Title of Invention	METHOD FOR EXTRACTING TOTAL LIPIDS FRACTIONS FROM MARINE AND AQUATIC ANIMAL MATERIAL
Abstract	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 preferably acetone, 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 is selected from the group of solvents consisting of alcohol preferable ethanol, isopropanol, t-butanol and esters of acetic acid preferably 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)

Title of Invention

METHOD FOR EXTRACTING TOTAL LIPIDS FRACTIONS FROM MARINE AND AQUATIC ANIMAL MATERIAL

Abstract

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 preferably acetone, 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 is selected from the group of solvents consisting of alcohol preferable ethanol, isopropanol, t-butanol and esters of acetic acid preferably 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)

Full Text	FORM 2 THE PATENTS ACT 1970 [39 OF 1970] COMPLETE SPECIFICATION [See Section 10] T "METHOD OF EXTRACTING LIPIDS FROM MARINE AND AQUATIC ANIMAL UNIVERSITE DE SHERBROOKE, of University Boulevard, Sherbrooke, Quebec J1K 2R1, Canada The following specification particularly describes and ascertain the nature of the invention and the manner in which it is to be performed :- 1 BACKGROUND OF THE INVENTION This invention relates to the extraction of lipid fractions from marine and aquatic animals such as krill, Calanus, fish and sea mammals. More specifically, this invention relates to an improved method of extracting lipid fractions by dehydration with solvents and recovering a solid residue rich in active enzymes. Lipid fractions obtained from marine and aquatic animals such as krill, Calanus, fish and sea mammals have various applications: Medical applications Marine and aquatic animal oils and fractions thereof contain various therapeutic agents. For example, it is reported that various marine and aquatic animal oils have anti-inflammatory properties. Marine and aquatic animal oils are also reported as helpful in reducing the incidence of cardiovascular disease. Also, some marine and aquatic animal oils are reported as suppressing the development of certain forms of lupus and renal diseases. As a further example, krill may be used as a source of enzymes for debridement of ulcers and wounds or to facilitate food digestion. Also marine and aquatic oils contain various antioxidants, which may have potential therapeutic properties. Nutraceuticals Considering the beneficial effects of omega-3 fatty acids, oils from krill, Calanus and fish could be used as dietary supplements to human diet. These fatty acids are essential for proper development of the brain and the eye. Marine and aquatic animal oils are also rich in liposoluble vitamins A, D and E and carotenoids. Cosmetics Various marine and aquatic animal oils are used for the production of moisturizing creams. 2 Fish farming Among the lipids found in krill, Calanus and fish, high concentrations of fatty acids 20:5 (eicosapentaenoic acid) and 22:6 (docosahexaenoic acid) are present. These fatty acids are essential nutrients and are beneficial as fish feed. Furthermore, these essential nutrients are carried over in human diet by eating the fish grown on such diets. Animal feed Animal feed diets rich in omega-3 fatty acids may increase the level of unsaturated fatty acids and decrease cholesterol levels of meat. This property is already exploited in the poultry industry to improve the quality of eggs. Various methods for extracting marine and aquatic animal oils are known. For example, it is 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 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 divided animal products. The extracted oils are then made to precipitate under high pressure and elevated temperatures of 50 to 200°C. However, hexane is a poor extraction solvent for marine animals such as krill. Furthermore, the high temperatures used in the precipitation step negatively alters the lipids. Canadian Patent Application 2,115,571 describes a method for extracting oils from various brown and(i^aa)algae species. The method provides for example Soxhlet extraction using nearly pure ethanol for 40 hours. USP 5,006,281 describes a method for extracting oil from marine and aquatic animals such as fish. The marine and aquatic animal is first treated with an antioxidant compound, finely divided and centrifuged to separate the oil phase from 3 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 and separate recovery of a valuable protein rich solid residue that comprises active enzymes. Other objects and further scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that this detailed description, while indicating preferred embodiments of the invention, is given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Gas-liquid chromatography 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 (f-butanol) 4 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 chromatography of neutral lipids of G. galeus Figure 11. Thin-layer chromatography of neutral lipids of Angel Shark Figure 12. Thin-layer chromatography of phospholipids of Calanus sp. and M. norvegica Figure 13. Thin-layer chromatography of phospholipids of E. pacifica Figure 14. Thin-layer chromatography of phospholipids of M. schmitti Figure 15. Thin-layer chromatography of phospholipids of G. galeus Figure 16. Thin-layer chromatography 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 and not limitation. The method of the invention comprises suspending freshly collected marine and aquatic material in acetone. Lipids are extracted with a ketone such as acetone. This allows a rapid dehydration of animal tissue and a migration of the lipid fraction to the solvent. The dry residue is a valuable product rich in active enzymes. 5 In a preferred embodiment, the extraction is carried out by successive acetone and alcohol treatments. Preferred alcohols are isopropanol, and f-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 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, Calanus, 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 zooplancton such as krill and Calanus and in the case of fish-filleting by-products such as fish viscera, it is noted that extraction with acetone alone may 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 6 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 used. 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, f-butanol or alternatively with ethyl acetate, preferably two volumes (original volume of material). The filtrate is evaporated leaving a second fraction of lipids (identified 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 organic solvents, except f-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 (Folch et al. 1957) using chloroform and methanol was applied to krill. this method is the reference for measuring efficiency of the extraction process. Another comparison has been made with a technique using hexane as the extraction solvent. Lipid recovery 7 was estimated by suspending lipid fractions in small volumes of their original solvents and measuring by gravimetry small aliquots after evaporation. For all examples provided herein, the method of the present invention involving acetone extraction followed by extraction with a second solvent (ethyl acetate, for example) gave a translucent oil having appearance and properties more attractive than any oil obtained by the classical technique of Folch et al. (1957). To analyze lipid composition, 780 ug of each extract was loaded on silica-gel plates and fractionated by thin layer chromatography, TLC (Bowyer et al. 1962) with the following solvents. Neutral lipids: hexane, ethyl ether, acetic acid (90:10:1, v/v) and phospholipids: chloroform, methanol, water (80:25:2, v/v). Fatty acid composition of E. pacifica was analyzed by gas liquid chromatography, GLC (Bowyer et al. 1962, see bibliography) including some modifications to the original technique: 2h at 65°C instead of 1 h at 80°C, three washes with hexane instead of two and no wash with water. To get rid of traces of organic solvents, lipid fractions I and II are warmed to about 125°C for about 15 minutes under inert atmosphere. Fat was analyzed according to the American Oil Chemist'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 content has also been determined by the method of Plummer 1987 (see bibliography). The same analyzes and others have been made by an independent laboratory under Professor Robert Ackman's supervision (Canadian Instituteof Fisheries Technology, DalTech, Dalhousie University, Halifax, Nova Scotia, Canada). This includes Wijs iodine index, peroxide and anisidine values, lipid class composition, fatty acid composition, free fatty acid FAME, cholesterol, tocopherol, a\\-trans retinol, cholecalciferol, asthaxanthin and canthaxantin contents. 8 Table 1 shows that higher levels of lipids are extracted from dry krill by acetone followed by ethanol as compared to the classical procedure of Folch et al. (1957). Table 2 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, f-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, f-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. This 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 solid 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 Bacto™ beef extract 0,3%, Bacto™ peptone 0,5% and Bacto™ agar 1,5% (Difco Laboratories, Detroit, USA) then incubated at room temperature or 4°C for 18 days. No significant bacterial growth was observed at a ratio of 1 volume of acetone per gram of krill. At higher proportions of acetone (2 volumes and 5 volumes), there was no bacterial growth at all, which means that acetone preserves krill samples. Acetone is known as an efficient bactericidal and viricidal agent (Goodman et al. 1980). 9 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 experiments 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 method. Indeed small amounts of lipids from mackerel (0.52% from viscera and 1,45% from tissues) have been extracted by the method of Folch after a first extraction with acetone and ethanol as described in the present invention. Comparative extractions with the method described in the present invention carried out in parallel with the method of Folch on trout and herring show superior recovery with the latter. However, it is noteworthy that the Folch method can not be applied for the recovery of lipids for commercial uses (because of toxicity). 10 In Tables 9 to 11, are shown results of lipids extraction from shark liver tissues. There is no marked difference in results between techniques within a species. Table 12 shows the fatty acid composition of krill oil (e. pacifica) following extraction in various solvents. Tables 13 shows some characteristics features of fraction I (acetone) and fraction II (alcohol or ethyl acetate) for krill oil (e. pacifica). First, the saponification index of fraction I (130,6) indicates that this fraction contains fatty acids with longer chains, compared to fraction II (185,7). The Wijs iodine index of fraction I shows that this 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 fraction II which has a iodine index of 127,2. The fatty acid composition shown in Table 12 corroborates these iodine indexes: fraction I has a high percentage (30,24%) of polyunsaturated fatty acids (pentaenes+hexaenes) and so fraction II (22,98%). Finally, Table 13 shows also that fraction I is comprised of 10,0% of volatile matter and humidity after evaporation of the solvent. 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 krill oils obtained in accordance with the method of the present invention (fraction I extracted with acetone and fraction II extracted with ethyl acetate) are provided in Tables 13, 14, 15, 16, 17 and 18. It is noteworthy to mention that in Table 18, the carotenoids content was significantly high as measured in terms of two carotenoids namely asthaxanthin and canthaxanthin. indeed, duplicates analyzes revealed values of 92 to 124 pg/g of lipid fraction for asthaxanthin and 262 to 734 pg/g for canthaxanthin. Thus, for the purpose of the present invention it may be said that the krill extract comprises asthaxanthin at least 75 and preferably at least 90 ug/g of lipid fraction. In the case of canthaxanthin, at least 250 and preferably at least 270 pg/g of lipid fraction. Low values for peroxide and anisidine are advantageous and are due to the presence of high levels of natural antioxidants 11 (astaxanthin and canthaxanthin). These compounds are indicative of favourable pharmaceutical or cosmetological properties of the krill extract whereby high levels of carotenoids indicate excellent transdermal migration characteristics. Thus, krill extract is a good candidative for transdermal delivery of medicines. Table 19 shows the best mode of the method in accordance with the present invention for lipid extraction of aquatic animal tissues. Table 20 shows that the enzyme activity of the solid fraction is maintained following the method of the present invention. Indeed, the demonstration was completed for solid krill residue obtained after successive acetone and ethyl acetate extraction. Proteolytic activities were measure by the liberation of amino groups by spectrophotometric assay using o-pthaldialdehyde as reagent. Protein concentrations were measured by the Bradford method. 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 50mM potassium phosphate buffer, trichloroacetic acid was added and the amount of NH3 group was measured in the supernatant according to the method of Church et al. [1983, J Dairy Sci 66: 1219-1227]. Figures 1 to 6 show chromatograms of fatty acid composition of E. pacifica lipids. On each of them, high proportions of 20:5 and 22:6 fatty acids (characteristic of marine and aquatic oils) are noticeable and represented by two distinct peaks. Data are shown in Table 12. Variations in lipid patterns 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) 12 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. 13 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 comparison with column chromatography. BBA. 70:423-431. Chandrasekar, B., Troyer, D.A., Venkatraman, J.T. and Femandes, G. 1996. Tissue specific regulation of transforming growth factor beta by omega-3 lipid-rich krill oil in autoimmune murine lupus. Nutr Res. 16(3): 489-503. Christensen, M.S., 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. 10th 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 Macmilian Canada ltd, Toronto. Harwood, HJ. 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): 102-103 14 Plummer, D.T. 1987. An introduction to practical biochemistry. 3th ed. McGraw-Hill Book Company, London. Rawn, J.D. 1990. Traite de biochimie. De Boeck-Wesmael, Bruxelles. Runge, J.A. and Joly, P. 1994. Rapport sur I'etat des invertebres en 1994: 7:0 Zooplancton (Euphausiaces et Calanus) de I'Estuaire et du Golfe du Saint-Laurent. Sargent JR. 1997. Fish oils and human diet Br J Nutr.78 Suppl 1: S5-S13. 15 TABLE 1. EXTRACTION OF DRY KRILL LIPIDS (E. pacifica) Exp. No. Technique Yield (%) Total (%) Mean (%) ± s.d. 1- acetonea) ethanolb) 8,00 7,60 15,60 2- tt 19,70 6,90 26,60 3- it 8,15 11,20 19,35 4- tt 6,80 13,60 20,40 20,49+3,95 5- chlor: MeOHc) 15,50 6- it 14,90 15,20+0,30 Determinations in triplicates (variation 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 at4°C, following a first extraction with acetone. c) :Fo!ch et al. 1957. TABLE 2. EXTRACTION OF FROZEN KRILL LIPIDS (E. pacifica) Exp. No. Technique Yield (%) Total (%) Mean (%) + s.d. 1- acetonea) 1,17 ethanolb) 1,23 2,40 2- tt 3,05 1,09 4,14 3- tt 1,53 1,26 2,79 3,11+0,91 4- acetonea) 2,45 isopropanolb) 0,70 3,15 5- . " 1,80 0,80 2,60 6- t" 1,60 0,80 2,40 2,72±0,39 16 TABLE 2 (continued). EXTRACTION OF FROZEN KRILL LIPIDS (E. pacifica) Exp. No. Technique Yield (%) Total (%) Mean (%) ± s.d. 7- acetonea) t-butanolc> 2,15 0,47 2,62 8- tf 2,11 0,40 2,51 9-10- rr acetonea) ethyl acetateb) 2,37 0,45 2,28 0,21 2,82 2,49 2,65+0,16 11- rr 1,09 0,16 1,25 12- rr 2,54 0,09 2,63 2,12+0,76 13- combined acetone-ethanol ^ 3,28 14- " 3,02 15- " 3,25 3,18±0,14 16- ethyl acetatee) 1,32 17- " 1,49 18- " 1,31 1,37+0,10 19- hexanee) 0,31 20- " 0,18 21- " 0,20 22- chlonMeOH ° 2,37 17 0,23±0,07 TABLE 2 (continued). EXTRACTION OF FROZEN KR1LL LIPIDS (E. pacifica) Exp. No. Technique Yield (%) Total (%) Mean (%) ± s.d. 23- " 2,07 24- " 2,62 2,35±0,28 Determinations in triplicates (variation 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 sample-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. 0: Folch et al. 1957. TABLE 3. EXTRACTION OF FROZEN KRILL LlPIDS(M. norvegica) Exp. No. Technique Yield (%) Total (%) Mean (%) ± s.d. 1- acetonea) ethanolb) 1,82 1,82 3,64 2- rt 1,15 2,35 3,50 3- ft 1,68 2,19 3,87 3,67±0,15 Determinations in triplicates (variation 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. 18 TABLE 4. INFLUENCE OF GRINDING ON EXTRACTION OF FROZEN KRILL LIPIDS (M. norvegica) yes no yes yes yes acetone ' ethano!b) 2- 3- 4-5- chlor:MeOHc> Exp. No. Technique Krill ground before 1st extraction Yield (%) Total (%) 3,10 1,07 4,17 2,14 1,39 3,53 3,32 1,14 4,46 3,30 3,26 Determinations in triplicates (variation a) .Extraction made with a sample-solvent ratio of 1.6, incubated 2 h at4°C. b> Extraction made with a sample-solvent ratio of 1:2, incubated 30 min at 4°C, following a first extraction with acetone. c) .Folch et al. 1957. TABLE 5. EXTRACTION OF FROZEN Calanus LIPIDS (Calanus sp.) Exp. No. 1- 2- Technique acetonea) ethanolb> Yield (%) Total (%) Mean (%) ± s.d. 6,18 2,04 8,22 8,64 2,26 10,90 9,56+1,34 Determinations in triplicates (variation 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. 19 WO U0y23i>46 FCT/CA99/00987' ^rET" TABLE 6. EXTRACTION OF FRESH FISH LIPIDS (Mackerel) Exp. No. Technique Yield (%) Total (%) 1-viscera acetonea) 6,11 fish 1 ethanolb> 0,59 6,70 2- tissues tt 3,78 fish 1 0,91 4,69 3- viscera it 10,46 fish 2 0,57 11,03 4- issues IT 6,65 fish 2 1,41 8,06 5- viscera tt 8,39 fish 3 0,66 9,05 6- tissues It 5,27 fish 3 0,97 6,24 7- viscera II 8,47 fish 4 0,69 9,16 8- tissues ft 8,40 fish 4 1,02 9,42 9- viscera chlonMeOH c) 0,52 fish 1 10-tissues // 1,45 fish 1 3) :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 1h at 4°C, following a first extraction with acetone. b) :Folch et al. 1957, following extractions with acetone, then ethanol.. 20 TABLE 7. EXTRACTION OF FRESH FfSH LIPIDS (Trout) Exp. No. Technique Yield (%) Total (%) 1- viscera acetonea) 34,70 ethanolb) 2,18 36,88 2- tissues tt 5,53 1,17 6,70 3- viscera chlor.MeOH c) 39,81 4- tissues tt 14,93 Determinations in triplicates (variation a) :Extraction made with a sample-solvent ratio of 1:9 (w/v), incubated 1 night at4°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 acetonea) 2,09 ethanol b) 0,68 2,77 2-tissues and viscera chlor:MeOH c) 5,95 Determination in triplicates (variation 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) Eolch et al. 1957. TABLE 9. EXTRACTION OF FRESH SHARK LIVER LIPIDS (M. schmitti) _ Exp. No. Technique Yield (%) Total (%) 1- acetonea) 36,39 ethyl acetateb) 4,48 40,87 2- ethyl acetatec) 36,68 3- chlor: MeOHd) 41,86 Determinations in triplicates (variations a) Extraction made with a sample-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. c) Extraction made with a sample-solvent ratio of 1 .9 (w/v), incubated 2h at 4°C. d) Eolch et al. 1957. 21 TABLE 10. EXTRACTION OF FRESH SHARK LIVER LIPIDS (G. galeus). Determinations in triplicates (variations a) Extraction made with a sample-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. c) Extraction made with a sample-solvent ratio of 1 :9 (w/v), incubated 2h at 4°C. d) :Fo!ch et al. 1957. TABLE 11. EXTRACTION OF FRESH SHARK LIVER LIPIDS (Angel Shark) Exp. No. Technique Yield (%) Total (%) 1- 2- 3- acetonea) ethyl acetateb) ethyl acetatec) chlor: MeOH d) 19,23 8,98 28,21 39,22 39,23 Determinations in triplicates (variations a) Extraction made with a sample-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. c> Extraction made with a sample-solvent ratio of 1 :9 (w/v), incubated 2h at 4°C. d) Eolch et al. 1957. TABLE 12. FATTY ACID COMPOSITION (E. pacifica) Data expressed in percentage of total fatty acids (%). 22 TABLE 13. CHARACTERISTICS OF KRILL OIL (E. pacffica) Saponification index Fraction Ic) Fraction IId) Olive oil Wijs iodine index Fraction I c) Fraction IId) Olive oil Cholesterol content (%) Fraction Ic) Fraction II d> Olive oil Volatile matter and moisture levels (%) Fraction Ic) Fraction II d> Peroxide value (meg peroxide/kg oil) Fraction I c) Fraction IId) p-Anisidine value (g"1 absorption) Fraction Ic) Fraction IId) independent laboratorya) handbook 130,6 185,7 192,0 e) — 189,7 185,2 127,2 85,3 e) 172.5 139,2 81,1 2,1 3,7 0,2 e) 1,9 3,0 — 10,0 6,8 — — — 0,0 0,0 — — 0,1 5,5 — a): Professor Robert Ackman's laboratory, Canadian Institute of Fisheries Technology, Halifax, Nova Scotia. b): Harwood and Geyer 1964. c): Extraction made with a sample-acetone ratio of 1:6 (w/v), incubated 2h at 4°C. d): 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. e): Extra virgin olive oil cold compressed from Bertolli ™. 23 TABLE 14. LIPID CLASS COMPOSITION OF KRILL OIL (AREA %) (E. pacifica) Triglycerides Fraction Ia) 19,0±0,7 Fraction IIb) 66,5+2,3 Hydrocarbons Fraction Ia) trace Fraction IIb) 1,3+0,1 Free fatty acids Fraction Ia) 23,7±1,1 Fraction IIb} 20,3+ 0,3 Monoglycerides Fraction I a) 1,4+0,3 Fraction IIb) 0,5+0,1 Phospholipids or other polar material Fraction Ia) 54,1 ±6.1 Fraction IIb) 8,5+1,6 Data from Professor Robert Ackman's laboratory, Canadian Institute of Fisheries Technology, 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. 24 TABLE 15 (continued). FATTY ACID COMPOSITION OF KRILL OIL (WT/WT%) (£. pacifica) Fatty acids Fraction Ia) Fraction IIb) 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 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 20:1 n-7 0,3 0,4 20:1 n-5 0,3 0,4 22:1 n-11+13 0,1 0,2 Monoenes 21,6 26,3 16:2 n-6 0,6 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 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 18:3 n-4 0,2 0,2 18:3 n-3 3,2 3,0 18:3 n-1 0,1 0,1 20:3 n-3 0,1 0,1 Trienes 5,4 4,9 16:4 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 n-6 0,2 0,1 22:5 n-3 0,5 0,3 Pentaenes 18,8 9,5 25 TABLE 15 (continued). FATTY ACID COMPOSITION OF KRILL OIL (WT/WT%) {E. pacifica) ___„ Fatty acids Fraction I a) Fraction IIb) 22:6 n-3 Hexaenes Iodine value calculated 13,2 214,8 6,6 145,1 Data from Professor Robert Ackman's laboratory, Canadian Institute of Fisheries Technology, 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 16, KRILL LIPID FREE FATTY ACID FAME (WT/WT%) (E. pacifica) 12:0 13:0 ISO14:0 14:0 ISO 15:0 ANT 15:0 15:0 ISO 16:0 ANT 16:0 16:0 7MH ANT 17:0 Phytanic 17:0 18:0 20:0 22:0 Saturates 14:1 15:1 16:1 n-9 16:1 n-7 16:1 n-5+H7:0 17.1 18:1 n-9 18:1 n-7 18:1 n-5 20:1 n-11 20:1 n-9 22:1 n-11+13 24:1 n-9 Monoenes Fatty acids Fraction Ia) Fraction IIb) 0,5 0,1 0,2 0,0 0,2 0,2 1,3 2,6 0,3 0,3 0,1 0,1 0,2 0,5 0,1 0,2 0,2 0,1 3.3 10,6 0,6 0,8 0,2 0,2 0,2 0,0 0,5 0,8 0,2 0,6 0,3 0,2 0,0 0,1 8.3 17,4 0,2 0,2 0,2 0,1 0,5 0,0 5,2 " 6,8 0,1 0,1 0,6 0,7 7,0 11,4 4,9 9,3 0,1 0,3 0,2 0,3 0,1 0,3 0,1 0,2 0,0 ' 0,1 19,2 29,8 26 TABLE 16 (continued). KRtLL LIPID FREE FATTY ACID FAME (WT/WT%) (g. pacifica) Fatty acids Fraction Ia) Fraction IIb) 16:2 n-6 0,4 0,9 16:2 n-4 . 1,2 1,0 18:2 n-7 0,1 0,2 18:2 n-6 2,4 2,6 18:2 n-4 0,1 0,1 20:2 n-6 0,1 0,1 Dienes 4,3 4,9 16:3 n-4+l 17:1 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 0,1 20:3 n-6 0,1 0,1 20:3 n-3 0,1 0,2 Trienes 5,7 5,6 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-1 0,1 0,1 20:4n-6 1,7 1,4 20:4 n-3 0,6 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-6 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 14,4 Iodine value calculated 291,6 220,3 Data from Professor Robert Ackman's laboratory, Canadian Institute of Fisheries Technology, Halifax, Nova Scotia. a): Extraction made with a sample-acetone ratio of 1:6 (w/v), incubated 2h at4°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. 27 TABLE 17. TOCOPHEROL, ALL-frans RETINOL AND CHOLECALCIFEROL CONTENT IN KRILL OIL (g. pacifica) ajpha-tocopherol by HPLC (IU) Fraction I a) 0,91 Fraction II b> 0.83 gamma-tocopherol by HPLC uq/g Fraction Ia) Tr Fraction II b) Tr delta-tocopherol by HPLC uq/g Fraction I a) N.D. Fraction II b) N.D. a\\-trans retinol by HPLC (IU) Fraction I a> 395,57 Fraction II b) 440,47 cholecalciferol by HPLC (IU) Fraction Ia) N.D. Fraction IIb> 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 min 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 M-trans retinol ug/g -r 0,3 = International Unit TABLE 18. ASTAXANTHIN AND CANTHAXANTHIN CONTENT OF KRILL OIL (£. pacifica) Asthaxantin (ug/q oil) Fraction I a) 93,1 Fraction II b) 121,7 Canthaxanthin (uq/g oil) Fraction Ia> 270,4 Fraction II b) 733,0 Data from Professor Robert Ackman's laboratory, Canadian Institute of Fisheries Technology, 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. 28 ^8- TABLE 19. OPTIMAL CONDITIONS FOR LIPID EXTRACTION OF AQUATIC ANIMAL TISSUES (suggested procedure) STEP Grinding (if particles > 5mm) Lipid extraction Filtration Washing Filtration Evaporation Oil-water separation Lipid extraction Filtration Evaporation CONDITIONS 4°C sample-acetone ratio of 1:6 (w/v) 2h (including swirling 20 min) 4°C organic solvent resistant filter under reduced pressure sample-acetone ratio of 1:2 (w/v) pure and cold acetone organic solvent resistant filter under reduced pressure under reduced pressure 4°C sample: ethyl acetate ratio of 1:2 (w/v)a> 30 min 4°Cb> organic solvent resistant filter under reduced pressure under reduced pressure a): Ethanol can be replaced by isopropanol, f-butanoi or ethyl acetate. b>: 25 °C when using f-butanol. TABLE 20: 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 (min) Amino acids released (gmoles) Enzymatic rate (umoles/min) 1.917 0.999 0.322 0.308 Specific enzymatic activity 28.76 43.74 98.51 177.26 - (umoles/min/mq*) 15 30 170 255 0.164 0.125 0.050 0.060 total quantity of enzymes in hydrolysis media 29 Claim: 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 preferably acetone, 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 is selected from the group of solvents consisting of alcohol preferable ethanol, isopropanol, t-butanol and esters of acetic acid preferably 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) 2. A method as claimed in claim 1, wherein steps (a) and (d) are conducted under inert atmosphere. 3. A method as claimed in any of the preceding claims, wherein steps (b) and (e) are effected by techniques selected from filtration, centrifugation and sedimentation. 30 4. A method as claimed in any of the preceding claims, wherein steps (c) and (f) are effected by techniques selected from evaporation under reduced pressure, flash evaporation and spray drying. 5. A method as claimed in any of the preceding claims, 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). 6. A method as claimed in any of the preceding claims, 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). 7. A method as claimed in anyone of the preceding claims, wherein said marine and aquatic animal is zooplancton. 8. A method as claimed in claim 7, wherein said zooplancton is krill. 9. A method as claimed in claim 7, wherein said zooplancton is Calanus. 10. A method as in claimed in anyone of claims 1 to 7, wherein said marine and aquatic animal is fish. 11. 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, to achieve an extraction of the soluble lipid fraction from said marine and aquatic animal material; 31 (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. 12. 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. 13. A method as claimed in claim 11 or 12, wherein the animal material is krill. 14. A method as claimed in claim 11 or 12, wherein the animal material is Calanus. 15. A method as claimed in anyone of the preceding claims, wherein the extraction of step (a) is conducted under agitation after the animal material has been ground. 16. A method as claimed in anyone of claims 11 to 15, wherein step (a) conducted under inert atmosphere. 32 17. A method as claimed in anyone of claims 11 to 16, wherein step (b) is effected by a technique selected from filtration, centrifugation and sedimentation. 18. A method as in claimed anyone of claims 11 to 17, wherein step (c) is effected by a technique selected from vacuum evaporation, flash evaporation and spray drying. 19. A method as claimed in anyone of claims 11 to 18, 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). 20. A method as claimed in anyone of the preceding claims, wherein steps (a) and (b) are conducted at solvent temperatures of 5°C or less. 21. A method as claimed in claim 7 or 20, wherein the animal material is divided to an average particle size of less than 5mm. 22. A method of lipid extraction as claimed in any of the preceding claims, wherein the resulting solid contents is recovered and consists of a dehydrated residue containing active enzymes. Dated this 17th Day of April, 2001. [RANJNA MEHTA-DUTT] OF REMFRY & SAGAR ATTORNEY FOR THE APPLICANTS 33

Full Text

FORM 2
THE PATENTS ACT 1970
[39 OF 1970]
COMPLETE SPECIFICATION
[See Section 10]
T "METHOD OF EXTRACTING LIPIDS FROM MARINE AND AQUATIC ANIMAL
UNIVERSITE DE SHERBROOKE, of University Boulevard, Sherbrooke, Quebec J1K 2R1, Canada
The following specification particularly describes and ascertain the nature of the invention and the manner in which it is to be performed :-

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BACKGROUND OF THE INVENTION
This invention relates to the extraction of lipid fractions from marine and aquatic animals such as krill, Calanus, fish and sea mammals. More specifically, this invention relates to an improved method of extracting lipid fractions by dehydration with solvents and recovering a solid residue rich in active enzymes.
Lipid fractions obtained from marine and aquatic animals such as krill, Calanus, fish and sea mammals have various applications:
Medical applications Marine and aquatic animal oils and fractions thereof contain various therapeutic agents. For example, it is reported that various marine and aquatic animal oils have anti-inflammatory properties. Marine and aquatic animal oils are also reported as helpful in reducing the incidence of cardiovascular disease. Also, some marine and aquatic animal oils are reported as suppressing the development of certain forms of lupus and renal diseases. As a further example, krill may be used as a source of enzymes for debridement of ulcers and wounds or to facilitate food digestion. Also marine and aquatic oils contain various antioxidants, which may have potential therapeutic properties.
Nutraceuticals Considering the beneficial effects of omega-3 fatty acids, oils from krill, Calanus and fish could be used as dietary supplements to human diet. These fatty acids are essential for proper development of the brain and the eye. Marine and aquatic animal oils are also rich in liposoluble vitamins A, D and E and carotenoids.
Cosmetics Various marine and aquatic animal oils are used for the production of moisturizing creams.
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Fish farming Among the lipids found in krill, Calanus and fish, high concentrations of fatty acids 20:5 (eicosapentaenoic acid) and 22:6 (docosahexaenoic acid) are present. These fatty acids are essential nutrients and are beneficial as fish feed. Furthermore, these essential nutrients are carried over in human diet by eating the fish grown on such diets.
Animal feed Animal feed diets rich in omega-3 fatty acids may increase the level of unsaturated fatty acids and decrease cholesterol levels of meat. This property is already exploited in the poultry industry to improve the quality of eggs.
Various methods for extracting marine and aquatic animal oils are known. For example, it is 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 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 divided animal products. The extracted oils are then made to precipitate under high pressure and elevated temperatures of 50 to 200°C. However, hexane is a poor extraction solvent for marine animals such as krill. Furthermore, the high temperatures used in the precipitation step negatively alters the lipids.
Canadian Patent Application 2,115,571 describes a method for extracting oils from various brown and(i^aa)algae species. The method provides for example Soxhlet extraction using nearly pure ethanol for 40 hours.
USP 5,006,281 describes a method for extracting oil from marine and aquatic animals such as fish. The marine and aquatic animal is first treated with an antioxidant compound, finely divided and centrifuged to separate the oil phase from
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the aqueous phase and solid phase. The oil phase is then further treated with 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 and separate recovery of a valuable protein rich solid residue that comprises active enzymes.
Other objects and further scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that this detailed description, while indicating preferred embodiments of the invention, is given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Gas-liquid chromatography 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 (f-butanol)

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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 chromatography of neutral lipids of G. galeus Figure 11. Thin-layer chromatography of neutral lipids of Angel Shark Figure 12. Thin-layer chromatography of phospholipids of Calanus sp. and
M. norvegica Figure 13. Thin-layer chromatography of phospholipids of E. pacifica Figure 14. Thin-layer chromatography of phospholipids of M. schmitti Figure 15. Thin-layer chromatography of phospholipids of G. galeus Figure 16. Thin-layer chromatography 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 and not limitation.
The method of the invention comprises suspending freshly collected marine and aquatic material in acetone. Lipids are extracted with a ketone such as acetone. This allows a rapid dehydration of animal tissue and a migration of the lipid fraction to the solvent. The dry residue is a valuable product rich in active enzymes.
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In a preferred embodiment, the extraction is carried out by successive acetone and alcohol treatments. Preferred alcohols are isopropanol, and f-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 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, Calanus, 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 zooplancton such as krill and Calanus and in the case of fish-filleting by-products such as fish viscera, it is noted that extraction with acetone alone may 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

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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 used.
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, f-butanol or alternatively with ethyl acetate, preferably two volumes (original volume of material). The filtrate is evaporated leaving a second fraction of lipids (identified 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 organic solvents, except f-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 (Folch et al. 1957) using chloroform and methanol was applied to krill. this method is the reference for measuring efficiency of the extraction process. Another comparison has been made with a technique using hexane as the extraction solvent. Lipid recovery
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was estimated by suspending lipid fractions in small volumes of their original solvents and measuring by gravimetry small aliquots after evaporation.
For all examples provided herein, the method of the present invention involving acetone extraction followed by extraction with a second solvent (ethyl acetate, for example) gave a translucent oil having appearance and properties more attractive than any oil obtained by the classical technique of Folch et al. (1957).
To analyze lipid composition, 780 ug of each extract was loaded on silica-gel plates and fractionated by thin layer chromatography, TLC (Bowyer et al. 1962) with the following solvents. Neutral lipids: hexane, ethyl ether, acetic acid (90:10:1, v/v) and phospholipids: chloroform, methanol, water (80:25:2, v/v). Fatty acid composition of E. pacifica was analyzed by gas liquid chromatography, GLC (Bowyer et al. 1962, see bibliography) including some modifications to the original technique: 2h at 65°C instead of 1 h at 80°C, three washes with hexane instead of two and no wash with water.
To get rid of traces of organic solvents, lipid fractions I and II are warmed to about 125°C for about 15 minutes under inert atmosphere.
Fat was analyzed according to the American Oil Chemist'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 content has also been determined by the method of Plummer 1987 (see bibliography). The same analyzes and others have been made by an independent laboratory under Professor Robert Ackman's supervision (Canadian Instituteof Fisheries Technology, DalTech, Dalhousie University, Halifax, Nova Scotia, Canada). This includes Wijs iodine index, peroxide and anisidine values, lipid class composition, fatty acid composition, free fatty acid FAME, cholesterol, tocopherol, a\\-trans retinol, cholecalciferol, asthaxanthin and canthaxantin contents.

8

Table 1 shows that higher levels of lipids are extracted from dry krill by acetone followed by ethanol as compared to the classical procedure of Folch et al. (1957).
Table 2 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, f-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, f-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. This 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 solid 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 Bacto™ beef extract 0,3%, Bacto™ peptone 0,5% and Bacto™ agar 1,5% (Difco Laboratories, Detroit, USA) then incubated at room temperature or 4°C for 18 days. No significant bacterial growth was observed at a ratio of 1 volume of acetone per gram of krill. At higher proportions of acetone (2 volumes and 5 volumes), there was no bacterial growth at all, which means that acetone preserves krill samples. Acetone is known as an efficient bactericidal and viricidal agent (Goodman et al. 1980).

9

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 experiments 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 method. Indeed small amounts of lipids from mackerel (0.52% from viscera and 1,45% from tissues) have been extracted by the method of Folch after a first extraction with acetone and ethanol as described in the present invention. Comparative extractions with the method described in the present invention carried out in parallel with the method of Folch on trout and herring show superior recovery with the latter. However, it is noteworthy that the Folch method can not be applied for the recovery of lipids for commercial uses (because of toxicity).

10

In Tables 9 to 11, are shown results of lipids extraction from shark liver tissues. There is no marked difference in results between techniques within a species. Table 12 shows the fatty acid composition of krill oil (e. pacifica) following extraction in various solvents.
Tables 13 shows some characteristics features of fraction I (acetone) and fraction II (alcohol or ethyl acetate) for krill oil (e. pacifica). First, the saponification index of fraction I (130,6) indicates that this fraction contains fatty acids with longer chains, compared to fraction II (185,7). The Wijs iodine index of fraction I shows that this 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 fraction II which has a iodine index of 127,2. The fatty acid composition shown in Table 12 corroborates these iodine indexes: fraction I has a high percentage (30,24%) of polyunsaturated fatty acids (pentaenes+hexaenes) and so fraction II (22,98%). Finally, Table 13 shows also that fraction I is comprised of 10,0% of volatile matter and humidity after evaporation of the solvent. 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 krill oils obtained in accordance with the method of the present invention (fraction I extracted with acetone and fraction II extracted with ethyl acetate) are provided in Tables 13, 14, 15, 16, 17 and 18. It is noteworthy to mention that in Table 18, the carotenoids content was significantly high as measured in terms of two carotenoids namely asthaxanthin and canthaxanthin. indeed, duplicates analyzes revealed values of 92 to 124 pg/g of lipid fraction for asthaxanthin and 262 to 734 pg/g for canthaxanthin. Thus, for the purpose of the present invention it may be said that the krill extract comprises asthaxanthin at least 75 and preferably at least 90 ug/g of lipid fraction. In the case of canthaxanthin, at least 250 and preferably at least 270 pg/g of lipid fraction. Low values for peroxide and anisidine are advantageous and are due to the presence of high levels of natural antioxidants

11

(astaxanthin and canthaxanthin). These compounds are indicative of favourable pharmaceutical or cosmetological properties of the krill extract whereby high levels of carotenoids indicate excellent transdermal migration characteristics. Thus, krill extract is a good candidative for transdermal delivery of medicines.
Table 19 shows the best mode of the method in accordance with the present invention for lipid extraction of aquatic animal tissues.
Table 20 shows that the enzyme activity of the solid fraction is maintained following the method of the present invention. Indeed, the demonstration was completed for solid krill residue obtained after successive acetone and ethyl acetate extraction. Proteolytic activities were measure by the liberation of amino groups by spectrophotometric assay using o-pthaldialdehyde as reagent. Protein concentrations were measured by the Bradford method. 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 50mM potassium phosphate buffer, trichloroacetic acid was added and the amount of NH3 group was measured in the supernatant according to the method of Church et al. [1983, J Dairy Sci 66: 1219-1227].
Figures 1 to 6 show chromatograms of fatty acid composition of E. pacifica lipids. On each of them, high proportions of 20:5 and 22:6 fatty acids (characteristic of marine and aquatic oils) are noticeable and represented by two distinct peaks. Data are shown in Table 12.
Variations in lipid patterns 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)

12

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.

13

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 comparison with column chromatography. BBA. 70:423-431.
Chandrasekar, B., Troyer, D.A., Venkatraman, J.T. and Femandes, G. 1996. Tissue specific regulation of transforming growth factor beta by omega-3 lipid-rich krill oil in autoimmune murine lupus. Nutr Res. 16(3): 489-503.
Christensen, M.S., 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. 10th 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 Macmilian Canada ltd, Toronto.
Harwood, HJ. 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): 102-103

14

Plummer, D.T. 1987. An introduction to practical biochemistry. 3th ed. McGraw-Hill Book Company, London.
Rawn, J.D. 1990. Traite de biochimie. De Boeck-Wesmael, Bruxelles.
Runge, J.A. and Joly, P. 1994. Rapport sur I'etat des invertebres en 1994: 7:0 Zooplancton (Euphausiaces et Calanus) de I'Estuaire et du Golfe du Saint-Laurent. Sargent JR. 1997. Fish oils and human diet Br J Nutr.78 Suppl 1: S5-S13.

15

TABLE 1. EXTRACTION OF DRY KRILL LIPIDS (E. pacifica)
Exp. No. Technique Yield (%) Total (%) Mean (%) ± s.d.
1- acetonea) ethanolb) 8,00 7,60 15,60
2- tt 19,70 6,90 26,60
3- it 8,15 11,20 19,35
4- tt 6,80 13,60 20,40 20,49+3,95
5- chlor: MeOHc) 15,50
6- it 14,90 15,20+0,30
Determinations in triplicates (variation 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 at4°C, following
a first extraction with acetone.
c) :Fo!ch et al. 1957.
TABLE 2. EXTRACTION OF FROZEN KRILL LIPIDS (E. pacifica)
Exp. No. Technique Yield (%) Total (%) Mean (%) + s.d.
1- acetonea) 1,17
ethanolb) 1,23 2,40
2- tt 3,05 1,09 4,14
3- tt 1,53 1,26 2,79 3,11+0,91
4- acetonea) 2,45
isopropanolb) 0,70 3,15
5- . " 1,80 0,80 2,60
6- t" 1,60 0,80 2,40 2,72±0,39
16

TABLE 2 (continued). EXTRACTION OF FROZEN KRILL LIPIDS (E. pacifica)
Exp. No. Technique Yield (%) Total (%) Mean (%) ± s.d.
7- acetonea) t-butanolc> 2,15 0,47 2,62
8- tf 2,11 0,40 2,51
9-10- rr
acetonea) ethyl acetateb) 2,37 0,45
2,28 0,21 2,82 2,49 2,65+0,16
11- rr 1,09 0,16 1,25
12- rr 2,54 0,09 2,63 2,12+0,76
13- combined acetone-ethanol ^ 3,28
14- " 3,02
15- " 3,25
3,18±0,14
16- ethyl acetatee) 1,32
17- " 1,49
18- " 1,31
1,37+0,10

19- hexanee) 0,31
20- " 0,18
21- " 0,20
22- chlonMeOH ° 2,37

17

0,23±0,07

TABLE 2 (continued). EXTRACTION OF FROZEN KR1LL LIPIDS (E. pacifica)
Exp. No. Technique Yield (%) Total (%) Mean (%) ± s.d.
23- " 2,07
24- " 2,62
2,35±0,28
Determinations in triplicates (variation 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 sample-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.
0: Folch et al. 1957.
TABLE 3. EXTRACTION OF FROZEN KRILL LlPIDS(M. norvegica)
Exp. No. Technique Yield (%) Total (%) Mean (%) ± s.d.
1- acetonea) ethanolb) 1,82 1,82 3,64
2- rt 1,15 2,35 3,50
3- ft 1,68 2,19 3,87 3,67±0,15
Determinations in triplicates (variation 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.
18

TABLE 4. INFLUENCE OF GRINDING ON EXTRACTION OF FROZEN KRILL LIPIDS
(M. norvegica)
yes
no
yes
yes yes
acetone ' ethano!b)
2-
3-
4-5-
chlor:MeOHc>
Exp. No. Technique Krill ground before 1st extraction
Yield (%) Total (%)
3,10 1,07 4,17
2,14 1,39 3,53
3,32 1,14 4,46 3,30 3,26
Determinations in triplicates (variation a) .Extraction made with a sample-solvent ratio of 1.6, incubated 2 h at4°C.
b> Extraction made with a sample-solvent ratio of 1:2, incubated 30 min at 4°C, following a first
extraction with acetone. c) .Folch et al. 1957.
TABLE 5. EXTRACTION OF FROZEN Calanus LIPIDS (Calanus sp.)

Exp. No. 1-
2-

Technique
acetonea) ethanolb>

Yield (%) Total (%) Mean (%) ± s.d.
6,18 2,04 8,22
8,64 2,26 10,90
9,56+1,34

Determinations in triplicates (variation 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.

19

WO U0y23i>46

FCT/CA99/00987'

^rET"
TABLE 6. EXTRACTION OF FRESH FISH LIPIDS (Mackerel)
Exp. No. Technique Yield (%) Total (%)
1-viscera acetonea) 6,11
fish 1 ethanolb> 0,59 6,70
2- tissues tt 3,78
fish 1 0,91 4,69
3- viscera it 10,46
fish 2 0,57 11,03
4- issues IT 6,65
fish 2 1,41 8,06
5- viscera tt 8,39
fish 3 0,66 9,05
6- tissues It 5,27
fish 3 0,97 6,24
7- viscera II 8,47
fish 4 0,69 9,16
8- tissues ft 8,40
fish 4 1,02 9,42
9- viscera chlonMeOH c) 0,52
fish 1
10-tissues // 1,45
fish 1
3) :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 1h at 4°C, following a first
extraction with acetone.
b) :Folch et al. 1957, following extractions with acetone, then ethanol..

20

TABLE 7. EXTRACTION OF FRESH FfSH LIPIDS (Trout)
Exp. No. Technique Yield (%) Total (%)
1- viscera acetonea) 34,70
ethanolb) 2,18 36,88
2- tissues tt 5,53 1,17 6,70
3- viscera chlor.MeOH c) 39,81
4- tissues tt 14,93
Determinations in triplicates (variation a) :Extraction made with a sample-solvent ratio of 1:9 (w/v), incubated 1 night at4°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 acetonea) 2,09
ethanol b) 0,68 2,77
2-tissues and viscera chlor:MeOH c) 5,95
Determination in triplicates (variation 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) Eolch et al. 1957.
TABLE 9. EXTRACTION OF FRESH SHARK LIVER LIPIDS (M. schmitti) _
Exp. No. Technique Yield (%) Total (%)
1- acetonea) 36,39
ethyl acetateb) 4,48 40,87
2- ethyl acetatec) 36,68
3- chlor: MeOHd) 41,86
Determinations in triplicates (variations a) Extraction made with a sample-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.
c) Extraction made with a sample-solvent ratio of 1 .9 (w/v), incubated 2h at 4°C.
d) Eolch et al. 1957.

21

TABLE 10. EXTRACTION OF FRESH SHARK LIVER LIPIDS (G. galeus).

Determinations in triplicates (variations a) Extraction made with a sample-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.
c) Extraction made with a sample-solvent ratio of 1 :9 (w/v), incubated 2h at 4°C.
d) :Fo!ch et al. 1957.
TABLE 11. EXTRACTION OF FRESH SHARK LIVER LIPIDS (Angel Shark)
Exp. No. Technique Yield (%) Total (%)

1-
2-
3-

acetonea) ethyl acetateb)
ethyl acetatec)
chlor: MeOH d)

19,23 8,98

28,21 39,22 39,23

Determinations in triplicates (variations a) Extraction made with a sample-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.
c> Extraction made with a sample-solvent ratio of 1 :9 (w/v), incubated 2h at 4°C. d) Eolch et al. 1957.
TABLE 12. FATTY ACID COMPOSITION (E. pacifica)

Data expressed in percentage of total fatty acids (%).

22

TABLE 13. CHARACTERISTICS OF KRILL OIL (E. pacffica)

Saponification index
Fraction Ic) Fraction IId) Olive oil
Wijs iodine index
Fraction I c) Fraction IId) Olive oil
Cholesterol content (%)
Fraction Ic) Fraction II d> Olive oil
Volatile matter and moisture levels (%)
Fraction Ic) Fraction II d>
Peroxide value (meg peroxide/kg oil)
Fraction I c) Fraction IId)
p-Anisidine value (g"1 absorption)
Fraction Ic) Fraction IId)

independent laboratorya) handbook
130,6 185,7 192,0 e) — 189,7
185,2 127,2 85,3 e) 172.5 139,2 81,1
2,1 3,7 0,2 e) 1,9 3,0 —
10,0 6,8 — —
— 0,0 0,0 —
— 0,1 5,5 —

a): Professor Robert Ackman's laboratory, Canadian Institute of Fisheries Technology,
Halifax, Nova Scotia. b): Harwood and Geyer 1964.
c): Extraction made with a sample-acetone ratio of 1:6 (w/v), incubated 2h at 4°C. d): 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. e): Extra virgin olive oil cold compressed from Bertolli ™.
23

TABLE 14. LIPID CLASS COMPOSITION OF KRILL OIL (AREA %) (E. pacifica)
Triglycerides
Fraction Ia) 19,0±0,7
Fraction IIb) 66,5+2,3
Hydrocarbons
Fraction Ia) trace
Fraction IIb) 1,3+0,1
Free fatty acids
Fraction Ia) 23,7±1,1
Fraction IIb} 20,3+ 0,3
Monoglycerides
Fraction I a) 1,4+0,3
Fraction IIb) 0,5+0,1
Phospholipids or other polar material
Fraction Ia) 54,1 ±6.1
Fraction IIb) 8,5+1,6
Data from Professor Robert Ackman's laboratory, Canadian Institute of Fisheries Technology, 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.
24

TABLE 15 (continued). FATTY ACID COMPOSITION OF KRILL OIL (WT/WT%)
(£. pacifica)
Fatty acids Fraction Ia) Fraction IIb)
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
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
20:1 n-7 0,3 0,4
20:1 n-5 0,3 0,4
22:1 n-11+13 0,1 0,2
Monoenes 21,6 26,3
16:2 n-6 0,6 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
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
18:3 n-4 0,2 0,2
18:3 n-3 3,2 3,0
18:3 n-1 0,1 0,1
20:3 n-3 0,1 0,1
Trienes 5,4 4,9
16:4 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 n-6 0,2 0,1
22:5 n-3 0,5 0,3
Pentaenes 18,8 9,5
25

TABLE 15 (continued). FATTY ACID COMPOSITION OF KRILL OIL (WT/WT%)
{E. pacifica) ___„

Fatty acids

Fraction I a)

Fraction IIb)

22:6 n-3 Hexaenes
Iodine value calculated

13,2
214,8

6,6 145,1

Data from Professor Robert Ackman's laboratory, Canadian Institute of Fisheries Technology, 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 16, KRILL LIPID FREE FATTY ACID FAME (WT/WT%) (E. pacifica)
12:0
13:0
ISO14:0
14:0
ISO 15:0
ANT 15:0
15:0
ISO 16:0
ANT 16:0
16:0
7MH
ANT 17:0
Phytanic
17:0
18:0
20:0
22:0
Saturates
14:1
15:1
16:1 n-9
16:1 n-7
16:1 n-5+H7:0
17.1
18:1 n-9
18:1 n-7
18:1 n-5
20:1 n-11
20:1 n-9
22:1 n-11+13
24:1 n-9
Monoenes
Fatty acids Fraction Ia) Fraction IIb)
0,5 0,1
0,2 0,0
0,2 0,2
1,3 2,6
0,3 0,3
0,1 0,1
0,2 0,5
0,1 0,2
0,2 0,1
3.3 10,6 0,6 0,8 0,2 0,2 0,2 0,0 0,5 0,8 0,2 0,6 0,3 0,2 0,0 0,1
8.3 17,4
0,2 0,2
0,2 0,1
0,5 0,0
5,2 " 6,8
0,1 0,1
0,6 0,7
7,0 11,4
4,9 9,3
0,1 0,3
0,2 0,3
0,1 0,3
0,1 0,2
0,0 ' 0,1
19,2 29,8
26

TABLE 16 (continued). KRtLL LIPID FREE FATTY ACID FAME (WT/WT%) (g. pacifica)
Fatty acids Fraction Ia) Fraction IIb)

16:2 n-6 0,4 0,9
16:2 n-4 . 1,2 1,0
18:2 n-7 0,1 0,2
18:2 n-6 2,4 2,6
18:2 n-4 0,1 0,1
20:2 n-6 0,1 0,1
Dienes 4,3 4,9
16:3 n-4+l 17:1 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 0,1
20:3 n-6 0,1 0,1
20:3 n-3 0,1 0,2
Trienes 5,7 5,6
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-1 0,1 0,1
20:4n-6 1,7 1,4
20:4 n-3 0,6 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-6 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 14,4
Iodine value calculated 291,6 220,3
Data from Professor Robert Ackman's laboratory, Canadian Institute of Fisheries Technology, Halifax, Nova Scotia.
a): Extraction made with a sample-acetone ratio of 1:6 (w/v), incubated 2h at4°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.
27

TABLE 17. TOCOPHEROL, ALL-frans RETINOL AND CHOLECALCIFEROL CONTENT
IN KRILL OIL (g. pacifica)
ajpha-tocopherol by HPLC (IU)
Fraction I a) 0,91
Fraction II b> 0.83
gamma-tocopherol by HPLC uq/g
Fraction Ia) Tr
Fraction II b) Tr
delta-tocopherol by HPLC uq/g
Fraction I a) N.D.
Fraction II b) N.D.
a\\-trans retinol by HPLC (IU)
Fraction I a> 395,57
Fraction II b) 440,47
cholecalciferol by HPLC (IU)
Fraction Ia) N.D.
Fraction IIb> 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 min 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
M-trans retinol ug/g -r 0,3 = International Unit
TABLE 18. ASTAXANTHIN AND CANTHAXANTHIN CONTENT OF KRILL OIL
(£. pacifica)
Asthaxantin (ug/q oil)
Fraction I a) 93,1
Fraction II b) 121,7
Canthaxanthin (uq/g oil)
Fraction Ia> 270,4
Fraction II b) 733,0
Data from Professor Robert Ackman's laboratory, Canadian Institute of Fisheries Technology, 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.
28

^8-
TABLE 19. OPTIMAL CONDITIONS FOR LIPID EXTRACTION OF AQUATIC ANIMAL
TISSUES (suggested procedure)

STEP
Grinding (if particles > 5mm)
Lipid extraction
Filtration
Washing
Filtration
Evaporation Oil-water separation Lipid extraction
Filtration
Evaporation

CONDITIONS
4°C
sample-acetone ratio of 1:6 (w/v) 2h (including swirling 20 min) 4°C
organic solvent resistant filter under reduced pressure
sample-acetone ratio of 1:2 (w/v) pure and cold acetone
organic solvent resistant filter under reduced pressure
under reduced pressure
4°C
sample: ethyl acetate ratio of 1:2 (w/v)a>
30 min 4°Cb>
organic solvent resistant filter under reduced pressure
under reduced pressure

a): Ethanol can be replaced by isopropanol, f-butanoi or ethyl acetate. b>: 25 °C when using f-butanol.
TABLE 20: 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 (min) Amino acids released (gmoles) Enzymatic rate (umoles/min)
1.917 0.999 0.322 0.308 Specific enzymatic activity
28.76 43.74 98.51 177.26
- (umoles/min/mq*)
15 30 170 255

0.164 0.125 0.050 0.060
total quantity of enzymes in hydrolysis media
29

Claim:
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 preferably acetone, 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 is selected from the group of solvents consisting of alcohol preferable ethanol, isopropanol, t-butanol and esters of acetic acid preferably 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)

2. A method as claimed in claim 1, wherein steps (a) and (d) are conducted under inert atmosphere.
3. A method as claimed in any of the preceding claims, wherein steps (b) and (e) are effected by techniques selected from filtration, centrifugation and sedimentation.

30

4. A method as claimed in any of the preceding claims, wherein steps (c) and (f) are effected by techniques selected from evaporation under reduced pressure, flash evaporation and spray drying.
5. A method as claimed in any of the preceding claims, 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).
6. A method as claimed in any of the preceding claims, 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).
7. A method as claimed in anyone of the preceding claims, wherein said marine and aquatic animal is zooplancton.
8. A method as claimed in claim 7, wherein said zooplancton is krill.
9. A method as claimed in claim 7, wherein said zooplancton is Calanus.
10. A method as in claimed in anyone of claims 1 to 7, wherein said marine and aquatic animal is fish.
11. 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, to achieve an extraction of the soluble lipid fraction from said marine and aquatic animal material;

31

(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.
12. 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.
13. A method as claimed in claim 11 or 12, wherein the animal material is krill.
14. A method as claimed in claim 11 or 12, wherein the animal material is Calanus.
15. A method as claimed in anyone of the preceding claims, wherein the extraction of step (a) is conducted under agitation after the animal material has been ground.
16. A method as claimed in anyone of claims 11 to 15, wherein step (a) conducted under inert atmosphere.

32

17. A method as claimed in anyone of claims 11 to 16, wherein step (b) is
effected by a technique selected from filtration, centrifugation and
sedimentation.
18. A method as in claimed anyone of claims 11 to 17, wherein step (c) is
effected by a technique selected from vacuum evaporation, flash
evaporation and spray drying.
19. A method as claimed in anyone of claims 11 to 18, 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).
20. A method as claimed in anyone of the preceding claims, wherein steps
(a) and (b) are conducted at solvent temperatures of 5°C or less.
21. A method as claimed in claim 7 or 20, wherein the animal material is divided to an average particle size of less than 5mm.
22. A method of lipid extraction as claimed in any of the preceding claims,
wherein the resulting solid contents is recovered and consists of a
dehydrated residue containing active enzymes.
Dated this 17th Day of April, 2001.
[RANJNA MEHTA-DUTT]
OF REMFRY & SAGAR
ATTORNEY FOR THE APPLICANTS

33

Documents:

abstract1.jpg

in-pct-2001-00408-mum-cancelled pages(17-4-2001).pdf

in-pct-2001-00408-mum-claims(granted)-(17-4-2001).pdf

in-pct-2001-00408-mum-correspondence(25-4-2005).pdf

in-pct-2001-00408-mum-correspondence(ipo)-(23-7-2007).pdf

in-pct-2001-00408-mum-drawing(17-4-2001).pdf

in-pct-2001-00408-mum-form 1(17-4-2001).pdf

in-pct-2001-00408-mum-form 19(26-3-2004).pdf

in-pct-2001-00408-mum-form 1a(6-4-2005).pdf

in-pct-2001-00408-mum-form 2(granted)-(17-4-2001).pdf

in-pct-2001-00408-mum-form 3(15-4-2005).pdf

in-pct-2001-00408-mum-form 3(17-4-2001).pdf

in-pct-2001-00408-mum-form 5(17-4-2001).pdf

in-pct-2001-00408-mum-form-pct-isa-210(17-4-2001).pdf

in-pct-2001-00408-mum-petition under rule 137(15-4-2005).pdf

in-pct-2001-00408-mum-petition under rule 138(15-4-2005).pdf

in-pct-2001-00408-mum-power of authority(17-4-2001).pdf

in-pct-2001-00408-mum-power of authority(6-4-2005).pdf

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Patent Number

208339

Indian Patent Application Number

IN/PCT/2001/00408/MUM

PG Journal Number

32/2007

Publication Date

10-Aug-2007

Grant Date

23-Jul-2007

Date of Filing

17-Apr-2001

Name of Patentee

UNIVERSITE DE SHERBROOKE

Applicant Address

UNIVERSITY BOULEVARD, SHERBROOKE, QUEBEC J1K 2R1,

Inventors:

#	Inventor's Name	Inventor's Address
1	ADRIEN BEAUDOIN	748 BOULEVARD DES VETERANS, ROCK FOREST QUEBEC J1N 1Z7,
2	GENEVIEVE MARTIN	797 MC MANAMY, SHERBROOKE, QUEBEC J1H 2NI,

PCT International Classification Number

C11B 1/10

PCT International Application Number

PCT/CA99/00987

PCT International Filing date

1999-10-21

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	2,251,265	1998-10-21	Canada