NZ243042A - Cholesterol free lipid extracts extracted from mammalian brains, food compositions containing them and preparatory processes - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £43042 &'■***' ...7,G-91.

■ ■ • ., L 1 f;OQ.;.ft.Z3L.y^/^a.^ j A.ZXCtt^oo; C'J 1 Ba^,; ^ SSy/^o 2 6 OCT 1994 Ifl v .f j >. ; N.z. patent or;," : ;;. -5 JUM 1992 RECEIVED Li MUUBL II ■ Jlll|l'1 11 1 1 Patents Form No. 5 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION BRAIN LIPID EXTRACTS AND METHOD FOR THE PRODUCTION AND USE THEREOF WE, MARTEK CORPORATION, a corporation under the state of Maryland, U.S.A. of 6480 Dobbin Road, Columbia, Maryland 21045, U.S.A. hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: (followed by page la) r\ T«J 496-126A TE:TT/051: 2 la BRAIN LIPID EXTRACTS AND METHOD FOR THE PRODUCTION AND USE THEREOF This invention relates to the obtention of brain lipid extracts, to compositions containing brain lipid 5 extracts and to uses thereof. ! "Brain lipid extracts" as used herein refers to a lipid mixture extracted from the brain tissue of animals. It has been found that such brain lipid extracts contain a mixture of long chain 10 polyunsaturated fatty acids (PUFAs) that have four or more double bonds and closely resemble the mixture of PUFAs found in human breast milk. The mixture of PUFAs typically includes arachidonic acid (ARA), docosahexaenoic acid (DHA) and docosatetraenoic acid 15 (DTA). These PUFAs are important structural lipids that are required for normal development of human infants. See, e.g., U.S. Patent No. 4,670,285 (Clandinin et al.), incorporated herein by reference. Moreover, these PUFAs cannot be synthesized de novo by 20 humans and, accordingly, must be either provided in the diet or synthesized from dietary sources of linolenic acid (LNA) or linoleic acid (LOA). These latter essential fatty acids are poorly converted into DHA. See N. Salem, et al. , Health Effects of Polyunsaturated 25 Fatty Acids in Seafoods. (1986) chapter 15, p.203-317. Moreover, commercially available infant formula is not supplemented with either LNA or LOA.

The first year of a human's life is a period of very rapid growth. During this period of growth, the (followed by page 2) 2 infant requires PUFAs in an amount sufficient to facilitate that growth. Typically, the infant's requirement for PUFAs has, in the past, been satisfied by providing the infant with human breast milk for the 5 duration of the period of rapid growth. However, many mothers now choose either not to nurse their infants or to nurse for a period of time less than the period of rapid infant growth. Infants who are not breast fed typically are provided a commercially available infant 10 formula. As used herein, "infant formula" means a dietary substitute for human breast milk which is fed to infants in an attempt to fulfill their nutbitional needs. Unfortunately, commercially available infant formulas do not contain long chain PUFAs. Accordingly, 15 it would be desirable to provide either an infant formula or a supplement to infant formula which would provide PUFAs to the infant.

Jackson, et al., Am. J. Clin. Nutr., 50:980-2 (1989), teach that the lipid content of brains is so 20 low that it would be virtually impossible to supplement the diet of formula-fed infants to match the long-chain PUFA intake of breast-fed infants.

Additionally, vegetarian women who are pregnant or nursing do not receive PUFAs in their diet, as PUFAs 25 are not available from plants. In turn, the developing fetus or infant of such mothers will receive less than optimal amounts of PUFAs. Accordingly, a dietary supplement of purified PUFAs for such women would be especially desirable.

It is an object of the present invention to provide a commercially feasible solution to the above described problems. This and other objects of the invention are disclcscd herein. 3 ft g -■=}! n, /, 0 Z ^ o 0 "<• r.

SUMMARY OF THE INVENTION This invention relates to the obtention and use of brain lipid extracts and to compositions containing such extracts. Brain lipids are extracted from brain 5 tissue using an appropriate extraction method. The extracted brain lipids are separated from the remaining tissue and recovered from the extracting medium. The brain lipids then can be used to prepare a variety of useful compositions such as infant formula, dietary 10 supplements and the like.

DETAILED DESCRIPTION OF PRACTICING T*HE BEST MODE OF THE INVENTION The present invention concerns the unexpected discovery that the lipid content of mammalian brain 15 tissue is quite similar to the lipid content of human breast milk with respect to certain long chain PUFAs, including ARA, DHA and DTA.

ARA is an omega-6 fatty acid that is 20 carbons in length and has 4 double bonds. DHA is an omega-3 fatty 20 acid, 22 carbons in length, having 6 double bonds. DTA is an omega-6 fatty acid, 22 carbons in length, having 4 double bonds. As noted above, these PUFAs are present in human breast milk and brain tissue, but not in commercially available infant formula. Brain lipid 25 extracts containing these PUFAs can be recovered by extraction from brain tissues. Desirably, mammalian brain tissue is used. Most preferably, the mammal will be a large mammal. Such brain tissue conveniently can be obtained from, for example, slaughterhouses. Cow 30 brain in particular is a preferred source, due to its typically low cost. Additionally, the PUFA profile of bovine brain is remarkably similar to that of human breast milk. This similarity in PUFA profiles is 24 3 0 4 2 4 completely unexpected since the PUFA content of cow's milk differs substantially from that of human breast milk. Of course, sheep, goat or pig brains also can be used where available.

One aspect of the present invention concerns the preparation of brain lipids for use in infant formula. In this method, the brain lipids are extracted from the brain tissue. The extraction process produces a brain lipid extract which can be combined with, or used to 10 make, an infant formula. As used herein, "brain lipid extract" includes both "crude" and "pure" brain lipid extracts, both of which are further defined h'erein.

The lipids are extracted using a suitable extractant. The extraction can be carried out under 15 non-oxidizing conditions if desired. The non-oxidizing conditions can be maintained by any suitable method, such as performing the extraction under a layer of nitrogen. Additionally, antioxidants can be used in the extraction procedure. The presence of an 20 antioxidant is desirable if the extraction and recovery process is not carried out promptly or if the recovered extract is to be stored for a period of time prior to use. Suitable antioxidants include butylated hydroxy toluene, ascorbic acid and (3-carotene. Of these (3-25 carotene is preferred. While any effective amount of P-carotene can be used, an amount equal to about 0.1% by weight of the total extractable lipid in the brain tissue is especially preferred.

A variety of extractants can be used in the 30 present invention. In a preferred embodiment of the invention, the brain tissue is homogenized in an effective amount of an extractant which is a food-grade solvent. Such solvents are known to those of skill in the art and include ethanol and isopropanol (IPA). n Isopropanol is especially preferred. Preferably, from about 0.25 to about 5.0 liters of IPA can be used per kilogram of fresh brain tissue. Most preferably, about 1.0 liter IPA/kg brain tissue can be used. To this 5 homogenized mixture, hexane can be added. Typically, a volume of hexane which is equivalent to the volume of extracting solution is added after homogenization. The homogenized mixture, including the hexane, is equilibrated for an effective amount of time to permit 10 extraction of the brain lipids. Typically, this time will be about one hour. Most of the lipids initially extracted with the IPA will partition into thjb hexane fraction and can be removed easily by decanting the hexane layer of the mixture. Definition of this phase 15 separation can be aided by centrifugation if desired. After removal of the hexane layer, the aqueous IPA layer can be further diluted with water and reextracted with additional volumes of hexane. This latter step can increase the lipid yield by varying amounts, 20 usually about 10%.

In an alternative extraction procedure, supercritical fluids, such as carbon dioxide or nitrous oxide, can be used as the extractant to extract the brain lipids from wet or dry brain tissue. The use of 25 supercritical fluids to extract fatty acids from tissues is known to those of skill in the art and can be applied to the present invention.

In yet another embodiment, the brain tissue can be dried, milled and extracted with an effective amount of 30 a nonpolar solvent. Such solvents are known to those of skill in the art and include, for example, hexane. In this embodiment it is preferred to use from about two to about ten liters of hexane per kilogram of dried 9 L "I U * W tissue. The use of four liters/kg is especially preferred.

After the brain lipids have been extracted from the brain tissue into the extracting medium, the 5 extracted brain tissue is removed from the extractant. Any antioxidants present remain in the lipid fraction. Removal of the tissue can be by filtration or centrifugation and results in a cake of solid material and a liquor of extractant. The liquor contains the 10 brain lipids and antioxidants. Optionally, this cake can be extracted again. Yield often can be improved by another 10% by utilizing a second extraction.,1 The cake then can be used without further modification as an animal feed. The residual brain tissue in the cake is 15 an excellent source of dietary protein. Thus, one embodiment of the present invention is a method of providing protein to an animal by including in that animal's diet brain tissue from which the brain lipids have been extracted as described herein. 20 Techniques for removing solvents are known to those of skill in the art and include, for example, evaporation. A rotary evaporator of the type known to those of skill in the art is useful for evaporating the extractant because the evaporation step can be done 25 rapidly and at reduced temperatures. Preferably, residual water can be removed from the solvent using molecular sieves or sodium sulfate prior to evaporation. Removal of the extractant produces what is termed a "crude" brain lipid extract. This extract 30 can be directly used in the methods and compositions of the present invention. The extract can be used as is or converted into the corresponding ethyl esters prior to use. The conversion of fatty acids to their ethyl O !■ "7 O /■ll , : »-..A <J 4L. "T "J \-i 1 iL 7 esters is known to those of skill in the art and does not comprise any part of this invention.

If total brain tissue has been extracted, a considerable amount of cholesterol (up to 40% by 5 weight) will co-extract with the other lipids. This is due primarily to a contribution from the cholesterol-rich white matter of the brain. If only grey matter of the brain is used in the extraction, the cholesterol content typically is considerably lower. To further 10 reduce the cholesterol levels in the crude brain lipid extract, refining and purification techniques familiar to those of skill in the art of vegetable oil! refining can be applied. For example, counter-current phase partitioning between hexane and 87% aqueous ethanol 15 will result in the separation of the polar lipid fraction (rich in the desired fatty acids) from the neutral lipid fractions (rich in cholesterol). This step results in what is termed a "pure" brain lipid extract. Typically, the pure brain lipid extract 20 contains about 30 - 40 g of polar lipids per kg fresh weight of whole brain tissue. Again, as in all embodiments herein, the corresponding ethyl glycerol esters of the lipids can be used if desired. Table 1 compares the typical fatty acid composition of porcine 25 brain lipid and bovine brain lipid.

Table 1. Fatty acid composition of porcine brain lipid (PBL) and bovine brain lipid (BBL) in % by weight.

Fattv Acid PBL BBL 16:0 17. 70 19. 27 16 :1 1. 00 1. 33 18:0 22. 46 19. 86 18 : 1 38. 71 31. 74 :1 3. 85 3. 96 :4 n6 (ARA) 8. 79 7 . 22:4 n6 (DTA) 4. . 17 22:6 n3 (DHA) 4 . 16 8. 86 O " 7 * 't Alternatively, if desired, cholesterol and other non-saponifiable fatty acids can be removed by saponifying the brain tissue, again optionally under non-oxidizing conditions. Preferably, the brain tissue 5 is saponified by homogenization in a non-polar solvent, such as ethanol or isopropanol, which contains a saponifying agent. Such agents are known and include potassium hydroxide (KOH). The amount of KOH used can vary but should be sufficient to produce from about a 10 0.1M to 10M solution. A 1M solution of 95% ethanol containing KOH is especially preferred. Typifally, about 2 ml of such a solution per gram of brain tissue can be used. More or less, of course, can be used as desired.

The homogenized brain tissue in the saponification solution is heated to reflux to complete the saponification. Thirty minutes of applied heat generally is sufficient to completely saponify the brain tissue. Following the saponification, the 20 mixture can be diluted with water. Typically, a volume of water equal to that of the saponification solution is preferred. A first extraction with hexane can be used to remove the non-saponifiable fatty acids. After this removal, the saponified lipids can be acidified by 25 treatment with acid and extracted with hexane.

Preferably, the pH of the saponified lipids will be lowered to less than about 5, most preferably to about 2. Hydrochloric acid can be used to lower the pH. The resulting product will be substantially free of non-30 saponifiable fatty acids, including cholesterol.

Either the pure or crude brain lipid extract, optionally in combination with an antioxidant, can be used directly as a supplement for commercially 9 available infant formula. Typically about 1-2 grams of brain lipid extract per liter of formula are sufficient to provide ARA, DHA and DTA levels similar to those found in human breast milk. As reported by Carlson, et 5 al. Amer. J. Clin. Nutr. 45:798-804 ( 1986 ) (incorporated herein by reference), those levels are about 0.59% ± 0.04, 0.19% ± 0.03 and 0.21% + 0.01 by weight respectively. As an alternative to a supplement, the brain lipid extract can be added 10 directly to the vegetable oils typically used in manufacturing infant formula. Thus, both infant formulas and infant formula supplements comprising brain lipid extract are expressly contemplated to be within the scope of this invention.

Another aspect of the present invention relates to the treatment of various pathologies which can arise from long chain PUFA deficiencies in humans. These types of deficiencies have been reported in certain instances of total parenteral nutrition (TPN) or long 20 term tube feeding of hospitalized patients.

Simopoulos, A.P. Omega-3 Fatty Acids in Health and Disease, pp. 115-156 (1990). Such deficiencies can be treated by the administration of a pharmaceutically effective amount of the brain lipid extract of the 25 present invention to a human in need of such treatment. Doctors and others of skill in the treatment of pathologies can easily determine the effective amount to be administered to each patient. All forms of administration, including parenteral, enteral and 30 topical administration, are encompassed by this aspect of the present invention. A preferred embodiment is an encapsulated enteral composition. Gelatin capsules, or the like, can be used to contain the brain lipid extract. 9 / v»^ The encapsulated brain lipid extract is a particularly palatable form of a dietary supplement for humans due to its ease of ingestion. Of course, the brain lipid extract could be utilized by itself as a 5 dietary supplement. In particular, such supplements provide needed PUFAs to pregnant or nursing females, especially to vegetarians.

The present invention having been generally described, reference now is made to the following 10 illustrative, non-limiting examples.

EXAMPLE 1 ! Extraction of porcine brain lipid with ethanol. Fifty grams of fresh porcine brain tissue were homogenized in a blender with 100 ml of 95% ethanol. 15 The resulting mixture was filtered to separate the extracted brain tissue from the lipid-containing filtrate. The filtrate was dried using a rotary evaporator to remove the ethanol. The fatty acid profile of the crude lipid fraction is that illustrated 20 in Table 1, above. This extract was mixed with infant formula in a ratio of one part brain lipid extract per 20 parts formula. With a formula containing 30 g lipid per liter, this corresponded to the addition of 1.5 g of extract per liter of formula.

The effect of the addition of extract in this ratio to the fatty acid profile of a commercial formula (Similac, manufactured by Ross Laboratories) is shown in Table 2.

EXAMPLE 2 Extraction of bovine brain lipid with IPA and hexane. Two hundred milliliters of IPA and 100 mg of fj-carotene were added to 34 0 g (fresh weight) of bovine brain. A blender was used to homogenize the brain. Two hundred milliliters of hexane then were added and the extract was stirred for 30 minutes at room temperature. This mixture then was centrifuged at 5,000 RPM for 10 minutes and the upper (hexane) layer was decanted. The pellets were washed once with a mixture of hexane and IPA (1:1) and the hexane layer again was decanted following centrifugation. The combined aqueous fractions were diluted with water (1:1 v/v) and extracted with an equal volume of hexane to remove any remaining lipids in the aqueous fraction. Ten grams of anhydrous sodium sulfate were ad'ded to the combined hexane fractions to remove any residual water before rotary evaporation. The crude bovine brain lipid (18.2 g) was collected following removal of the hexane by rotary evaporation. This crude extract was placed in 200 ml of hexane/87% ethanol (1:1) and transferred to a separatory funnel where the phases were allowed to separate. The upper layer was washed twice with 87% ethanol and the lower phase was washed twice with hexane using a counter-current approach. The solvents were evaporated. The hexane fraction yielded about 11 g of polar lipids while the ethanol fraction yielded about 7.2 g of cholesterol-rich material. The fatty acid profile of the polar lipid extract is shown in Table 1, above. The polar lipid extract then was added to the commercial infant formula, Similac, at a ratio of one part brain lipid extract to 20 parts formula lipid. With a formula containing 30 g lipid per liter, this corresponds to the addition of 1.5 g of extract per liter of formula as indicated in Table 2. 43 0 12 Table 2. Comparison of the percent by weight lipid composition of infant formula, infant formula with added PBL, infant formula with added BBL, and human mother's milk. PBL and BBL were added at 1.5 g/1.

Infant formula formula breast Fatty Acid formula + BBL + PBL milk 8:0 + 10 : 0 41.8 39 . 71 39 .81 1.74 12:0 + 14:0 .7 19 .66 19 .72 14.95 16:0 6.8 7 .42 7 .32 19 .82 16:1 0.2 0 . 26 0 .24 3.20 18:0 2.3 3 .18 3 .26 .91 18:1 .0 11 .09 11 .37 34.82 18:2 n6 17.4 16 .53 16 .57 16.00 18:3 n3 0.9 0 .86 0 .86 0.62 :1 0.1 0 .29 0 .28 1.10 :2 n6 — 0 .00 0 .00 0.61 . :3 n6 __ 0 .00 0 .00 0.42 ■ :4 n6 (ARA) -- 0 .36 0 .42 0.59 :5 n3 — 0 .00 0 .00 0.03 22:4 n6 (DTA) — 0 .26 0 .20 0.21 22:6 n 3 (DHA) — 0 .44 0 .20 0.19 EXAMPLE 3 Extraction of porcine brain fatty acids. Fifty grams of porcine brain are homogenized in 100 ml of a 25 mixture of 95% ethanol and 5.6 g of potassium hydroxide. The sample is heated to reflux for 30 minutes to complete the saponification. When cool, the mixture is diluted with one volume of water and washed two times with 100 ml hexane to remove nonsaponifiable 30 lipids (especially cholesterol). The alkaline extract then is filtered and the filtrate acidified to pH 2.0 with HC1 before reextraction of the free fatty acids with hexane. The hexane extracts of the acidified aqueous fraction are pooled, 50 mg of ^-carotene is 35 added, and the hexane is removed by rotary evaporation yielding the free fatty acids rich in DHA, DTA and ARA. The polar lipid extract then either is added directly to infant formula at a ratio of one part brain lipid extract to 20 parts formula lipid, or converted into

Claims (43)

0 k ^ f'1 ^ 9 t i J U ' L■ 13 ethyl or glycerol esters before addition. With a formula containing 30 g lipid per liter, this corresponds to the addition of 1.5 g of extract per liter of formula. I i 24 3 0 4 2 14 YVHAI tyWE CLAIM IS:

1. Infant formula comprising a lipid extract from animal brains, wherein said extract comprises DHA, DTA and ARA and is essentially free of cholesterol.

2. The infant formula of claim l, wherein the animal brains are cow or pig brains.

3. A method for preparing a lipid extract from mammalian brains which comprises extracting brain lipids from mammalian brain tissue with an extractant, separating said extracted brain lipids from said tissue, treating said lipid extract to render it essentially cholesterol free and recovering said extracted brain lipids.

4. The method of claim 3, wherein said extraction step comprises homogenizing said brain tissue in a food-grade solvent.

5. The method of claim 4, wherein said solvent comprises ethanol.

6. The method of claim 4, wherein said solvent comprises isopropanol.

7. The method of claim 6, wherein said isopropanol is added to said brain tissue in amounts of from 0.25 to 5 liters per kilogram of brain, tissue.

8. The method of claim 3, wherein said extract step includes adding an antioxidant to said extractant to aid in the prevention of oxidation of said brain lipids.

9. The method of claim 8, wherein said antioxidant comprises butylated hydroxy toluene, ascorbic acid or /3-carotene.

10. The method of claim 4, wherein said separation step comprises filtering or centrifuging said homogenized material to produce a cake of brain tissue material and a liquor of solvent containing said brain lipids.

11. The method of claim 10, further comprising removing said solvent from said lipids.

12. The method of claim 3, wherein said lipids are extracted using a supercritical fluid, j / 7 n £ 'J V 15

13. The method of claim 12, wherein said supercritical fluid comprises carbon dioxide or nitrous oxide.

14. The method of claim 3, wherein said extraction comprises applying a non-polar solvent to said brain tissue.

14. The method of claim 14, wherein Baid solvent comprises hexane. ,

16. The method of claim 3, wherein the mammalian brains are cow or pig' brains.

17. A method for preparing a lipid extract from mammalian brains which comprises: a) homogenizing mammalian brain tissue in an extracting solution comprising a food grade solvent; b) mixing hexane with said extracting solution containing said homogenized brain tissue; c) equilibrating said mixture such that said hexane and said solvent form separate phases; d) recovering said hexane phase containing lipids; • e) evaporating said hexane; and f) removing cholesterol from said lipids and recovering the resultant lipid extract.

18. The method of claim 17, wherein said extracting solution further comprises an anti-oxidant.

19. The method of claim 18, wherein said anti-oxidant comprises /3-carotene.

20. The method of claim 17, wherein said food grade solvent comprises isopropanol.

21. A method for preparing a lipid extract from animal brains which comprises: a) saponifying mammalian brain tissue containing brain lipids in a saponification solution; b) treating said Baponified brain tissue with a first treatment of hexane; c) removing said hexane from said saponified brain lipids; d) acidifying said saponified brain lipids to produce free fatty acids; e) solubilizing said free fatty acids with a second treatment of hexane; f) separating said hexane from said saponification solution; and g) recovering said free fatty acids from said second treatment of hexane.

22. The method of claim 21, wherein said free fatty acids are converted to their corresponding ethyl esters or glycerol esters.

23. The method of claim 21, wherein said saponification solution comprises ethanol and potassium hydroxide.

24. A method of obtaining a composition comprising an extract of brain lipids comprising free fatty acids which is essentially cholesterol free, which comprises a) saponifying mammalian brain tissue comprising lipids in a saponification solution,- b) heating to reflux said saponification solution containing brain lipids; c) extracting cholesterol from said saponification solution by treating said solution with hexane; and d) recovering said essentially cholesterol-free fatty acids.

25. The method of claim 24, wherein said saponification step comprises homogenizing said brain tissue in a solvent comprising a saponifying agent.

26. The method of claim 25, wherein said solvent comprises ethanol and isopropanol.

27. The method of claim 26, wherein said saponifying agent comprises potassium hydroxide.

28. The method of claim 24, further comprising diluting the refluxed saponification solution with water prior to the extracting step,

29. The method of claim 24, wherein each step is performed under anti-oxidizing conditions.

30. A method of obtaining an infant formula or food supplement comprising brain lipids extracted from brain tissue which comprises: a) homogenizing mammalian brain tissue in isopropanol to extract lipids comprising polyunsaturated fatty acids from Baid brain tissue into said isopropanol; b) removing Baid extracted brain tissue from said isopropanol; c) evaporating said isopropanol from said extracted lipids; d) treating said extracted lipids with a mixture o£ hexane and aqueous ethanol; e) separating said brain lipids into said aqueous ethanol by the application of counter-current phase partitioning techniques; f) extracting said brain lipids from said aqueous ethanol; and g) adding said lipids to an infant formula or food supplement.

31. A composition comprising a combination of an antioxidant and a lipid extract from animal brains, wherein said extract comprises DHA, DTA and ARA and is essentially free of Cholesterol.

32. The composition of claim 31, wherein the lipid extract was obtained from the brain of a cow or a pig.

33. The composition of claim 31, which is provided in a daily dosage form suitable for administration to pregnant or nursing women.

34. A method for providing polyunsaturated fatty acids to infant formula comprising adding to said formula the composition of claim 31 in an amount sufficient to provide the fatty acids in desired amounts.

35. A method for providing polyunsaturated fatty acids to food products comprising adding to a food product the composition of claim 31 in an amount sufficient to provide the fatty acids in desired amounts. 243 04? 18

36. A method of providing infant formula with a polyunsaturated fatty acid-containing lipid composition, wherein said fatty acids comprise DHA, DTA and ARA, which comprises: a) homogenizing animal brain tissue in an extracting solution comprising a solvent and an antioxidant, thereby extracting lipids from said brain tissue into said solvent; b) removing said extracted brain tissue from said solvent; c) removing cholesterol and solvent from said extracted lipids; and d) adding said lipids to an infant formula.

37. An animal feed comprising brain tissue from which brain lipids have been extracted according to the method of any one of claims 3 to 30.

38. Infant formula of claim 1 substantially as herein described.

39. A method for preparing a lipid extract from mammalian brains according to any one of claims 3, 17 and 21, substantially as herein described.

40. A method of claim 3, substantially as herein described with reference to any one of the Examples.

41. A method of claim 24 substantially as herein described.

42. A method of either claim 30 or claim 36, substantially as herein described.

43. A composition of claim 31, substantially as herein described. 1^17 MAR 1994^ MARTEK CORPORATION By their attorneys BALDWIN, SON St CAREY O '/ «■ >