HK1038765B - Method of extracting lipids from marine and aquatic animal tissues - Google Patents

Description

FIELD 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.

BACKGROUND OF THE INVENTION

Lipid fractions obtained from marine and aquatic animals such as krill, Calanus, fish and sea mammals have various applications:

Medical applications

Marine and aquatic animal oils and fractions thereof contain various therapeutic agents. For example, it is reported that various marine and aquatic animal oils have anti-inflammatory properties. Marine and aquatic animal oils are also reported as helpful in reducing the incidence of cardiovascular disease. Also, some marine and aquatic 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.

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 read algae species. The method provides for example Soxhlet extraction using nearly pure ethanol for 40 hours.

USP 5,006,281 describes a method for extracting oil from marine and aquatic animals such as fish. The marine and aquatic animal is first treated with an antioxidant compound, finely divided and centrifuged to separate the oil phase from the aqueous phase and solid phase. The oil phase is then further treated with antioxidant to remove undesirable odour or taste.

Canadian Patent 1,098,900 describes a method for extracting oils from krill. The method involves emulsifying fresh or defrosted krill in an aqueous medium. The oil fraction is recovered by centrifugation.

Folch in the article published in the year 1957 in J. biol. Chem. 226: 497-509 "A simple method for the isolation and purification of total lipids from animal tissues " proposes an extraction method using chloroform and methanol. This method is not commercially feasible because of the toxicity of the solvents involved.

Chem Abs. 177859 describes a procedure for extraction of the dye astaxanthine, which is used as a source of colour in salmon fish farming, from shrimp waste or krill, comprising mixing the raw material with triglyeride oil and pressing the raw material to extract the astaxanthine-enriched oil.

However, prior art processes are generally commercially unfeasible, or provide low quantitative yields.

The present invention provides 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 selected from the group of solvents consisting of alcohol preferably ethanol, isopropanol or t-butanol, and ester 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); and (f) recovering a second total lipid rich fraction by evaporation of the solvent from the liquid contents of e).

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.

The present invention also provides a method for extracting an astaxanthin-andcanthaxantin-containing total lipid fraction from a marine and aquatic animal material selected from zooplankton and fish, said method comprising the steps of: (a) placing said animal material in a ketone solvent, preferably acetone to achieve an extraction of the soluble lipid fraction from said marine and aquatic animal material; (b) separating liquid and solid contents resulting from step (a); (c) recovering a lipid rich fraction from the liquid contents by evaporation of the solvent present in the liquid contents; whereby an astaxanthin-and-canthaxantin-containing total liquid fraction is obtained.

The present invention also provides a method for extracting a total lipid fraction from a marine and aquatic animal material selected from zooplankton 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.

The present invention also provides a total krill lipid extract characterized in that it is devoid of toxic solvent and in that the carotenoid content in asthaxathin is 75µg/g or more and preferably at least about 90 µg/g or more of krill extract, and the carotenoid content in canthaxanthin is 250 µg/g or more and preferably at least about 270 µg/g of krill extract or more.

The present invention also provides a krill lipid extract characterised in that it is edible, and in that the carotenoid in astaxanthin is 75ug/g or more and preferably at least about 90 µg/g or more of krill extract, and the carotenoid content in canthaxanthin is 250 µg/g or more and preferably at least about 270µg/g of krill extract or more.

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 scope of the invention will become apparent to those skilled in the art.

Figure 1.

Gas-liquid chromatography of fatty acids from dry krill (chloroformmethanol)

Figure 2.

Gas-liquid chromatography of fatty acids from dry krill (acetone)

Figure 3.

Gas-liquid chromatography of fatty acids from frozen krill (acetone)

Figure 4.

Gas-liquid chromatography of fatty acids from frozen krill (ethanol)

Figure 5.

Gas-liquid chromatography of fatty acids from frozen krill (t-butanol)

Figure 6.

Gas-liquid chromatography of fatty acids from frozen krill (ethyl acetate)

Figure 7.

Thin-layer chromatography of neutral lipids of Calanus sp. and M. norvegica

Figure 8.

Thin-layer chromatography of neutral lipids of E. pacifica

Figure 9.

Thin-layer chromatography of neutral lipids of M. schmitti

Figure 10.

Thin-layer 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.

In a preferred embodiment, the extraction is carried out by successive acetone and alcohol treatments. Preferred alcohols are isopropanol, and t-butanol. The alcohol may also be substituted with an ester of acetic acid such as ethyl acetate. The procedure produces two successive lipid fractions and a dry residue enriched in 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 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, t-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 t-butanol, and temperature of the sample are not critical parameters, but it is preferable to be as cold as possible. However, in the case of t-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 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 µg 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) including some modifications to the original technique: 2h at 65°C instead of 1h at 80°C, three washes with hexane instead of two and no wash with water.

To get rid of 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. The same analyzes and others have been made by an independent laboratory under Professor Robert Ackman's supervision (Canadian Institute of Fisheries Technology, DalTech, Dalhousie University, Halifax, Nova Scotia. Canada). This includes Wijs iodine index, peroxide and anisidine values, lipid class composition, fatty acid composition, free fatty acid FAME, cholesterol, tocopherol, all-trans retinol, cholecalciferol, astaxanthin and canthaxantin contents.

Table 1 shows that higher levels of lipids are extracted from dry krill by acetone followed by ethanol as compared to the classical procedure of Folch et al. (1957).

Table 2 shows the results of lipid extraction from frozen Euphausia pacifica, a species of krill from Pacific Ocean. Assuming an eighty percent content of water, the lipid content is comparable to dry krill as shown in Table 1. Isopropanol, t-butanol and ethyl acetate, as solvent for the second extraction, give a yield less important than ethanol, but are not necessarily less effective in lipid recovery since ethanol carries more impurities than isopropanol, t-butanol or ethyl acetate. Then, they can be used as second solvent after acetone as well. Variations between results from acetone extractions are mainly due to the water-oil separations. These separations are influenced by the quantity of residual acetone in the water-oil solution after acetone evaporation. 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 BactoTM 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).

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).

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.

Tables 12 shows some characteristics features of fraction I (acetone) and fraction II (alcohol or ethyl acetate) for krill oil (e. pacifica). First, the saponification index 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 14 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 12 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 12, 13, 14, 15, 16 and 17. It is noteworthy to mention that in Table 17, the carotenoids content was significantly high as measured in terms of two carotenoids namely astaxanthin and canthaxanthin. Indeed, duplicates analyzes revealed values of 92 to 124 µg/g of lipid fraction for astaxanthin and 262 to 734 µg/g for canthaxanthin. Thus, for the purpose of the present invention it may be said that the krill extract comprises astaxanthin at least 75 and preferably at least 90 ug/g of lipid fraction. In the case of canthaxanthin, at least 250 and preferably at least 270 µg/g of lipid fraction. Low values for peroxide and anisidine are advantageous and are due to the presence of high levels of natural antioxidants (astaxanthin and canthaxanthin). These compounds are indicative of favourable pharmaceutical or cosmetological properties of the krill extract whereby high levels of carotenoids indicate excellent transdermal migration characteristics. Thus, krill extract is a good candidative for transdermal delivery of medicines.

Table 18 shows the best mode of the method in accordance with the present invention for lipid extraction of aquatic animal tissues.

Table 19 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 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.

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) and commercial cod-liver oil (sample available from Uniprix drugstores, Province of Québec, Canada) are mainly composed of neutral lipids as opposed to phospholipids.

The influence of the volume of solvent and incubation time on the efficiency of the acetone to extract lipids from E. pacifica is illustrated in Figures 17 and 18, respectively. A ratio of 1:6 (w/v) produced optimal yield with near complete extraction after 2h. The second extraction step has been experimented with ethanol. The volume of this solvent does not appear to be critical since the same yield was obtained with different volumes of ethanol (Figure 19), but incubations time in ethanol should be at least 30 minutes as indicated by the results on Figure 20.

One of the inventors, Dr. Adrien Beaudoin, has ingested the different lipid fractions of krill. No side effect profile was observed.

Although the invention has been described above with respect with one specific form, it will be evident to a person skilled in the art that it may be modified and refined in various ways. It is therefore wished to have it understood that the present invention should not be limited in scope, except by the terms of the following claims.

Demonstration that krill residue, obtained after acetone and ethyl acetate extraction, contains enzyme proteolytic activities. Proteolytic activities were measured by the liberation of amino groups by spectrophotometric assay using o-phthaldialdehyde as reagent. Protein concentrations were measured by the Bradford method.

The enzyme source was the residue obtained after acetone and ethyl acetate extractions of lipids. Soluble proteins were extracted with water and added to a 10% lactoserum protein concentrate obtained by ultrafiltration.

At the end of incubation at 37°C in 50 mM potassium phosphate buffer, trichloroacetic acid was added and the amount of NH₃ groups were measured in the supernatant according to Church and al. 1983.

BIBLIOGRAPHY

Bowyer, D.E., Leat, W.M.F., Howard, A.N. and Gresham, G.A. 1962. The determination of the fatty acid composition of serum lipids separated by thin-layer chromatography; and a comparison with column chromatography. BBA. 70: 423-431.

Chandrasekar, B., Troyer, D.A., Venkatraman, J.T. and Fernandes, G. 1996. Tissue specific regulation of transforming growth factor beta by omega-3 lipid-rich krill oil in autoimmune murine lupus. Nutr Res. 16(3): 489-503.

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 Macmillan Canada Itd, Toronto.

Harwood, H.J. and Geyer, R.P. 1964. Biology Data Book. The Federation of American Societies for Experimental Biology, Washington.

Hellgren, L., Karlstam, B., Mohr, V. and Vincent, J. 1991. Krill enzymes. A new concept for efficient debridement of necrotic ulcers. Int J Dermatol. 30(2): 102-103

Plummer, D.T. 1987. An introduction to practical biochemistry. 3th ed. McGraw-Hill Book Company, London.

Rawn, J.D. 1990. Traité de biochimie. De Boeck-Wesmael, Bruxelles.

Runge, J.A. and Joly, P. 1994. Rapport sur l'état des invertébrés en 1994: 7:0 Zooplancton (Euphausiacés et Calanus) de l'Estuaire et du Golfe du Saint-Laurent. Sargent, J.R. 1997. Fish oils and human diet. Br J Nutr.78 Suppl 1: S5-S13.

Exp. No.	Technique	Yield (%)	Total (%)	Mean (%) ±s.d.
1-		8,00
	7,60
2-	"	19,70
	6,90
3-	"	8,15
	11,20
4-	"	6,80
	13,60
5-
6-	"

Exp. No.	Technique	Yield (%)	Total (%)	Mean (%)± s.d.
1-	1,17
1,23
2-	"	3,05
1,09
3-	"	1,53
1,26
4-	2,45
0,70
5-	"	1,80
0,80
6-	"	1,60
0,80
7-	2,15
0,47
8-	"	2.11
0,40
9-	"	2,37
0,45
10-	2,28
0,21
11-	"	1,09
0,16
12-	"	2,54
0,09
13-
14-	"
15-	"
16-
17-	"
18-	"
19-
20-	"
21-	"
22-
23-	"
24-	"

Exp. No.	Technique	Yield (%)	Total (%)	Mean (%) ± s.d.
1-		1,82
	1,82
2-	"	1,15
	2,35
3-	"	1,68
	2,19

1-		yes	3,10
		1,07	4,17
2-	"	no	2,14
		1,39	3,53
3-	"	yes	3,32
		1,14	4,46
4-		yes		3,30
5-	"	yes		3,26

Exp. No.	Technique	Yield (%)	Total (%)	Mean (%) ± s.d.
1-		6,18
	2,04
2-	"	8,64
	2,26

	Technique	Yield (%)	Total (%)
1- viscera		6,11
fish 1		0,59
2- tissues	"	3,78
fish 1		0,91
3- viscera	"	10,46
fish 2		0,57
4- issues	"	6,65
fish 2		1.41
5- viscera	"	8.39
fish 3		0,66
6- tissues	"	5,27
fish 3		0,97
7- viscera	"	8,47
fish 4		0,69
8- tissues	"	8,40
fish 4		1,02
9- viscera
fish 1
10- tissues	"
fish 1

Exp. No.	Technique	Yield (%)	Total (%)
1- viscera		34,70
	2,18
2- tissues	"	5,53
	1,17
3- viscera
4- tissues	"

Exp. No.	Technique	Yield (%)	Total (%)
1-tissues and viscera	2,09
0,68
2-tissues and viscera

Exp. No	Technique	Yield (%)	Total (%)
1-
2-
3-

Exp. No.	Technique	Yield (%)	Total (%)
1-		21,39
		5,27
2-
3-

Exp. No.	Technique	Yield (%)	Total (%)
1-		19,23
		8,98
2-
3-

Saponification index
130,6	--	--
185,7	--	--
Olive oil		--	189,7
Wijs iodine index
185,2	172,5	--
127,2	139,2	--
Olive oil		--	81,1
Cholesterol content (%)
2,1	1,9	--
3,7	3,0	--
Olive oil		-	--
Volatile matter and moisture levels (%)
10,0	--	--
6,8	--	--
Peroxide value (meq peroxide/kg oil)
--	0,0	--
--	0,0	--
--	0,1	--
--	5,5	--

Triglycerides
19,0±0,7
66.5± 2,3
Hydrocarbons
trace
1,3± 0.1
Free fatty acids
23.7± 1,1
20,3± 0,3
Monoglycerides
1,4± 0,3
0.5± 0,1
Phospholipids or other polar material
54,1± 6,1
8,5 ±1.6

Fatty acids
12 :0	0,0	0,1
13 :0	0,2	0,1
ISO 14 :0	0,4	0,6
14:0	4,2	7,6
ISO 15:0	0,5	0,7
ANT 15:0	0,2	0,2
15:0	0,6	1,0
ISO 16:0	0,2	0,3
ANT 16:0	0,2	0,2
16:0	14,1	21,6
7MH	0,6	0.9
ANT 17:0	0,1	0,3
17:0	2,8	3,7
18:0	1,0	1,6
20:0	0,1	0,3
Saturates	25,2	39,2
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
16:3n-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
22:6 n-3 Hexaenes	13.2	6,6
Iodine value calculated	214,8	145,1

Fatty acids
12:0	0,5	0,1
13:0	0,2	0,0
IS014:0	0,2	0,2
14:0	1,3	2,6
ISO 15:0	0,3	0,3
ANT 15:0	0,1	0,1
15:0	0,2	0,5
ISO 16:0	0,1	0,2
ANT 16:0	0,2	0,1
16:0	3,3	10,6
7MH	0,6	0.8
ANT 17:0	0,2	0,2
Phytanic	0,2	0,0
17:0	0,5	0,8
18:0	0,2	0,6
20:0	0,3	0,2
22:0	0,0	0,1
Saturates	8,4	17,4
14:1	0,2	0,2
15:1	0,2	0,1
16:1 n-9	0,5	0,0
16:1 n-7	5,2	6,8
16:1 n-5+117:0	0,1	0,1
17:1	0,6	0,7
18:1 n-9	7,0	11,4
18:1 n-7	4,9	9,3
18:1 n-5	0,1	0,3
20:1 n-11	0,2	0,3
20:1 n-9	0,1	0,3
22:1 n-11+13	0,1	0,2
24:1 n-9	0,0	0,1
Monoenes	19,2	29,8
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+117:1	1,4	0,9
16:3 n-3+1 18:0	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

	0,01
0,83
gamma-tocopherol bv HPLC µg/g
Tr
Tr
delta-tocopherol by HPLC µg/g
N.D.
N.D.
395,57
440,47
cholecalciferol by HPLC (IU)
N.D.
N.D.

Asthaxantin (µg/g oil)
93.1
121,7
Canthaxanthin (µg/g oil)
270,4
733,0

STEP	CONDITIONS
Grinding (if particles > 5mm)	4°C
Lipid extraction	sample-acetone ratio of 1:6 (w/v) 2h (including swirling 20 min) 4°C
Filtration	organic solvent resistant filter under reduced pressure
Washing	sample-acetone ratio of 1:2 (w/v) pure and cold acetone
Filtration	organic solvent resistant filter under reduced pressure
Evaporation	under reduced pressure
Oil-water separation	4°C
Lipid extraction
Filtration	organic solvent resistant filter under reduced pressure
Evaporation	under reduced pressure

Time (min)	Amino acids released (µmoles)	Enzymatic rate (µmoles/min)	Specific activity	enzymatic
			(µmoles min/mg*)
15	28.76	1.917	0.164
30	43.74	0.999	0.125
170	98.51	0.322	0.050
255	177.26	0.308	0.060

* total quantity of enzymes in hydrolysis media

Claims (35)

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 selected from the group of solvents consisting of alcohol, preferably ethanol, isopropanol or 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); and

   (f) recovering a second total lipid rich fraction by evaporation of the solvent from the liquid contents of e).
2. A method according to Claim 1, wherein the extraction of step (a) is conducted under agitation after the animal material has been ground.
3. A method according to any of Claims 1 and 2, wherein the extraction of step (d) is conducted under agitation after the animal material has been ground.
4. A method according to any of Claims 1 to 3, wherein steps (a) and (d) are conducted under inert atmosphere.
5. A method according to any of Claims 1 to 4, wherein steps (b) and (e) are effected by techniques selected from filtration, centrifugation and sedimentation.
6. A method according to any of Claims 1 to 5, wherein steps (c) and (f) are effected by techniques selected from evaporation under reduced pressure, flash evaporation and spray drying.
7. A method according to any of Claims 1 to 6, wherein after step (b) and before step (c), the method additionally comprises the intervening step of washing the solid contents with the solvent and adding the resulting washing solution to the liquid contents of step (b).
8. A method according to any of Claims 1 to 7, wherein after step (e) and before step (f), the method additionally comprises the intervening step of washing the solid contents with the organic solvent selected in step (d).
9. A method according to any of Claims 1 to 8, wherein prior to step (a) the marine and aquatic animal material is finely divided, preferably to an average particle size of 5mm or less.
10. A method according to any of Claims 1 to 9, wherein the organic solvent is selected from the group of solvents consisting of ethanol or isopropanol and esters of acetic acid, preferably ethyl acetate, and wherein said method is conducted at a temperature of 5°C or less.
11. A method according to any of Claims 1 to 10, wherein said marine and aquatic animal is zooplankton.
12. A method according to Claim 11, wherein said zooplankton is krill.
13. A method according to Claim 11, wherein said zooplankton is Calanus.
14. A method according to any of Claims 1 to 10, wherein said marine and aquatic animal is fish.
15. A method according to any of Claims 1-9, wherein the organic solvent is t-butanol, and wherein said method is conducted at a temperatures of 25°C.
16. A method for extracting an astaxanthin-and-canthaxantin-containing total lipid fraction from a marine and aquatic animal material selected from zooplankton and fish, said method comprising the steps of:

    (a) placing said animal material in a ketone solvent, preferably acetone to achieve an extraction of the soluble lipid fraction from said marine and aquatic animal material;

    (b) separating liquid and solid contents resulting from step (a);

    (c) recovering a lipid rich fraction from the liquid contents by evaporation of the solvent present in the liquid contents;

    whereby an astaxanthin-and-canthaxantin-containing total lipid fraction is obtained.
17. A method for extracting a total lipid fraction from a marine and aquatic animal material selected from zooplankton 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) seperating 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.
18. A method according to Claim 16 or 17, wherein the animal material is krill.
19. A method according to Claim 16 or 18, wherein the animal material is Calanus.
20. A method as in any of Claims 16 to 19, wherein the extraction of step (a) is conducted under agitation after the animal material has been ground.
21. A method according to any of Claims 16 to 20, wherein step (a) is conducted under inert atmosphere.
22. A method according to any of Claims 16 to 21, wherein step (b) is effected by a technique selected from filtration, centrifugation and sedimentation.
23. A method according to any one of Claims 16 to 22, wherein step (c) is effected by a technique selected from vacuum evaporation, flash evaporation and spray drying.
24. A method according to any one of Claims 16 to 23, 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).
25. A method according to any of Claims 16 to 24, wherein prior to step (a) the marine and aquatic animal material is finely divided, preferably to an average particle size of 5mm or less.
26. A method according to any of Claims 16 to 25, wherein said method is conducted at a temperature of 5°C or less.
27. A method of lipid extraction according to any one of Claims 1 to 26, wherein the resulting solid contents are recovered and consist of a dehydrated residue containing active enzymes.
28. A total krill lipid extract characterized in that it is devoid of toxic solvent and in that the carotenoid content in asthaxathin is 75µg/g or more and preferably at least about 90 µg/g or more of krill extract, and the carotenoid content in canthaxanthin is 250 µg/g or more and preferably at least about 270 µg/g of krill extract or more.
29. A lipid extract according to Claim 28, obtainable from an aquatic or marine animal by a method according to any of Claims 1-27.
30. Use of a krill lipid extract according to Claim 28 or 29, in an application selected from the group consisting of nutraceuticals, cosmetics, fish farming and animal feeding applications.
31. Use of a krill lipid extract according to Claim 28 or 29, for the manufacture of a medicament for treating a disease selected from the group consisting of an inflammatory disease, cardiovascular disease, lupus and renal disease.
32. A krill lipid extract characterised in that it is edible, and in that the carotenoid content in astaxanthin is 75µg/g or more and preferably at least about 90µg/g or more of krill extract, and the carotenoid content in canthaxanthin is 250µg/g or more and preferably at least about 270µg/g of krill extract or more.
33. A krill lipid extract according to Claim 32, obtainable from an aquatic or marine animal by a method according to any of Claims 1-27.
34. Use of a krill lipid extract according to Claim 32 or 33, in an application selected from the group consisting of nutraceuticals, cosmetics, fish farming and animal feeding applications.
35. Use of a krill lipid extract according to Claim 32 or 33, for the manufacture of a medicament for treating a disease selected from the group consisting of an inflammatory disease, cardiovascular disease, lupus and renal disease.