmosphere and at a temperature in the order of about 5 C or less.
Preferably, the beginning of the extraction will be conducted under agitation for about 10 to 40 minutes, preferably 20 minutes. Although extraction time is not critical, it 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 preferably useci.
After separation by filtration on an organic solvent resistant filter (metal, glass or paper) the residue is optionally washed with pure acetone, preferably two volumes (original volume of material) to recover yet more lipids. The combined filtrates are evaporated under reduced pressure. Optionally, flash evaporation or spray drying may be used. The water residue obtained after evaporation is allowed to separate from the oil phase (fraction I) at low temperature.
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 volurrie of material). The filtrate is evaporated leaving a second fraction of lipids (identifiecl as fraction II). Although the extraction period is not critical, it was found that an extraction time of about 30 minutes is sufficient at temperatures below about 5 C.
Temperature of the orgariic solvents, except t-butanol, and temperature of the sample are not critical parameters, but it is preferable to be as cold as possible.
However, in the case of f-butanol which is solid at room temperature, it is important to warm it before using it and to perform the extraction at 25 C immediately.
Comparative examples To compare the efficiency of the extraction process, a classical technique (Foich et al. 1957) using chloroforrri and methanol was applied to krill. This method is the reference for measuring efficiency of the extraction process. Another 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 pnosent invention invoiving acetone extraction followed by extracbon with a second solvent (ethyl acetate, for example) gave a translucent oil havinp appearance and properties more attractive than any oil obtained by the classical tecnnique of Folch et al. (1957).
To analyze lipid aamposition. 780 Ng of each extract was loaded on afl'ica-gel pMates and fractionated by thin layer chromatography, TLC (Bowyer et al. 1962) with the following solvents. Neutral lipids: hsxane, ethyl ether. aosticc acid (90:10:1, v/v) and phospholipids: chlorofonn, methand, water (80:25:2, v/v). Fatty acid camposiUon of E. pacifica was analyzed by gas liquid chromatography, GLC (Bowyer et al.
1962) including some modifications to the origlnal technique: 2h at 65 C instead of 1h at 80 C, three washes with hexane instead of two and no wash with water_ To get rid of traees of organic solvent$, lipid fraictions I and !I are warmed to about 125 C for about 15 minutes under inert atmosphere.
Fat was analyzed according to the American Oil ChemiBt's Society (AOCS). The following criteria have been used to analyze the lipids extracted:
saponification and Wijs iodine indexes and moisture-volatile matter levels. Cholesterol corltent has also been determined by the method of Plummer 1987. The same anayyzes and others have been made by an independent laboratory under Professor Robert Ackman's supervision (Canadian Institute of Fisheries Technology, palTech, Dalhousie University. Halifax, Nova Scotia, Canada). This includes Wijs iodine index, peroxide and anisidine values, lipid class c:omposition, fatty acid composition, free fatty acid FAME, cholesterol, tocopherol, aN-trans retinol. cholecalcderol, astaxanthin and canthaxantin contents.
r~ ~~ x ~ AMENDED SHEET
Table I shows that higl-ier levels of lipids are extracted from dry krill by acetone followed by ethanol as compared to the classical procedure of Folch et al.
(1957).
Table 2 shows the results of lipid extraction from frozen Euphausia pacifica, a 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 acetone extractions are mainly due to the water-oil separations. These separations are influenced by the quantity of residual acetone in the water-oil solution after acetone evaporation. Th'is quantity of acetone varies from an experiment 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, compared to acetone alone (1,86% extraction rate, and even greater extraction rates with an efficient acetone evaporation system).
One of the main advantages of the procedure is the removal of bacteria from extracts (lipid fraction and solicl protein-rich material). Indeed, samples of E.
pacifica incubated in different ratios of acetone at 4 C for 112 days have been inoculated on NA medium containing BactoTM beef extract 0,3%, BactoTM peptone 0,5% and BactoTM
agar 1,5% (Difco Laboratories, Detroit, USA) then incubated at room temperature or 4 C for 18 days. No sigriificant bacterial growth was observed at a ratio of 1 volume of acetone per gram of krill. At higher proportions of acetone (2 voiumes and 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).
(1957).
Table 2 shows the 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 acetone extractions are mainly due to the water-oil separations. These separations are influenced by the quantity of residual acetone in the water-oil solution after acetone evaporation. Th'is quantity of acetone varies from an experiment 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, compared to acetone alone (1,86% extraction rate, and even greater extraction rates with an efficient acetone evaporation system).
One of the main advantages of the procedure is the removal of bacteria from extracts (lipid fraction and solicl protein-rich material). Indeed, samples of E.
pacifica incubated in different ratios of acetone at 4 C for 112 days have been inoculated on NA medium containing BactoTM beef extract 0,3%, BactoTM peptone 0,5% and BactoTM
agar 1,5% (Difco Laboratories, Detroit, USA) then incubated at room temperature or 4 C for 18 days. No sigriificant bacterial growth was observed at a ratio of 1 volume of acetone per gram of krill. At higher proportions of acetone (2 voiumes and 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 shows the yield of lipids from M. norvegica. The percentage of lipids (3,67%) is comparable to the one obtained with E. pacifica (3,11%) shown in Table 2.
Variations can be attributable to diet and time (season) of collection, which are different for those two species.
Table 4 shows the influence of grinding on the efficiency of extraction of M.
norvegica lipids. These extractions were carried out under optimal conditions and show the definite advantage of the procedure over the classical method (4,46 %
versus 3,30 %). It also shows that grinding may be an important factor when the species is large (4,46% versus 3,53 %).
Table 5 reports on lipid extraction from Calanus. Considerable quantities of lipids were obtained. Some variations in Calanus species composition may explain the variations between experinients 1 and 2 (8,22 % and 10,90 % of fresh weight).
Tables 6-8 report the total amount of lipids extracted from fish tissue. The method of the present invention was demonstrated on mackerel, trout and herring. The method was demonstrated on peripheral tissues (mainly muscles) and viscera.
Advantageously, the present method would permit the recovery of valuable lipid fractions from parts of fish that are usually wasted after the withdrawal of fillets of the fish. Those fish tissues not used after the transformation of the fish for human consumption could be stored in acetone, and lipids extracted therefrom in accordance with the present invention even if the method Folch [1957] recovers more lipid than our methocl. Indeed small amounts of lipids from mackerel (0.52%
12 5 from viscera and 1,45% from tissues) have been extracted by the method of Folch after a first extraction with acetone and ethanol as described in the present invention.
Comparative extractions with the method described in the present invention carried out in parallel with the method of Folch on trout and herring show superior recovery with the latter. However, it is noteworthy that the Folch method can not be applied for the recovery of lipids for commercial uses (because of toxicity).
II VAL LL .1L. f,.~ ,rpJõa.. ~
CA 02346979 2001 LVVt 04 12 D ,~, ~
--- -----_ __ ----- : ...., 47 .
In Tables 9 to 11, are shown results of lipids extraction from shark liver tissues.
There is no marked difference in results between technlques within a species.
Tables 12 shows some characteristics features of fraction I(ac:etone) and fraction iI (alcohol or ethyl acetate) for krill oil (e. pacifica). First, the saponificabon index of fraction 1(130,6) indicates that this frac#ion contains fatty acids wiih longer chains, compared to fraction 11 (185,7). The Wijs iodine index of fraction I shows that this 10 fraction contains high levels of polyunsaturated fatty acids. As compared to olive oil which has an index of 81.1. it explains why fraction I is liquid at room temperature.
It is well known that unsaturated fatty acids have a fusion point inferior to the one of their saturated homologues. The same observations are made for fracxion II
which has a iodine inbex of 127,2. The fatty acid composidon shown in Table 14 corroborates these iodine indexes: frection I has a high percentage (30,24%) of polyunsaturated fatty acids (pentaenes+hexaenes) and so fraCtion 11 (22,98%).
Finally, Table 12 shows also that fnsction I is comprised of 10.0% of volatile matEer and humidity after evaporation of the $olvent. For the same test, the fraction II gives a value of 6,8%. To get rid of traces of solvents, it is important to briefly heat (to about 125 C, for about 15 min) the oil under nitrogen.
Results on kriil 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 measuneci in terms of two carotenolds namely astaxanthin and canthaxanthin. Indeed, duplicates analyzes revealed values of 92 to 124 Ng/g of Opid fraction for astaxanthin and 262 to 734 pglg 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 uglg of lipid fraction. In the case of canthaxanthin, at least 250 and preferably at least 270 pg/g of lipid fracdon. Low values for peroxide and anisidine are advantageous and are due to the presence of high levels of natural antioxidants (astaxanthin and Printed:4 2-01-2001 AN-ENDED SHEET
. ~. v i .w L a. . - !.. . -4 I .l CA 02346979 2 sd001 04 12 ~;=~
canthaxanthin). These compounds are indicative of favourable pharmaceutical or cosmetological properties of the kryq extract whereby high levels of carotenoids indicate excellent transdermal migration characteristics. Thus, krili extract is a good candidative for transdermal delivery of inedicines.
Table 18 shows the best mode of the method in accordance with the present invention for lipid extracdon of aquatic animal tissues.
Table 19 shows that the enzyme act'rvity of the solid fraction is msintained following the method of the present invenoon. Indeed, the demonstration was completed for solid krilf residue obtained after successive acetone and ethyl acetate extraction.
Proteotytic activipes were measure by the liberation of amino groups by spectrophotometric assay using o-pthaldialdehyda as reagent. Protein conoentraitions were measured by the Bradford method. Soluble proteins were extracted with water and added to a 10% lactoserum protein concentrate obtained by uhrafittration. At the end of incubation at 37 C in 50mM potassium phosphate buffer, trichloroacetic acid was added and the amount of NH, group was measured in the supematant according to the method of Church et ai. [1983, J Dairy Sci 86:
1219-1221.
Figures 1 to 6 show chromatogmms of fatty acid composition of E. pacifica dplds. On each of them, high proportions of 20:5 and 22:6 fatty acids (characpenstic of marine and aquatic oils) are noticeable and represented py two distinct peaks.
Variations in lipid pattems of neutral lipids (from Figure 7 to Figure 11) from one species to another are attributable to the differences in food sources. Within a species (E. pacifica, for example) there is no marked variation between lipid patterns obtained from different techniques of lipid extraction. Concerning phospholipids (Figure 12 to Figure 16), the opposite is observed: variations are explained by the different extraction processes of lipids since the same species do not lead to the same lipid pattern. Lipids from shark species (extracted by the mentioned methods) AFMENl:iED SHEET
and commercial cod-liver oil (sample available from Uniprix drugstores, Province of Quebec, Canada) are mainly composed of neutral lipids as opposed to phospholipids.
The influence of the volume of solvent and incubation time on the efficiency of the acetone to extract lipids from E. pacifica is illustrated in Figures 17 and 18, respectively. A ratio of 1:6 (w/v) produced optimal yield with near complete extraction after 2h. The second extraction step has been experimented with ethanol. The volume of this solvent does not appear to be critical since the same yield was obtained with different volumes of ethanol (Figure 19), but incubations time in ethanol should be at least 30 minutes as indicated by the results on Figure 20.
One of the inventors, Dr. Adrien Beaudoin, has ingested the different lipid fractions of krill. No side effect profile was observed.
Although the invention has been described above with respect with one specific form, it will be evident to a person skilled in the art that it may be modified and refined in various ways. It is therefore wished to have it understood that the present invention should not be limited in scope, except by the terms of the following claims.
Demonstration that krill residue, obtained after acetone and ethyl acetate extraction, contains enzyme proteolytic activities. Proteolytic activities were measured by the liberation of amino groups by spectrophotometric assay using o-phthaldialdehyde as reagent. Protein concentrations were measured by the Bradford method.
The enzyme source was the residue obtained after acetone and ethyl acetate extractions of lipids. Soluble proteins were extracted with water and added to a 10%
lactoserum protein concentrate obtained by ultrafiltration.
At the end of incubation at 37 C in 50 mM potassium phosphate buffer, trichloroacetic acid was added and the amount of NH3 groups were measured in the supernatant according to Church and al. 1983.
BIBLIOGRAPHY
Bowyer, D.E., Leat, W.M.F., Howard, A.N. and Gresham, G.A. 1962. The determination of the fatty acid composition of serum lipids separated by thin-layer chromatography; and a cornparison with column chromatography. BBA. 70: 423-431.
Chandrasekar, B., Troyer, D.A., Venkatraman, J.T. and Fernandes, G. 1996.
Tissue specific regulation of transforming growth factor beta by omega-3 lipid-rich krill oil in autoimmune murine lupus. Nutr Res. 16(3): 489-503.
'10 Christensen, M.S., Hoy, C-E. and Redgrave, T.G. 1994. Lymphatic absorption of n-3 polyunsaturated fatty acids from marine oils with different intramolecular fatty acid distributions. BBA. 1215: '198-204.
Church, F.C., Swaisgood, H.E., Porter, D.H. and Catignani, G.L. 1983.
Spectrophotometric assay using o- Phthaldialdehyde for determination of proteolysis in milk and isolated milk proteins. J Dairy Sci. 66: 1219-1227.
Difco laboratories. 1984. Difco Manual Dehydrated Culture Media and Reagents for Microbiology. 10t" ed. Detroit.
Folch, J., Lees, M. and Sloane-Stanley, G.H. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226: 497-509.
Goodman Gilman, A., Goodman, L.L. and Gilman, A. 1980. The Pharmacological Basis of Therapeutics. 6th' ed. Collier Macmillan Canada ltd, Toronto.
Harwood, H.J. and Geyer, R.P. 1964. Biology Data Book. The Federation of American Societies for Experimental Biology, Washington.
Hellgren, L., Karlstam, B., Mohr, V. and Vincent, J. 1991. Krill enzymes. A
new concept for efficient debridement of necrotic ulcers. Int J Dermatol. 30(2):
Plummer, D.T. 1987. An iritroduction to practical biochemistry. 3th ed. McGraw-Hill Book Company, London.
Rawn, J.D. 1990. Trait6 dE: biochimie. De Boeck-Wesmael, Bruxelles.
Runge, J.A. and Joly, P. 1994. Rapport sur 1'etat des invertebres en 1994: 7:0 Zooplancton (EuphausiacE;s et Calanus) de I'Estuaire et du Golfe du Saint-Laurent.
Sargent, J.R. 1997. Fish oils and human diet. Br J Nutr.78 Suppl 1: S5-S13.
'10 TABLE 1. EXTRACTION OF DRY KRILL LIPIDS (E. pacifica) Exp. No. Technigue Yield % Total % Mean (%) s.d.
5 1- acetone a) 8,00 ethanol b) 7,60 15,60 2- " 19,70 6,90 26,60 3- " 8,15 11,20 19,35 4- " 6,80 13,60 20,40 20,49 3,95 5- chlor: MeOH 15,50 6- " 14,90 15,20 0,30 Determinations in triplicates (variation < 5 %).
a) :Extraction made with a sample-solvent ratio of 1:9 (w/v), no incubation.
b) :Extraction made with a sample-solvent ratio of 1:4 (w/v), incubated 1 night at 4 C, following a first extraction with acetone.
`) :Folch et al. 1957.
TABLE 2. EXTRACTION OF FROZEN KRILL LIPIDS (E. pacifica) Exp. No. Technique Yield % Total % Mean (%) s.d.
1- acetone 1,17 ethanol b~' 1,23 2,40 2- " 3,05 1,09 4,14 3- " 1,53 1,26 2,79 3,11 0,91 4- acetone 2,45 isopropanol b) 0,70 3,15 5- " 1,80 0,80 2,60 6- " 1,60 0,80 2,40 2,72 0,39 TABLE 2 (continued). EXTRACTION OF FROZEN KRILL LIPIDS (E. pacifica) Exp. No. Technique Yield % Total % Mean (%) t s.d.
7- acetone a~ 2,15 t-butanol r~ 0,47 2,62 8- " 2,11 0,40 2,51 9- 2,37 0,45 2,82 2,65 0,16 10- acetone a~ 2,28 ethyl acetate b) 0,21 2,49 11- " 1,09 0,16 1,25 12- " 2,54 0,09 2,63 2,12 0,76 13- combinecl acetone-ethanol d) 3,28 14- " 3,02 15- " 3,25 3,18 0,14 16- ethyl acetate e) 1,32 17- " 1,49 18- " 1,31 1,37 0,10 19- hexane e) 0,31 20- " 0,18 21- " 0,20 0,23 0,07 22- chlor:MeOH 2,37 TABLE 2 (continued). EXTRACTION OF FROZEN KRILL LIPIDS (E. pacirica) Exp. No. Technique Yield % Total % Mean (%) t s.d.
Variations can be attributable to diet and time (season) of collection, which are different for those two species.
Table 4 shows the influence of grinding on the efficiency of extraction of M.
norvegica lipids. These extractions were carried out under optimal conditions and show the definite advantage of the procedure over the classical method (4,46 %
versus 3,30 %). It also shows that grinding may be an important factor when the species is large (4,46% versus 3,53 %).
Table 5 reports on lipid extraction from Calanus. Considerable quantities of lipids were obtained. Some variations in Calanus species composition may explain the variations between experinients 1 and 2 (8,22 % and 10,90 % of fresh weight).
Tables 6-8 report the total amount of lipids extracted from fish tissue. The method of the present invention was demonstrated on mackerel, trout and herring. The method was demonstrated on peripheral tissues (mainly muscles) and viscera.
Advantageously, the present method would permit the recovery of valuable lipid fractions from parts of fish that are usually wasted after the withdrawal of fillets of the fish. Those fish tissues not used after the transformation of the fish for human consumption could be stored in acetone, and lipids extracted therefrom in accordance with the present invention even if the method Folch [1957] recovers more lipid than our methocl. Indeed small amounts of lipids from mackerel (0.52%
12 5 from viscera and 1,45% from tissues) have been extracted by the method of Folch after a first extraction with acetone and ethanol as described in the present invention.
Comparative extractions with the method described in the present invention carried out in parallel with the method of Folch on trout and herring show superior recovery with the latter. However, it is noteworthy that the Folch method can not be applied for the recovery of lipids for commercial uses (because of toxicity).
II VAL LL .1L. f,.~ ,rpJõa.. ~
CA 02346979 2001 LVVt 04 12 D ,~, ~
--- -----_ __ ----- : ...., 47 .
In Tables 9 to 11, are shown results of lipids extraction from shark liver tissues.
There is no marked difference in results between technlques within a species.
Tables 12 shows some characteristics features of fraction I(ac:etone) and fraction iI (alcohol or ethyl acetate) for krill oil (e. pacifica). First, the saponificabon index of fraction 1(130,6) indicates that this frac#ion contains fatty acids wiih longer chains, compared to fraction 11 (185,7). The Wijs iodine index of fraction I shows that this 10 fraction contains high levels of polyunsaturated fatty acids. As compared to olive oil which has an index of 81.1. it explains why fraction I is liquid at room temperature.
It is well known that unsaturated fatty acids have a fusion point inferior to the one of their saturated homologues. The same observations are made for fracxion II
which has a iodine inbex of 127,2. The fatty acid composidon shown in Table 14 corroborates these iodine indexes: frection I has a high percentage (30,24%) of polyunsaturated fatty acids (pentaenes+hexaenes) and so fraCtion 11 (22,98%).
Finally, Table 12 shows also that fnsction I is comprised of 10.0% of volatile matEer and humidity after evaporation of the $olvent. For the same test, the fraction II gives a value of 6,8%. To get rid of traces of solvents, it is important to briefly heat (to about 125 C, for about 15 min) the oil under nitrogen.
Results on kriil 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 measuneci in terms of two carotenolds namely astaxanthin and canthaxanthin. Indeed, duplicates analyzes revealed values of 92 to 124 Ng/g of Opid fraction for astaxanthin and 262 to 734 pglg 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 uglg of lipid fraction. In the case of canthaxanthin, at least 250 and preferably at least 270 pg/g of lipid fracdon. Low values for peroxide and anisidine are advantageous and are due to the presence of high levels of natural antioxidants (astaxanthin and Printed:4 2-01-2001 AN-ENDED SHEET
. ~. v i .w L a. . - !.. . -4 I .l CA 02346979 2 sd001 04 12 ~;=~
canthaxanthin). These compounds are indicative of favourable pharmaceutical or cosmetological properties of the kryq extract whereby high levels of carotenoids indicate excellent transdermal migration characteristics. Thus, krili extract is a good candidative for transdermal delivery of inedicines.
Table 18 shows the best mode of the method in accordance with the present invention for lipid extracdon of aquatic animal tissues.
Table 19 shows that the enzyme act'rvity of the solid fraction is msintained following the method of the present invenoon. Indeed, the demonstration was completed for solid krilf residue obtained after successive acetone and ethyl acetate extraction.
Proteotytic activipes were measure by the liberation of amino groups by spectrophotometric assay using o-pthaldialdehyda as reagent. Protein conoentraitions were measured by the Bradford method. Soluble proteins were extracted with water and added to a 10% lactoserum protein concentrate obtained by uhrafittration. At the end of incubation at 37 C in 50mM potassium phosphate buffer, trichloroacetic acid was added and the amount of NH, group was measured in the supematant according to the method of Church et ai. [1983, J Dairy Sci 86:
1219-1221.
Figures 1 to 6 show chromatogmms of fatty acid composition of E. pacifica dplds. On each of them, high proportions of 20:5 and 22:6 fatty acids (characpenstic of marine and aquatic oils) are noticeable and represented py two distinct peaks.
Variations in lipid pattems of neutral lipids (from Figure 7 to Figure 11) from one species to another are attributable to the differences in food sources. Within a species (E. pacifica, for example) there is no marked variation between lipid patterns obtained from different techniques of lipid extraction. Concerning phospholipids (Figure 12 to Figure 16), the opposite is observed: variations are explained by the different extraction processes of lipids since the same species do not lead to the same lipid pattern. Lipids from shark species (extracted by the mentioned methods) AFMENl:iED SHEET
and commercial cod-liver oil (sample available from Uniprix drugstores, Province of Quebec, Canada) are mainly composed of neutral lipids as opposed to phospholipids.
The influence of the volume of solvent and incubation time on the efficiency of the acetone to extract lipids from E. pacifica is illustrated in Figures 17 and 18, respectively. A ratio of 1:6 (w/v) produced optimal yield with near complete extraction after 2h. The second extraction step has been experimented with ethanol. The volume of this solvent does not appear to be critical since the same yield was obtained with different volumes of ethanol (Figure 19), but incubations time in ethanol should be at least 30 minutes as indicated by the results on Figure 20.
One of the inventors, Dr. Adrien Beaudoin, has ingested the different lipid fractions of krill. No side effect profile was observed.
Although the invention has been described above with respect with one specific form, it will be evident to a person skilled in the art that it may be modified and refined in various ways. It is therefore wished to have it understood that the present invention should not be limited in scope, except by the terms of the following claims.
Demonstration that krill residue, obtained after acetone and ethyl acetate extraction, contains enzyme proteolytic activities. Proteolytic activities were measured by the liberation of amino groups by spectrophotometric assay using o-phthaldialdehyde as reagent. Protein concentrations were measured by the Bradford method.
The enzyme source was the residue obtained after acetone and ethyl acetate extractions of lipids. Soluble proteins were extracted with water and added to a 10%
lactoserum protein concentrate obtained by ultrafiltration.
At the end of incubation at 37 C in 50 mM potassium phosphate buffer, trichloroacetic acid was added and the amount of NH3 groups were measured in the supernatant according to Church and al. 1983.
BIBLIOGRAPHY
Bowyer, D.E., Leat, W.M.F., Howard, A.N. and Gresham, G.A. 1962. The determination of the fatty acid composition of serum lipids separated by thin-layer chromatography; and a cornparison with column chromatography. BBA. 70: 423-431.
Chandrasekar, B., Troyer, D.A., Venkatraman, J.T. and Fernandes, G. 1996.
Tissue specific regulation of transforming growth factor beta by omega-3 lipid-rich krill oil in autoimmune murine lupus. Nutr Res. 16(3): 489-503.
'10 Christensen, M.S., Hoy, C-E. and Redgrave, T.G. 1994. Lymphatic absorption of n-3 polyunsaturated fatty acids from marine oils with different intramolecular fatty acid distributions. BBA. 1215: '198-204.
Church, F.C., Swaisgood, H.E., Porter, D.H. and Catignani, G.L. 1983.
Spectrophotometric assay using o- Phthaldialdehyde for determination of proteolysis in milk and isolated milk proteins. J Dairy Sci. 66: 1219-1227.
Difco laboratories. 1984. Difco Manual Dehydrated Culture Media and Reagents for Microbiology. 10t" ed. Detroit.
Folch, J., Lees, M. and Sloane-Stanley, G.H. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. biol. Chem. 226: 497-509.
Goodman Gilman, A., Goodman, L.L. and Gilman, A. 1980. The Pharmacological Basis of Therapeutics. 6th' ed. Collier Macmillan Canada ltd, Toronto.
Harwood, H.J. and Geyer, R.P. 1964. Biology Data Book. The Federation of American Societies for Experimental Biology, Washington.
Hellgren, L., Karlstam, B., Mohr, V. and Vincent, J. 1991. Krill enzymes. A
new concept for efficient debridement of necrotic ulcers. Int J Dermatol. 30(2):
Plummer, D.T. 1987. An iritroduction to practical biochemistry. 3th ed. McGraw-Hill Book Company, London.
Rawn, J.D. 1990. Trait6 dE: biochimie. De Boeck-Wesmael, Bruxelles.
Runge, J.A. and Joly, P. 1994. Rapport sur 1'etat des invertebres en 1994: 7:0 Zooplancton (EuphausiacE;s et Calanus) de I'Estuaire et du Golfe du Saint-Laurent.
Sargent, J.R. 1997. Fish oils and human diet. Br J Nutr.78 Suppl 1: S5-S13.
'10 TABLE 1. EXTRACTION OF DRY KRILL LIPIDS (E. pacifica) Exp. No. Technigue Yield % Total % Mean (%) s.d.
5 1- acetone a) 8,00 ethanol b) 7,60 15,60 2- " 19,70 6,90 26,60 3- " 8,15 11,20 19,35 4- " 6,80 13,60 20,40 20,49 3,95 5- chlor: MeOH 15,50 6- " 14,90 15,20 0,30 Determinations in triplicates (variation < 5 %).
a) :Extraction made with a sample-solvent ratio of 1:9 (w/v), no incubation.
b) :Extraction made with a sample-solvent ratio of 1:4 (w/v), incubated 1 night at 4 C, following a first extraction with acetone.
`) :Folch et al. 1957.
TABLE 2. EXTRACTION OF FROZEN KRILL LIPIDS (E. pacifica) Exp. No. Technique Yield % Total % Mean (%) s.d.
1- acetone 1,17 ethanol b~' 1,23 2,40 2- " 3,05 1,09 4,14 3- " 1,53 1,26 2,79 3,11 0,91 4- acetone 2,45 isopropanol b) 0,70 3,15 5- " 1,80 0,80 2,60 6- " 1,60 0,80 2,40 2,72 0,39 TABLE 2 (continued). EXTRACTION OF FROZEN KRILL LIPIDS (E. pacifica) Exp. No. Technique Yield % Total % Mean (%) t s.d.
7- acetone a~ 2,15 t-butanol r~ 0,47 2,62 8- " 2,11 0,40 2,51 9- 2,37 0,45 2,82 2,65 0,16 10- acetone a~ 2,28 ethyl acetate b) 0,21 2,49 11- " 1,09 0,16 1,25 12- " 2,54 0,09 2,63 2,12 0,76 13- combinecl acetone-ethanol d) 3,28 14- " 3,02 15- " 3,25 3,18 0,14 16- ethyl acetate e) 1,32 17- " 1,49 18- " 1,31 1,37 0,10 19- hexane e) 0,31 20- " 0,18 21- " 0,20 0,23 0,07 22- chlor:MeOH 2,37 TABLE 2 (continued). EXTRACTION OF FROZEN KRILL LIPIDS (E. pacirica) Exp. No. Technique Yield % Total % Mean (%) t s.d.
23- " 2,07 24- " 2,62 2,35f0,28 Determinations in triplicates (variation < 5%).
a):Extraction made with a sample-solvent ratio of 1:6 (w/v), incubated 2 h at 4 C.
b) :Extraction made with a sample-solvent ratio of 1:2 (w/v), incubated 30 min at 4 C, following a first extraction with acetone.
C) :Extraction made with a sample-solvent ratio of 1:2 (w/v), incubated 30 min at 25 C, following a first extraction with acetone.
d) :Extraction made with a sampde-acetone-ethanol ratio of 1:5:5 (w/v/v), incubated 2 h at 4 C.
e) :Extraction made with a sample-solvent ratio of 1:9 (w/v), incubated 2 h at 4 C.
n: Folch et al. 1957.
TABLE 3. EXTRACTION OF FROZEN KRILL LIPIDS(M. norvegica) Exp. No. Technique Yield % Total % Mean (%) f s.d.
1- acetone'a) 1,82 ethanol b) 1,82 3,64 2- " 1,15 2,35 3,50 3- " 1,68 2,19 3,87 3,67f0,15 Determinations in triplicates (variation < 5%).
a) :Extraction made with a sample-solvent ratio of 1:9 (w/v), incubated 1 night at 4 C.
b) :Extraction made with a sample-solvent ratio of 1:4 (w/v), incubated 1 h at 4 C, following a first extraction with acetone.
TABLE 4. INFLUENCE OF GRINDING ON EXTRACTION OF FROZEN KRILL LIPIDS
(M. norvegica) Exp. No. Technique Krill ground before 1 stextraction Yield % Total %
1- acetone yes 3,10 ethanol b) 1,07 4,17 2- " no 2,14 1,39 3,53 3- " yes 3,32 1,14 4,46 4- chlor: MeOH yes 3,30 5- " yes 3,26 Determinations in triplicates (variation < 5 %).
a):Extraction made with a sample-solvent ratio of 1:6, incubated 2 h at 4 C.
b) :Extraction made with a sample-solvent ratio of 1:2, incubated 30 min at 4 C, following a first extraction with acetone.
`) :Folch et al. 1957.
TABLE 5. EXTRACTION OF FROZEN Calanus LIPIDS (Calanus sp.) Exp. No. Technigue Yield % Total % Mean (%) s.d.
1- acetone i3) 6,18 ethanol b' 2,04 8,22 2- " 8,64 2,26 10,90 9,56 1,34 Determinations in triplicates (variation < 5 %).
a) :Extraction made with a sample-solvent ratio of 1:9 (w/v), incubated 1 night at 4 C.
b) :Extraction made with a sample-solvent ratio of 1:4 (wlv), incubated 1 h at 4 C, following a first extraction with acetone.
TABLE 6. EXTRACTION OF FRESH FISH LIPIDS (Mackerel) Exp. No. Technigue Yield (%) Total (%L
1- viscera acetone a~ 6,11 fish 1 ethanol0,59 6,70 2- tissues " 3,78 fish 1 0,91 4,69 3- viscera " 10,46 fish 2 0,57 11,03 4- issues " 6,65 fish 2 1,41 8,06 5- viscera " 8,39 fish 3 0,66 9,05 6- tissues " 5,27 fish 3 0,97 6,24 7- viscera " 8,47 fish 4 0,69 9,16 8- tissues " 8,40 fish 4 1,02 9,42 9- viscera chlor:MeOH c) 0,52 fish 1 10- tissues " 1,45 fish 1 8:Extraction made with a sample-solvent ratio of 1:9 (w/v), incubation time:
= fish 1 viscera: 4h, fish 1 tissues: 23h = fish 2 viscera: 23h45, fish 2 tissues: 45h30 = fish 3 viscera: 8 days 2h20, fish 3 tissues: 8 days 22h30 = fish 4 viscera: 17 days 23h, fish 4 tissues: 18 days 2h25.
b) :Extraction made with a sample-solvent ratio of 1:4 (w/v), incubated 1 h at 4 C, following a first extraction with acetone.
`) :Folch et al. 1957, following extractions with acetone, then ethanol..
TABLE 7. EXTRACTION OF FRESH FISH LIPIDS (Trout) Exp. No. Technique Yield % Total %
5 1- viscera acetone a~ 34,70 ethanol 2,18 36,88 2- tissues " 5,53 1,17 6,70 3- viscera chlor:MeOH } 39,81 4- tissues " 14,93 Determinations in triplicates (variation < 5%).
a) :Extraction made with a sample-solvent ratio of 1:9 (w/v), incubated 1 night at 4 C.
b) :Extraction made with a sample-solvent ratio of 1:4 (w/v), incubated 1 h at 4 C, following a first extraction with acetone.
C) :Folch et al. 1957.
TABLE 8. EXTRACTION OF FRESH FISH LIPIDS (Herring) Exp. No. Technique Yield % Total %
1-tissues and viscera acetone a) 2,09 ethanol b) 0,68 2,77 2-tissues and viscera chlor:MeOH ) 5,95 Determination in triplicates (variation < 5 % ).
a) :Extraction made with a sample-solvent ratio of 1:9 (w/v), incubated 1 night at 4 .
b) :Extraction made with a sample-solvent ratio of 1:4 (w/v), incubated 1 h at 4 C, following a first extraction with acetone.
c) :Folch et al. 1957.
TABLE 9. EXTRACTION OF FRESH SHARK LIVER LIPIDS (M. schmitti) Exp. No. Technigue Yield % Total %
1- acetone a) 36,39 ethyl acetate b) 4,48 40,87 2- ethyl acetate c) 36,68 3- chlor : MeOH d) 41,86 Determinations in triplicates (variations <5 %).
a):Extraction made with a sampie-solvent ratio of 1:9 (w/v), incubated 2h at 4 C.
b) :Extraction made with a sample-solvent ratio of 1:2 (w/v), incubated 30 min at 4 C, following a first extraction with acetone.
) :Extraction made with a sample-solvent ratio of 1 :9 (w/v), incubated 2h at 4 C.
d) :Folch et al. 1957.
CA 02346979 2001 04 12 ` ~ " - ~` ' `v 1 -a first extracxion with acetone.
:Eztracpon made wob a sampUe-soivent rabo of 1 .9 (w!v), incubated 2h at 4C.
.Folch at al. 1957.
TABLE 10. EXTRACTION OF FRESH SHARK UYER UPIDS (G. gafsusj.
Exp. No. Technique Yield ~ Total t9G) 1- acetone =) 21,39 ethyl acetate 5.27 26,66 2- ethyl acetate 26,89 3- ch(or : MeOM ' 29,99 9b).
Determination9 in triplicates (variabons <5 ') :Extraction made with a sample-solvent ratio of 1:9 (w/v). incubated 2h at 01.Extraction made with a samplG-sohrent ratio of 1:2 (wlv), incubated 30 min at 4C, following a first extraction witn acetone.
'ExtracUon made wittt a samplersolvent rapo of 1:9 (w/v), incubated 2h at 4C.
'Fotch et al. 1957.
ark) TABLE 11. EXTRACTION OF FRESH SHARK LIVER UP1DS (Angel Shark) ..,, , ....._25 No. Technique Yeld 9b Total 1- acetone " 19,23 ethyl acetate ) 8.98 28,21 2- ethyl acetate c' 39,22 3- cntor : MsOH O 39,23 DeterminaGons in triplicates (vaiiadona c5 96).
":Extraotion made with a sample-soNrent rabo of 1:9 (w/v), incubated 2h at 4C.
Extraation made wah a sample-solvent ratio of 1:2 (w/v), incubated 30 min at 4C, fopowing a first extraction with stcetone.
c) :ExtracGon made with a sampl"olvent ratio of 1 :9 (w/v), incubated 2h at 4C.
' 'Folch et W. 1957.
CA 02346979 2001 04 12 ~~.
TABLE 12. CHAFiACTE.RIS7tCS OF KRIt.L OIL Lf-mdependent handbook"
Iaboratory =' Saponficato index Fraction 1 ) 130,6 --Ffaction 1141 185,7 .-Olive oil 192,04' - 189,7 Ms iodine index Fraction I " 185,2 172,5 --Fraction 11 ' 127,2 139,2 ---Olive oil 85.30) - 81.1 Cho esterof content M) Fraction 11' 2,1 1.9 --Fraotion 11,0) 3.7 3,0 --Olive oil 0,22' ----( 6) Volatiie matter and moisture levels Fraction l 1 10,0 --- ---Fraction 110 6.8 -- --Peroxge value (rn peroxide/kg oif) Fraction 1 ~' -- 0,0 .-Fraction 11 - -- 0,0 --p-6nisidine valo (9"' absorption) Fraction 1 " --- 0,1 ---Fraction 1! e -- 5,5 -'- Prafeessor Robert Ackman'a laporatory, Canadian insptute of Fshenes TecFmology, Halifax, Nova Scotia.
j . Harwood and Geyer 1964.
' - Extraction made with a sample-acetone ratio of 1:5 (w/v), incubated 2h at 4C.
Extraction macte with a sample-ethyl acetate ratio of 12(wh-), incubated 30 min at 4C, following a first extmckion with acetone ' : Extra virgin olive oil cold compressed from Sertolli T".
~~=] Ff T
TARLE 13. LIPID CLASS COMPO8ITION OF 1CRILL OIL (AREA% ~
r ri Trtiglycerides Fracbon I') 19,0$ 0,7 FracGon 11 j 66,5t 2,3 Hydrocar0ons Fraction I trace Frac:tion 11 1,3t 0,1 Free fatty acids Frafton t=' 23,7t 1,1 Fraction li 20,3t 0,3 Monotycendes Fraction I 1,4t 0,3 Fraction lI 0,5t 0,1 Phosgjolipid$ or other poiar matenal Fraction 1" 54,1t6,1 Fraction 110) 8r5 t1,B
Data from Profeasor Roben Aclanan's labomtory, Canadi.an Institute of Fisheries Technolosy, Halbx, Nova Scotia.
"' : Extracl<on made with a samplQ-acetone rqtio of 1:6 (wr+r), incupated 2h at 4C.
Extraction made with a sample-ethyl acetate ratio of 1:2 (wiv), incubated 30 min at 4C, following a first extraction vath aaetone.
LL.G.GVvV
Y i 3 .., :. , , .::. ~ ..: 24 TABL.614. FATTY ACID COMPOSITION OF KRlLL O!L (WTIYIl1''Ye) IFE pacilica) Fatty acids FraCtion I" Frwbon 11 12:0 0,0 0,1 13 :0 0,2 0,1 ISO 14 :0 0,4 0,15 14:0 4.2 7,6 ISO 15:0 0,5 0.7 ANT 15:0 0,2 0,2 15:0 0,6 1,0 ISO 16:0 0,2 0,3 ANT 16:0 012 0,2 16.0 14,1 21,6 7MH 0,6 0,9 ANT 17.0 0,1 0.3 77:0 2,8 3,7 18:0 1,0 1,6 20=0 0,1 0,3 Saturates 25,2 39,2 YV 11 Y= . . ,,, ,,, ,. ..
CA 02346979 2001-04-12 "~ -TABLE 14 (continued). FATTY ACfD CClMPOSITtON OF KRILL OIL. (WTNV11134) IE- ~~~~l-~ili 5 Fatty acids Fraction!'N F ction 1 ' 14:1 0,4 0,5 15-1 0,1 0,2 16.1 n-7 6,6 7,8 16:1 n-5 0,6 0,2 10 17*1 0,6 0,7 18:1 n-9 8,0 9,8 18:1 n-7 4,2 5,6 18:1 n-5 0,1 0,1 20:1 n-9 0.3 0,4 15 20:1 n-7 0,3 0,4 20:1 n-5 0,3 0,4 22:1 n-11 t13 0,1 0,2 Monoenes 21,6 26,3 20 16:2 n-6 O,fi 1,2 16:2 n-4 1,3 1,3 18=2 n-7 0,1 0,2 18:2 n-6 2.0 1,8 18:2 n-4 0,1 0,1 25 20.2 NMID 0,2 0,2 20:2 n-6 0,1 0,1 Dienes 4,4 4,9 16-3 n-4 1,4 1,2 18:3 n-6 0,4 0,3 1 8:3 n-4 0,2 0,2 18-3 n-3 3,2 3,0 1 8-3 n-1 0,9 0,1 20:3 n-3 0,1 0,1 Trienes 5,4 4,9 164 n-3 0,9 0.7 16:4 n-1 1,0 0,8 18:4 n-3 9,2 7,4 18.4 n-1 0,1 0,0 20:4 n-6 0,7 0,5 20:4 n-3 0,7 0,3 Tetraenes 12.6 9,7 20:5 n-3 17,4 8.6 21.5 n-3 0,7 0,5 22:5 r-6 0,2 0,1 22:5 n-3 0,5 0,3 Pentaenes 18,8 9,5 . . . , . .. .::r~ "7r .:~~;.....
a):Extraction made with a sample-solvent ratio of 1:6 (w/v), incubated 2 h at 4 C.
b) :Extraction made with a sample-solvent ratio of 1:2 (w/v), incubated 30 min at 4 C, following a first extraction with acetone.
C) :Extraction made with a sample-solvent ratio of 1:2 (w/v), incubated 30 min at 25 C, following a first extraction with acetone.
d) :Extraction made with a sampde-acetone-ethanol ratio of 1:5:5 (w/v/v), incubated 2 h at 4 C.
e) :Extraction made with a sample-solvent ratio of 1:9 (w/v), incubated 2 h at 4 C.
n: Folch et al. 1957.
TABLE 3. EXTRACTION OF FROZEN KRILL LIPIDS(M. norvegica) Exp. No. Technique Yield % Total % Mean (%) f s.d.
1- acetone'a) 1,82 ethanol b) 1,82 3,64 2- " 1,15 2,35 3,50 3- " 1,68 2,19 3,87 3,67f0,15 Determinations in triplicates (variation < 5%).
a) :Extraction made with a sample-solvent ratio of 1:9 (w/v), incubated 1 night at 4 C.
b) :Extraction made with a sample-solvent ratio of 1:4 (w/v), incubated 1 h at 4 C, following a first extraction with acetone.
TABLE 4. INFLUENCE OF GRINDING ON EXTRACTION OF FROZEN KRILL LIPIDS
(M. norvegica) Exp. No. Technique Krill ground before 1 stextraction Yield % Total %
1- acetone yes 3,10 ethanol b) 1,07 4,17 2- " no 2,14 1,39 3,53 3- " yes 3,32 1,14 4,46 4- chlor: MeOH yes 3,30 5- " yes 3,26 Determinations in triplicates (variation < 5 %).
a):Extraction made with a sample-solvent ratio of 1:6, incubated 2 h at 4 C.
b) :Extraction made with a sample-solvent ratio of 1:2, incubated 30 min at 4 C, following a first extraction with acetone.
`) :Folch et al. 1957.
TABLE 5. EXTRACTION OF FROZEN Calanus LIPIDS (Calanus sp.) Exp. No. Technigue Yield % Total % Mean (%) s.d.
1- acetone i3) 6,18 ethanol b' 2,04 8,22 2- " 8,64 2,26 10,90 9,56 1,34 Determinations in triplicates (variation < 5 %).
a) :Extraction made with a sample-solvent ratio of 1:9 (w/v), incubated 1 night at 4 C.
b) :Extraction made with a sample-solvent ratio of 1:4 (wlv), incubated 1 h at 4 C, following a first extraction with acetone.
TABLE 6. EXTRACTION OF FRESH FISH LIPIDS (Mackerel) Exp. No. Technigue Yield (%) Total (%L
1- viscera acetone a~ 6,11 fish 1 ethanol0,59 6,70 2- tissues " 3,78 fish 1 0,91 4,69 3- viscera " 10,46 fish 2 0,57 11,03 4- issues " 6,65 fish 2 1,41 8,06 5- viscera " 8,39 fish 3 0,66 9,05 6- tissues " 5,27 fish 3 0,97 6,24 7- viscera " 8,47 fish 4 0,69 9,16 8- tissues " 8,40 fish 4 1,02 9,42 9- viscera chlor:MeOH c) 0,52 fish 1 10- tissues " 1,45 fish 1 8:Extraction made with a sample-solvent ratio of 1:9 (w/v), incubation time:
= fish 1 viscera: 4h, fish 1 tissues: 23h = fish 2 viscera: 23h45, fish 2 tissues: 45h30 = fish 3 viscera: 8 days 2h20, fish 3 tissues: 8 days 22h30 = fish 4 viscera: 17 days 23h, fish 4 tissues: 18 days 2h25.
b) :Extraction made with a sample-solvent ratio of 1:4 (w/v), incubated 1 h at 4 C, following a first extraction with acetone.
`) :Folch et al. 1957, following extractions with acetone, then ethanol..
TABLE 7. EXTRACTION OF FRESH FISH LIPIDS (Trout) Exp. No. Technique Yield % Total %
5 1- viscera acetone a~ 34,70 ethanol 2,18 36,88 2- tissues " 5,53 1,17 6,70 3- viscera chlor:MeOH } 39,81 4- tissues " 14,93 Determinations in triplicates (variation < 5%).
a) :Extraction made with a sample-solvent ratio of 1:9 (w/v), incubated 1 night at 4 C.
b) :Extraction made with a sample-solvent ratio of 1:4 (w/v), incubated 1 h at 4 C, following a first extraction with acetone.
C) :Folch et al. 1957.
TABLE 8. EXTRACTION OF FRESH FISH LIPIDS (Herring) Exp. No. Technique Yield % Total %
1-tissues and viscera acetone a) 2,09 ethanol b) 0,68 2,77 2-tissues and viscera chlor:MeOH ) 5,95 Determination in triplicates (variation < 5 % ).
a) :Extraction made with a sample-solvent ratio of 1:9 (w/v), incubated 1 night at 4 .
b) :Extraction made with a sample-solvent ratio of 1:4 (w/v), incubated 1 h at 4 C, following a first extraction with acetone.
c) :Folch et al. 1957.
TABLE 9. EXTRACTION OF FRESH SHARK LIVER LIPIDS (M. schmitti) Exp. No. Technigue Yield % Total %
1- acetone a) 36,39 ethyl acetate b) 4,48 40,87 2- ethyl acetate c) 36,68 3- chlor : MeOH d) 41,86 Determinations in triplicates (variations <5 %).
a):Extraction made with a sampie-solvent ratio of 1:9 (w/v), incubated 2h at 4 C.
b) :Extraction made with a sample-solvent ratio of 1:2 (w/v), incubated 30 min at 4 C, following a first extraction with acetone.
) :Extraction made with a sample-solvent ratio of 1 :9 (w/v), incubated 2h at 4 C.
d) :Folch et al. 1957.
CA 02346979 2001 04 12 ` ~ " - ~` ' `v 1 -a first extracxion with acetone.
:Eztracpon made wob a sampUe-soivent rabo of 1 .9 (w!v), incubated 2h at 4C.
.Folch at al. 1957.
TABLE 10. EXTRACTION OF FRESH SHARK UYER UPIDS (G. gafsusj.
Exp. No. Technique Yield ~ Total t9G) 1- acetone =) 21,39 ethyl acetate 5.27 26,66 2- ethyl acetate 26,89 3- ch(or : MeOM ' 29,99 9b).
Determination9 in triplicates (variabons <5 ') :Extraction made with a sample-solvent ratio of 1:9 (w/v). incubated 2h at 01.Extraction made with a samplG-sohrent ratio of 1:2 (wlv), incubated 30 min at 4C, following a first extraction witn acetone.
'ExtracUon made wittt a samplersolvent rapo of 1:9 (w/v), incubated 2h at 4C.
'Fotch et al. 1957.
ark) TABLE 11. EXTRACTION OF FRESH SHARK LIVER UP1DS (Angel Shark) ..,, , ....._25 No. Technique Yeld 9b Total 1- acetone " 19,23 ethyl acetate ) 8.98 28,21 2- ethyl acetate c' 39,22 3- cntor : MsOH O 39,23 DeterminaGons in triplicates (vaiiadona c5 96).
":Extraotion made with a sample-soNrent rabo of 1:9 (w/v), incubated 2h at 4C.
Extraation made wah a sample-solvent ratio of 1:2 (w/v), incubated 30 min at 4C, fopowing a first extraction with stcetone.
c) :ExtracGon made with a sampl"olvent ratio of 1 :9 (w/v), incubated 2h at 4C.
' 'Folch et W. 1957.
CA 02346979 2001 04 12 ~~.
TABLE 12. CHAFiACTE.RIS7tCS OF KRIt.L OIL Lf-mdependent handbook"
Iaboratory =' Saponficato index Fraction 1 ) 130,6 --Ffaction 1141 185,7 .-Olive oil 192,04' - 189,7 Ms iodine index Fraction I " 185,2 172,5 --Fraction 11 ' 127,2 139,2 ---Olive oil 85.30) - 81.1 Cho esterof content M) Fraction 11' 2,1 1.9 --Fraotion 11,0) 3.7 3,0 --Olive oil 0,22' ----( 6) Volatiie matter and moisture levels Fraction l 1 10,0 --- ---Fraction 110 6.8 -- --Peroxge value (rn peroxide/kg oif) Fraction 1 ~' -- 0,0 .-Fraction 11 - -- 0,0 --p-6nisidine valo (9"' absorption) Fraction 1 " --- 0,1 ---Fraction 1! e -- 5,5 -'- Prafeessor Robert Ackman'a laporatory, Canadian insptute of Fshenes TecFmology, Halifax, Nova Scotia.
j . Harwood and Geyer 1964.
' - Extraction made with a sample-acetone ratio of 1:5 (w/v), incubated 2h at 4C.
Extraction macte with a sample-ethyl acetate ratio of 12(wh-), incubated 30 min at 4C, following a first extmckion with acetone ' : Extra virgin olive oil cold compressed from Sertolli T".
~~=] Ff T
TARLE 13. LIPID CLASS COMPO8ITION OF 1CRILL OIL (AREA% ~
r ri Trtiglycerides Fracbon I') 19,0$ 0,7 FracGon 11 j 66,5t 2,3 Hydrocar0ons Fraction I trace Frac:tion 11 1,3t 0,1 Free fatty acids Frafton t=' 23,7t 1,1 Fraction li 20,3t 0,3 Monotycendes Fraction I 1,4t 0,3 Fraction lI 0,5t 0,1 Phosgjolipid$ or other poiar matenal Fraction 1" 54,1t6,1 Fraction 110) 8r5 t1,B
Data from Profeasor Roben Aclanan's labomtory, Canadi.an Institute of Fisheries Technolosy, Halbx, Nova Scotia.
"' : Extracl<on made with a samplQ-acetone rqtio of 1:6 (wr+r), incupated 2h at 4C.
Extraction made with a sample-ethyl acetate ratio of 1:2 (wiv), incubated 30 min at 4C, following a first extraction vath aaetone.
LL.G.GVvV
Y i 3 .., :. , , .::. ~ ..: 24 TABL.614. FATTY ACID COMPOSITION OF KRlLL O!L (WTIYIl1''Ye) IFE pacilica) Fatty acids FraCtion I" Frwbon 11 12:0 0,0 0,1 13 :0 0,2 0,1 ISO 14 :0 0,4 0,15 14:0 4.2 7,6 ISO 15:0 0,5 0.7 ANT 15:0 0,2 0,2 15:0 0,6 1,0 ISO 16:0 0,2 0,3 ANT 16:0 012 0,2 16.0 14,1 21,6 7MH 0,6 0,9 ANT 17.0 0,1 0.3 77:0 2,8 3,7 18:0 1,0 1,6 20=0 0,1 0,3 Saturates 25,2 39,2 YV 11 Y= . . ,,, ,,, ,. ..
CA 02346979 2001-04-12 "~ -TABLE 14 (continued). FATTY ACfD CClMPOSITtON OF KRILL OIL. (WTNV11134) IE- ~~~~l-~ili 5 Fatty acids Fraction!'N F ction 1 ' 14:1 0,4 0,5 15-1 0,1 0,2 16.1 n-7 6,6 7,8 16:1 n-5 0,6 0,2 10 17*1 0,6 0,7 18:1 n-9 8,0 9,8 18:1 n-7 4,2 5,6 18:1 n-5 0,1 0,1 20:1 n-9 0.3 0,4 15 20:1 n-7 0,3 0,4 20:1 n-5 0,3 0,4 22:1 n-11 t13 0,1 0,2 Monoenes 21,6 26,3 20 16:2 n-6 O,fi 1,2 16:2 n-4 1,3 1,3 18=2 n-7 0,1 0,2 18:2 n-6 2.0 1,8 18:2 n-4 0,1 0,1 25 20.2 NMID 0,2 0,2 20:2 n-6 0,1 0,1 Dienes 4,4 4,9 16-3 n-4 1,4 1,2 18:3 n-6 0,4 0,3 1 8:3 n-4 0,2 0,2 18-3 n-3 3,2 3,0 1 8-3 n-1 0,9 0,1 20:3 n-3 0,1 0,1 Trienes 5,4 4,9 164 n-3 0,9 0.7 16:4 n-1 1,0 0,8 18:4 n-3 9,2 7,4 18.4 n-1 0,1 0,0 20:4 n-6 0,7 0,5 20:4 n-3 0,7 0,3 Tetraenes 12.6 9,7 20:5 n-3 17,4 8.6 21.5 n-3 0,7 0,5 22:5 r-6 0,2 0,1 22:5 n-3 0,5 0,3 Pentaenes 18,8 9,5 . . . , . .. .::r~ "7r .:~~;.....
TASI.F. 14 (conqnued). FATTY ACID COMPOSITION OF KRIL.1. OIL (WT11NT%) (E- pacHfca~
Fa aads Fraction flu Fracxion il 22,6 n-3 13,2 6,6 Hexaenes Ioaine value calculated 214,8 145,1 Data from Professor Robert Acicman's laboratory, Canadian institute of Fisheries Teechnofogy, Halifax, Nova Scotia.
'' : Extraction made vmth a sample-acetone ratio o11:6 (w/v), incuMed 2h at 4C.
Extrachon made with a sampte-ethyl acetate ratio of 1:2 (w/v), incubated 30 min at 4C, foiiowing a f,rst extraction with acetone TABLE 16. KRILL UPID FREE FATTY AC1D FAME (WTMITx) (E. paclflca) Fatty acids Fraction I'~ 11 A' 12:0 0,5 0,1 13:0 0,2 0,0 lS014:0 0,2 0,2 14:0 1.3 2.6 (S015.0 0,3 0,3 ANT 15:0 0.1 4,1 15:0 0,2 0,5 lSO16-0 0,1 0.2 ANT 16:0 0.2 0,1 16:0 3.3 10,6 7MM 0,6 0,8 ANT 17=0 0,2 0,2 Pi'tytanic 0,2 0,0 17:0 0,5 O,g 18.0 0,2 0,6 20-0 0,3 0,2 22.0 0,0 0.1 Satarates 8.4 17,4 14,1 0,2 0,2 15.1 0.2 0,1 16.1 n-9 0,5 0,0 16:1 rr7 5,2 618 16:1 rr5t117:0 0,1 0,1 17:1 0,6 0,7 18:1 n-9 7,0 11,4 18:1 n-7 4.9 9,3 18:1 n-5 0,1 0,3 20:1 n-11 0,2 0,3 20:1 n-9 0,1 0,3 CA 02346979 2001 04 12I ", ~
Fa aads Fraction flu Fracxion il 22,6 n-3 13,2 6,6 Hexaenes Ioaine value calculated 214,8 145,1 Data from Professor Robert Acicman's laboratory, Canadian institute of Fisheries Teechnofogy, Halifax, Nova Scotia.
'' : Extraction made vmth a sample-acetone ratio o11:6 (w/v), incuMed 2h at 4C.
Extrachon made with a sampte-ethyl acetate ratio of 1:2 (w/v), incubated 30 min at 4C, foiiowing a f,rst extraction with acetone TABLE 16. KRILL UPID FREE FATTY AC1D FAME (WTMITx) (E. paclflca) Fatty acids Fraction I'~ 11 A' 12:0 0,5 0,1 13:0 0,2 0,0 lS014:0 0,2 0,2 14:0 1.3 2.6 (S015.0 0,3 0,3 ANT 15:0 0.1 4,1 15:0 0,2 0,5 lSO16-0 0,1 0.2 ANT 16:0 0.2 0,1 16:0 3.3 10,6 7MM 0,6 0,8 ANT 17=0 0,2 0,2 Pi'tytanic 0,2 0,0 17:0 0,5 O,g 18.0 0,2 0,6 20-0 0,3 0,2 22.0 0,0 0.1 Satarates 8.4 17,4 14,1 0,2 0,2 15.1 0.2 0,1 16.1 n-9 0,5 0,0 16:1 rr7 5,2 618 16:1 rr5t117:0 0,1 0,1 17:1 0,6 0,7 18:1 n-9 7,0 11,4 18:1 n-7 4.9 9,3 18:1 n-5 0,1 0,3 20:1 n-11 0,2 0,3 20:1 n-9 0,1 0,3 CA 02346979 2001 04 12I ", ~
22:1 n-11*13 a,1 0,2 24:1 n-9 0.0 0.1 Monoenes 19,2 29,8 AMENDED SHEET
~~ ~.
CA 02346979 2001-04 ~12 = , = i~Rt..
~~ ~.
CA 02346979 2001-04 ~12 = , = i~Rt..
TABLE 15 (continued). KFtN.I- I.1PID FREE FATTY ACID FAME lWTNY'r'A) (F. pac, ,~ea) Fatty acit1s Frmqn I Fraction 11 16:2 n-S 0.4 0,9 16:2 n-4 1,2 1,0 18-2 n-7 0,1 0.2 18:2 n-$ 2,4 2,6 18:2 n-4 0.1 0,1 20:2 n-6 D,1 0,1 Dhenes 4.3 4,9 16:3 n-4+117:1 1.4 0.9 16-3 n-3+118:0 0,2 0.5 18:3 n-6 0,4 0,3 18:3 n-4 0.1 0,1 18:3 n-3 3,3 3,4 18:3 n-1 0,1 0,l 20:3 n-6 0,1 0,1 20.3 n-3 0,1 0,2 Tnenes 5,7 5,B
16:4 n-3 0,6 0,3 16:4 n-1 1.0 0,6 18:4 n-3 9,8 6,2 18:4 n-7 0,1 0,1 20:4n-6 1,7 1,4 20:4 n-3 0,8 0,5 22'4 n-3 0,3 0,3 Tetraenes 14,1 9.4 18.5 n-3 0.2 0,1 20:5 n-3 26,4 17.4 21:5 n-3 0.9 0,6 22:5 n-B 0,0 0,1 22:5 n-3 0,7 0,5 Pentaenes 28,2 18,7 22:6 n-3 20,5 14,4 Hexaenes 20,5 1e},e}
lodine value colculated 291,6 220,3 Data from Professor Robert Ackman's leboratory, Canadian inshtute of Fisheries Technology, Halifax, Nova Scotia.
Extraction macie with a sample-acetons ratio of 1.6 (wiv), inaubated 2h at 4C.
3 . Extraction maae with a sample-ethyl acetate ratio of 1:2 (w/v), incubated 30 min at 4C, following a first extraction with acetone.
~' ~~ AMENDED SHEET
16:4 n-3 0,6 0,3 16:4 n-1 1.0 0,6 18:4 n-3 9,8 6,2 18:4 n-7 0,1 0,1 20:4n-6 1,7 1,4 20:4 n-3 0,8 0,5 22'4 n-3 0,3 0,3 Tetraenes 14,1 9.4 18.5 n-3 0.2 0,1 20:5 n-3 26,4 17.4 21:5 n-3 0.9 0,6 22:5 n-B 0,0 0,1 22:5 n-3 0,7 0,5 Pentaenes 28,2 18,7 22:6 n-3 20,5 14,4 Hexaenes 20,5 1e},e}
lodine value colculated 291,6 220,3 Data from Professor Robert Ackman's leboratory, Canadian inshtute of Fisheries Technology, Halifax, Nova Scotia.
Extraction macie with a sample-acetons ratio of 1.6 (wiv), inaubated 2h at 4C.
3 . Extraction maae with a sample-ethyl acetate ratio of 1:2 (w/v), incubated 30 min at 4C, following a first extraction with acetone.
~' ~~ AMENDED SHEET
TABLE 16. TOCOPHEROL, ALL-trans RETINOL AND CHOLECALCIFEROL CONTENT
IN KRILL OIL (E. pacifica) alpha-tocopherol by HPLC (IU) Fraction I " 0,91 Fraction II )) 0,83 gamma-tocopherol by HPLC pg/q Fraction I a Tr Fraction II b~ Tr delta-tocopherol by HPLC ug/g Fraction I a~ N.D.
Fraction II b~ N.D.
all-trans retinol by HPLC (IU) Fraction I a) 395,57 Fraction II b) 440,47 cholecalciferol by HPLC (IU) Fraction I a) N.D.
Fraction II b) N. D.
Data from Professor Robert Ackman's laboratory, Canadian Institute of Fisheries Technology, Halifax, Nova Scotia.
Data expressed per gram of krill oil.
a) : Extraction made with a sample-acetone ratio of 1:6 (w/v), incubated 2h at 4 C.
b) : Extraction made with a sample-ethyl acetate ratio of 1:2 (w/v), incubated 30 rnin at 4 C, following a first extraction with acetone.
TR = trace N.D. = not detected Conversion : Vitamin alpha-tocopherol mg/g oil x 1,36 = International Unit All-trans retinol pg/g = 0,3 = International Unit TABLE 17. ASTAXANTHIN AND CANTHAXANTHIN CONTENT OF KRILL OIL
(E. pacifica) Asthaxantin (pg/g oil) Fraction I a) 93,1 Fraction II b~ 121,7 Canthaxanthin (uq/q oil) Fraction 1 a) 270,4 Fraction II b) 733,0 Data from Professor Robert Ackman's laboratory, Canadian Institute of Fisheries Technology, S0 Halifax, Nova Scotia.
a) : Extraction made with a sample-acetone ratio of 1:6 (w/v), incubated 2h at 4 C.
b) : Extraction made with a sample-ethyl acetate ratio of 1:2 (w/v), incubated 30 min at 4 C, following a first extraction with acetone.
TABLE 18. OPTIMAL CONDITIONS FOR LIPID EXTRACTION OF AQUATIC ANIMAL
TISSUES (suggested procedure) Grinding (if particles > 5mm) 4 C
Lipid extraction sample-acetone ratio of 1:6 (w/v) 10 2h (including swirling 20 min) Filtration organic solvent resistant filter under reduced pressure Washing sample-acetone ratio of 1:2 (w/v) pure and cold acetone Filtration organic solvent resistant filter under reduced pressure Evaporation under reduced pressure Oil-water separation 4 C
Lipid extraction sample: ethvl acetate ratio of 1:2 (w/v)a) pure ethvl acetate min 4 C b) Filtration organic solvent resistant filter under reduced pressure Evaporation under reduced pressure a: Ethanol can be replaced by isopropanol, t-butanol or ethyl acetate.
b): 25 C when using t-butanol.
TABLE 19: PROTEOLYTIC ACTIVITY OF KRILL RESIDU USING LACTOSERUM AS THE
SUBSTRATE, AT 37 C, PH 7.0 FOR A RATIO ENZYME:SUBSTRATE OF 1:43 Time Amino acids released Enzymatic rate Specific enzymatic min (pmoles) (umoles/min) activity (umoles/min/mg*) 15 28.76 1.917 0.164 30 43.74 0,999 0.125 170 98.51 0.322 0.050 255 177.26 0.308 0.060 * total quantity of enzymes in hydrolysis media
IN KRILL OIL (E. pacifica) alpha-tocopherol by HPLC (IU) Fraction I " 0,91 Fraction II )) 0,83 gamma-tocopherol by HPLC pg/q Fraction I a Tr Fraction II b~ Tr delta-tocopherol by HPLC ug/g Fraction I a~ N.D.
Fraction II b~ N.D.
all-trans retinol by HPLC (IU) Fraction I a) 395,57 Fraction II b) 440,47 cholecalciferol by HPLC (IU) Fraction I a) N.D.
Fraction II b) N. D.
Data from Professor Robert Ackman's laboratory, Canadian Institute of Fisheries Technology, Halifax, Nova Scotia.
Data expressed per gram of krill oil.
a) : Extraction made with a sample-acetone ratio of 1:6 (w/v), incubated 2h at 4 C.
b) : Extraction made with a sample-ethyl acetate ratio of 1:2 (w/v), incubated 30 rnin at 4 C, following a first extraction with acetone.
TR = trace N.D. = not detected Conversion : Vitamin alpha-tocopherol mg/g oil x 1,36 = International Unit All-trans retinol pg/g = 0,3 = International Unit TABLE 17. ASTAXANTHIN AND CANTHAXANTHIN CONTENT OF KRILL OIL
(E. pacifica) Asthaxantin (pg/g oil) Fraction I a) 93,1 Fraction II b~ 121,7 Canthaxanthin (uq/q oil) Fraction 1 a) 270,4 Fraction II b) 733,0 Data from Professor Robert Ackman's laboratory, Canadian Institute of Fisheries Technology, S0 Halifax, Nova Scotia.
a) : Extraction made with a sample-acetone ratio of 1:6 (w/v), incubated 2h at 4 C.
b) : Extraction made with a sample-ethyl acetate ratio of 1:2 (w/v), incubated 30 min at 4 C, following a first extraction with acetone.
TABLE 18. OPTIMAL CONDITIONS FOR LIPID EXTRACTION OF AQUATIC ANIMAL
TISSUES (suggested procedure) Grinding (if particles > 5mm) 4 C
Lipid extraction sample-acetone ratio of 1:6 (w/v) 10 2h (including swirling 20 min) Filtration organic solvent resistant filter under reduced pressure Washing sample-acetone ratio of 1:2 (w/v) pure and cold acetone Filtration organic solvent resistant filter under reduced pressure Evaporation under reduced pressure Oil-water separation 4 C
Lipid extraction sample: ethvl acetate ratio of 1:2 (w/v)a) pure ethvl acetate min 4 C b) Filtration organic solvent resistant filter under reduced pressure Evaporation under reduced pressure a: Ethanol can be replaced by isopropanol, t-butanol or ethyl acetate.
b): 25 C when using t-butanol.
TABLE 19: PROTEOLYTIC ACTIVITY OF KRILL RESIDU USING LACTOSERUM AS THE
SUBSTRATE, AT 37 C, PH 7.0 FOR A RATIO ENZYME:SUBSTRATE OF 1:43 Time Amino acids released Enzymatic rate Specific enzymatic min (pmoles) (umoles/min) activity (umoles/min/mg*) 15 28.76 1.917 0.164 30 43.74 0,999 0.125 170 98.51 0.322 0.050 255 177.26 0.308 0.060 * total quantity of enzymes in hydrolysis media
Claims (38)
1. A method for extracting total lipid fractions from marine and aquatic animal material, said method comprising the steps of:
(a) placing marine and aquatic animal material in a ketone solvent, to achieve extraction of the soluble lipid fraction from said marine and aquatic animal material;
(b) separating liquid and solid contents resulting from step (a);
(c) recovering a first total lipid rich fraction from the liquid contents of b) by evaporation of the solvent present in the liquid contents;
(d) placing said solid contents in an organic solvent selected from the group of solvents consisting of ethanol, isopropanol, t-butanol, and ethyl acetate, to achieve extraction of the remaining soluble lipid fraction from said marine and aquatic animal material;
(e) separating liquid and solid contents resulting from step (d);
(f) recovering a second total lipid rich fraction by evaporation of the solvent from the liquid contents of e); and (g) recovering the solid contents.
(a) placing marine and aquatic animal material in a ketone solvent, to achieve extraction of the soluble lipid fraction from said marine and aquatic animal material;
(b) separating liquid and solid contents resulting from step (a);
(c) recovering a first total lipid rich fraction from the liquid contents of b) by evaporation of the solvent present in the liquid contents;
(d) placing said solid contents in an organic solvent selected from the group of solvents consisting of ethanol, isopropanol, t-butanol, and ethyl acetate, to achieve extraction of the remaining soluble lipid fraction from said marine and aquatic animal material;
(e) separating liquid and solid contents resulting from step (d);
(f) recovering a second total lipid rich fraction by evaporation of the solvent from the liquid contents of e); and (g) recovering the solid contents.
2. A method as in claim 1, wherein the extraction of step (a) is conducted under agitation after the animal material has been ground.
3. A method as in any one of claims 1 and 2, wherein the extraction of step (d) is conducted under agitation after the animal material has been ground.
4. A method as in any one of claims 1 to 3, wherein steps (a) and (d) are conducted under inert atmosphere.
5. A method as in any one of claims 1 to 4, wherein steps (b) and (e) are effected by techniques selected from filtration, centrifugation and sedimentation.
6. A method as in any one of claims 1 to 5, wherein steps (c) and (f) are effected by techniques selected from evaporation under reduced pressure, flash evaporation and spray drying.
7. A method as in any one of claims 1 to 6, wherein after step (b) and before step (c), the method additionally comprises the intervening step of washing the solid contents with the solvent and adding the resulting washing solution to the liquid contents of step (b).
8. A method as in any one of claims 1 to 7, wherein after step (e) and before step (f), the method additionally comprises the intervening step of washing the solid contents with the organic solvent selected in step (d).
9. A method as in any one of claims 1 to 8, wherein prior to step (a) the marine and aquatic animal material is finely divided.
10. A method as in any one of claims 1 to 9, wherein steps (a) and (d) are conducted at solvent temperatures of 5°C or less and the organic solvent is not t-butanol.
11. A method as in any one of claims 1 to 10, wherein said marine and aquatic animal is zooplancton.
12. A method as in claim 11, wherein said zooplancton is krill.
13. A method as in claim 11, wherein said zooplancton is Calanus.
14. A method as in any one of claims 1 to 10, wherein said marine and aquatic animal is fish.
15. A method as in any one of claims 1 to 14, wherein the organic solvent is selected from the group consisting of ethanol, isopropanol and esters of acetic acid.
16. A method as in any one of claims 1-14, wherein the organic solvent is ethyl acetate.
17. A method as in any one of claims 1-9, wherein the organic solvent is t-butanol and wherein step (d) is conducted at a solvent temperature of 25°C.
18. A method for extracting an astaxanthin-and-canthaxantin-containing total lipid fraction from a marine and aquatic animal material selected from zooplancton and fish, said method comprising the steps of:
(a) placing said animal material in a ketone solvent at a solvent temperature of 5°C or less to achieve an extraction of the soluble lipid fraction from said marine and aquatic animal material;
(b) separating liquid and solid contents resulting from step (a);
(c) recovering a lipid rich fraction from the liquid contents by evaporation of the solvent present in the liquid contents;
whereby an astaxanthin-and-canthaxantin-containing total lipid fraction is obtained.
(a) placing said animal material in a ketone solvent at a solvent temperature of 5°C or less to achieve an extraction of the soluble lipid fraction from said marine and aquatic animal material;
(b) separating liquid and solid contents resulting from step (a);
(c) recovering a lipid rich fraction from the liquid contents by evaporation of the solvent present in the liquid contents;
whereby an astaxanthin-and-canthaxantin-containing total lipid fraction is obtained.
19. A method for extracting a total lipid fraction from a marine and aquatic animal material selected from zooplancton and fish, said method comprising the steps of:
(a) placing said animal material in a solvent mixture comprising acetone and ethanol to achieve an extraction of the soluble lipid fraction from said marine and aquatic animal material;
(b) separating liquid and solid contents resulting from step (a);
(c) recovering a lipid rich fraction from the liquid contents by evaporation of the solvents present in the liquid contents;
whereby a total lipid fraction is obtained
(a) placing said animal material in a solvent mixture comprising acetone and ethanol to achieve an extraction of the soluble lipid fraction from said marine and aquatic animal material;
(b) separating liquid and solid contents resulting from step (a);
(c) recovering a lipid rich fraction from the liquid contents by evaporation of the solvents present in the liquid contents;
whereby a total lipid fraction is obtained
20. A method as in claim 18 or 19, wherein the animal material is krill.
21. A method as in claim 18 or 19, wherein the animal material is Calanus
22. A method as in any one of claims 18 to 21, wherein the extraction of step (a) is conducted under agitation after the animal material has been ground.
23. A method as in any one of claims 18 to 22, wherein step (a) is conducted under inert atmosphere.
24. A method as in any one of claims 18 to 23, wherein step (b) is effected by a technique selected from filtration, centrifugation and sedimentation.
25. A method as in any one of claims 18 to 24, wherein step (c) is effected by a technique selected from vacuum evaporation, flash evaporation and spray drying.
26. A method as in any one of claims 18 to 25, wherein after step (b) and before step (c), the method additionally comprises a step of washing said solid contents with a solvent and adding the resulting washing solution to the liquid contents of step (b).
27. A method as in any one of claims 18 to 26, wherein prior to step (a) the marine and aquatic animal material is finely divided
28 A method as in any one of claims 19 to 27, wherein step (a) is conducted at a solvent temperature of 5°C or less.
29. A method of lipid extraction as in any one of claims 1 to 28, wherein the resulting solid contents is recovered and consists of a dehydrated residue containing active enzymes.
30. A method as in any one of claims 1 to 29, wherein the ketone solvent is acetone.
31. A method as in claim 9 or 27, wherein the animal material is divided to an average particle size of less than 5mm.
32. A total lipid fraction devoid of toxic solvents extracted from an aquatic or marine animal by the method of any one of claims 1 to 31.
33. Use of the lipid fraction of claim 32, in an application selected from the group consisting of medical, nutraceutical, cosmetic, fish farming and animal feeding applications.
34. Use of the lipid fraction of claim 32, in the manufacture of a medicament selected from the group consisting of medical, nutraceutical, cosmetic, fish farming and animal feeding products.
35. A total krill lipid extract characterized in that the carotenoid content in asthaxanthin is 75 or more of krill extract, and the carotenoid content in canthaxanthin is 250 µg/g or more of krill extract or more.
36. A krill lipid extract as recited in claim 35, wherein the carotenoid content in asthaxanthin is 90 µg/g or more and the carotenoid content in canthaxanthin is 270 µg/g of krill extract or more.
37. Use of the krill lipid extract of any one of claims 35 and 36, in an application selected from the group consisting of medical, nutraceutical, cosmetic, fish farming and animal feeding applications.
38. Use of the krill lipid extract of any one of claims 35 and 36, in the manufacture of a medicament selected from the group consisting of medical, nutraceutical, cosmetic, fish farming and animal feeding products.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002346979A CA2346979C (en) | 1998-10-21 | 1999-10-21 | Method of extracting lipids from marine and aquatic animal tissues |
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA2,251,265 | 1998-10-21 | ||
| CA002251265A CA2251265A1 (en) | 1998-10-21 | 1998-10-21 | Process for lipid extraction of aquatic animal tissues producing a dehydrated residue |
| CA002346979A CA2346979C (en) | 1998-10-21 | 1999-10-21 | Method of extracting lipids from marine and aquatic animal tissues |
| PCT/CA1999/000987 WO2000023546A1 (en) | 1998-10-21 | 1999-10-21 | Method of extracting lipids from marine and aquatic animal tissues |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2346979A1 CA2346979A1 (en) | 2000-04-27 |
| CA2346979C true CA2346979C (en) | 2009-07-07 |
Family
ID=25680590
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002346979A Expired - Fee Related CA2346979C (en) | 1998-10-21 | 1999-10-21 | Method of extracting lipids from marine and aquatic animal tissues |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2346979C (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3104711A1 (en) * | 2014-02-11 | 2016-12-21 | Enzymotec Ltd. | Krill oil preparations with optimal mineral and metal composition, low impurities and low and stable tma levels |
-
1999
- 1999-10-21 CA CA002346979A patent/CA2346979C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CA2346979A1 (en) | 2000-04-27 |
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