U.S. patent application number 09/758973 was filed with the patent office on 2002-02-28 for process for making an enriched mixture of polyunsaturated fatty acid esters.
Invention is credited to Luthria, Devanand L..
Application Number | 20020026063 09/758973 |
Document ID | / |
Family ID | 22640800 |
Filed Date | 2002-02-28 |
United States Patent
Application |
20020026063 |
Kind Code |
A1 |
Luthria, Devanand L. |
February 28, 2002 |
Process for making an enriched mixture of polyunsaturated fatty
acid esters
Abstract
This invention is directed to a process for making an enriched
mixture comprising a polyunsaturated fatty acid ester having the
Formula (V): 1 In one embodiment, this process comprises
transesterifying an oil from Schizochytrium sp. with an alcohol in
the presence of a base to form a saturated fatty acid ester and the
polyunsaturated fatty acid ester (the fatty acid esters are formed
from the alcohol and fatty acid residues of at least one glyceride
in the oil). Urea is subsequently dissolved in a medium comprising
the fatty acid esters to form a medium comprising the fatty acid
esters and dissolved urea. This medium is then cooled or
concentrated to form (a) a precipitate comprising urea and at least
a portion of the saturated fatty acid ester, and (b) a liquid
fraction comprising at least most of the polyunsaturated fatty acid
ester. Afterward, the precipitate and liquid fraction are
separated. In this embodiment, the alcohol is R.sub.3--OH, R.sub.3
is a hydrocarbyl or a substituted hydrocarbyl, and R.sub.4 is a
straight chain hydrocarbyl comprising 21 carbon atoms and at least
2 carbon-carbon double bonds.
Inventors: |
Luthria, Devanand L.;
(Ankeny, IA) |
Correspondence
Address: |
SENNIGER POWERS LEAVITT AND ROEDEL
ONE METROPOLITAN SQUARE
16TH FLOOR
ST LOUIS
MO
63102
US
|
Family ID: |
22640800 |
Appl. No.: |
09/758973 |
Filed: |
January 11, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60175583 |
Jan 11, 2000 |
|
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Current U.S.
Class: |
554/174 |
Current CPC
Class: |
C11B 1/025 20130101;
C11B 3/001 20130101; C11B 7/0083 20130101; C11C 3/003 20130101 |
Class at
Publication: |
554/174 |
International
Class: |
C11B 003/00 |
Claims
I claim:
1. A process for making a mixture comprising a polyunsaturated
fatty acid ester, said process comprising: transesterifying an oil
from Schizochytrium sp. with an alcohol in the presence of a base
to form a saturated fatty acid ester and said polyunsaturated fatty
acid ester, said fatty acid esters being formed from said alcohol
and fatty acid residues of at least one glyceride in said oil;
dissolving urea in a medium comprising said fatty acid esters to
form a medium comprising said fatty acid esters and dissolved urea;
cooling or concentrating said medium comprising said fatty acid
esters and dissolved urea to form (a) a precipitate comprising urea
and at least a portion of said saturated fatty acid ester, and (b)
a liquid fraction comprising at least most of said polyunsaturated
fatty acid ester; and separating said precipitate from said liquid
fraction, wherein said polyunsaturated fatty acid ester has Formula
(V): 9 the alcohol is R.sub.3--OH, R.sub.3 is a hydrocarbyl or a
substituted hydrocarbyl, and R.sub.4 is a straight chain
hydrocarbyl comprising 21 carbon atoms and at least 2 carbon-carbon
double bonds.
2. A process according to claim 1, wherein R.sub.4 comprises a
straight chain hydrocarbyl comprising 21 carbon atoms and 5
carbon-carbon double bonds.
3. A process according to claim 1, wherein R.sub.4 comprises a
straight chain hydrocarbyl comprising 21 carbon atoms and 6
carbon-carbon double bonds.
4. A process according to claim 1, wherein said glyceride(s)
comprises a triglyceride.
5. A process according to claim 1, wherein R.sub.3 comprises a
hydrocarbyl comprising from 1 to 4 carbon atoms.
6. A process according to claim 5, wherein R.sub.3 comprises
methyl.
7. A process according to claim 5, wherein R.sub.3 comprises
ethyl.
8. A process according to claim 1, wherein said base comprises NaOH
or KOH.
9. A process according to claim 1, wherein said base comprises
elemental sodium.
10. A process according to claim 1, wherein said
transesterification comprises hydrolysis of said glyceride(s) to
form free fatty acids, followed by esterification of said free
fatty acids.
11. A process according to claim 1, wherein said glyceride(s) is
contacted with said alcohol in the presence of said base in a
liquid that further comprises an organic solvent that can
solubilize said glyceride(s).
12. A process according to claim 11, wherein said organic solvent
comprises dichloromethane.
13. A process according to claim 11, wherein said glyceride(s) is
contacted with said alcohol in the presence of said base at a
temperature which is greater than the boiling point of said organic
solvent.
14. A process according to claim 1, wherein said glyceride(s) is
contacted with said alcohol in the presence of said base at a
temperature of at least about 40.degree. C.
15. A process according to claim 14, wherein said temperature is
from about 70 to about 150.degree. C.
16. A process according to claim 1, wherein said glyceride(s) is
contacted with said alcohol in the presence of said base to form a
mixture which is then heated under reflux to form said fatty acid
esters.
17. A process according to claim 1, wherein said fatty acid esters
are formed under a non-oxidizing atmosphere.
18. A process according to claim 1, wherein said medium comprising
said fatty acid esters and dissolved urea further comprises an
organic solvent that can solubilize said polyunsaturated fatty acid
ester.
19. A process according to claim 18, wherein said organic solvent
comprises an alkyl alcohol comprising from 1 to 4 carbon atoms.
20. A process according to claim 19, wherein said organic solvent
comprises ethanol.
21. A process according to claim 1, wherein said medium comprising
said fatty acid esters and dissolved urea is cooled to a
temperature of no less than about 15.degree. C. to form said
urea-containing precipitate.
22. A process according to claim 21, wherein said temperature is no
less than about 20.degree. C.
23. A process according to claim 21, wherein said temperature is
from about 15 to about 25.degree. C.
24. A process according to claim 21, wherein said temperature is
from about 20 to about 25.degree. C.
25. A process according to claim 1, wherein at least a portion of
said precipitate is formed under a non-oxidizing atmosphere.
26. A process according to claim 1, wherein said saturated fatty
acid ester has Formula (VI): 10wherein R.sub.5 is a hydrocarbyl
comprising no double bonds.
27. A process according to claim 26, wherein R.sub.5 comprises 13
or 15 carbon atoms.
28. A process for making a mixture comprising a polyunsaturated
fatty acid ester, said process comprising: transesterifying an oil
from Schizochytrium sp. with an alcohol in the presence of an acid
to form a saturated fatty acid ester and said polyunsaturated fatty
acid ester, said fatty acid esters being formed from said alcohol
and fatty acid residues of at least one glyceride in said oil;
dissolving urea in a medium comprising said fatty acid esters to
form a medium comprising said fatty acid esters and dissolved urea;
cooling said medium comprising said fatty acid esters and dissolved
urea to a temperature of no less than about 15.degree. C. to form
(a) a precipitate comprising urea and at least a portion of said
saturated fatty acid ester, and (b) a liquid fraction comprising at
least most of said polyunsaturated fatty acid ester; and separating
said precipitate from said liquid fraction, wherein said
polyunsaturated fatty acid ester has Formula (V): 11 the alcohol is
R.sub.3--OH, R.sub.3 is a hydrocarbyl or a substituted hydrocarbyl,
and R.sub.4 is a straight chain hydrocarbyl comprising 21 carbon
atoms and at least 2 carbon-carbon double bonds.
29. A process according to claim 28, wherein said acid comprises
HCl.
30. A process according to claim 28, wherein R.sub.4 comprises a
straight chain hydrocarbyl comprising 21 carbon atoms and 6
carbon-carbon double bonds.
31. A process according to claim 28, wherein R.sub.3 comprises
ethyl.
32. A process according to claim 28, wherein said polyunsaturated
fatty acid esters are formed under a non-oxidizing atmosphere.
33. A process according to claim 28, wherein said medium comprising
said fatty acid esters and dissolved urea further comprises an
organic solvent that can solubilize said polyunsaturated fatty acid
ester.
34. A process according to claim 33, wherein said organic solvent
comprises ethanol.
35. A process according to claim 33, wherein said medium comprising
said fatty acid esters and dissolved urea is cooled to a
temperature of no less than about 20.degree. C. to form said
precipitate.
36. A process according to claim 33, wherein said medium comprising
said fatty acid esters and dissolved urea is cooled to a
temperature of from about 15 to about 25.degree. C. to form said
precipitate.
37. A process according to claim 33, wherein said medium comprising
said fatty acid esters and dissolved urea is cooled to a
temperature of from about 20 to about 25.degree. C. to form said
precipitate.
38. A process according to claim 28, wherein at least a portion of
said precipitate is formed under a non-oxidizing atmosphere.
39. A process according to claim 28, wherein the saturated fatty
acid ester has Formula (VI): 12wherein R.sub.5 is a hydrocarbyl
comprising no double bonds.
40. A process according to claim 39, wherein R.sub.5 comprises 13
or 15 carbon atoms.
41. A process for making a mixture comprising a polyunsaturated
fatty acid ester, the process comprising: transesterifying an oil
from Schizochytrium sp. with an alcohol in the presence of an acid
to form a reaction mixture comprising a saturated fatty acid ester
and said polyunsaturated fatty acid ester, said fatty acid esters
being formed from said alcohol and fatty acid residues of at least
one glyceride in said oil; separating at least most of said
polyunsaturated fatty acid ester from said reaction mixture to form
a mixture comprising said polyunsaturated fatty acid and a residual
amount of said saturated fatty acid ester; dissolving urea in a
medium comprising said separated polyunsaturated fatty acid ester
and residual saturated fatty acid ester to form a medium comprising
said separated polyunsaturated fatty acid ester, residual saturated
fatty acid ester, and dissolved urea; cooling or concentrating said
medium comprising said separated polyunsaturated fatty acid ester,
residual saturated fatty acid ester, and dissolved urea to form (a)
a precipitate comprising urea and at least a portion of said
residual saturated fatty acid ester, and (b) a liquid fraction
comprising at least most of said separated polyunsaturated fatty
acid ester; and separating said precipitate from said liquid
fraction, wherein said polyunsaturated fatty acid ester has Formula
(V): 13 the alcohol is R.sub.3--OH, R.sub.3 is a hydrocarbyl or a
substituted hydrocarbyl, and R.sub.4 is a straight chain
hydrocarbyl comprising 21 carbon atoms and at least 2 carbon-carbon
double bonds.
42. A process according to claim 41, wherein R.sub.4 comprises a
straight chain hydrocarbyl comprising 21 carbon atoms and 6
carbon-carbon double bonds.
43. A process according to claim 41, wherein R.sub.3 comprises
ethyl.
44. A process according to claim 41, wherein said separation of
said polyunsaturated fatty acid ester from at least a portion of
said reaction mixture comprises extracting said polyunsaturated
fatty acid ester from said reaction mixture using a non-polar
organic solvent.
45. A process according to claim 44, wherein said non-polar solvent
comprises petroleum ether, pentane, hexane, cyclohexane, or
heptane.
46. A process according to claim 44, wherein said non-polar solvent
comprises hexane.
47. A process according to claim 41, wherein said separation of
said polyunsaturated fatty acid ester from at least a portion of
said reaction mixture comprises extracting said polyunsaturated
fatty acid ester from said reaction mixture using a non-polar
solvent and a polar organic solvent.
48. A process according to claim 47, wherein said polar solvent
comprises diethyl ether.
49. A process for making a mixture comprising a polyunsaturated
fatty acid ester, said process comprising: transesterifying an oil
from Schizochytrium sp. with an alcohol in the presence of an acid
to form a saturated fatty acid ester and said polyunsaturated fatty
acid ester, said fatty acid esters being formed from said alcohol
and fatty acid residues of at least one glyceride in said oil;
dissolving urea in a medium comprising said fatty acid esters to
form a medium comprising said fatty acid esters and dissolved urea;
cooling said medium comprising said fatty acid esters and dissolved
urea to a temperature of no less than 10.degree. C. to form (a) a
precipitate comprising urea and at least a portion of said
saturated fatty acid ester, and (b) a liquid fraction comprising at
least most of said polyunsaturated fatty acid ester; and separating
said precipitate from said liquid fraction, wherein said
polyunsaturated fatty acid ester has Formula (V): 14 the alcohol is
R.sub.3--OH, R.sub.3 comprises at least 2 carbon atoms and is a
hydrocarbyl or substituted hydrocarbyl, and R.sub.4 is a straight
chain hydrocarbyl comprising 21 carbon atoms and at least 2
carbon-carbon double bonds.
50. A process according to claim 49, wherein R.sub.3 comprises
ethyl.
51. A process according to claim 49, wherein said medium comprising
said fatty acid esters and dissolved urea is cooled to a
temperature of no less than about 15.degree. C. to form said
precipitate.
52. A process according to claim 51, wherein said temperature is no
less than about 20.degree. C.
53. A process according to claim 51, wherein said temperature is
from about 20 to about 25.degree. C.
54. A process according to claim 49, wherein said fatty acid esters
are formed under a non-oxidizing atmosphere.
55. A process according to claim 49, wherein at least a portion of
said precipitate is formed under a non-oxidizing atmosphere.
56. A process for making a mixture comprising a polyunsaturated
fatty acid ester, said process comprising: forming said
polyunsaturated fatty acid ester and a saturated fatty acid ester,
said fatty acid esters being derived from at least one glyceride
obtained from Schizochytrium sp., cooling a solvent comprising said
fatty acid esters to a temperature of no less than about
-30.degree. C. and no greater than about 0.degree. C. to form (a) a
precipitate comprising at least a portion of said saturated fatty
acid ester, and (b) a liquid fraction comprising at least most of
said polyunsaturated fatty acid ester; separating said precipitate
from said liquid fraction, where said polyunsaturated fatty acid
ester has Formula (V): 15 the saturated fatty acid ester has
Formula (VI): 16 R.sub.3 is a hydrocarbyl or a substituted
hydrocarbyl, R.sub.4 is a straight chain hydrocarbyl comprising 21
carbon atoms and at least 2 carbon-carbon double bonds, and R.sub.5
is a hydrocarbyl comprising no double bonds.
57. A process according to claim 56, wherein R.sub.3 comprises
ethyl.
58. A process according to claim 56, wherein said solvent
comprising said fatty acid esters comprises an organic solvent that
can solubilize said polyunsaturated fatty acid ester.
59. A process according to claim 58, wherein said organic solvent
comprises methanol.
60. A process according to claim 56, wherein at least a portion of
said precipitate is formed under a non-oxidizing atmosphere.
61. A process according to claim 56, wherein said temperature is no
greater than about -10.degree. C.
62. A process according to claim 56, wherein said temperature is no
greater than about -20.degree. C.
63. A process according to claim 56, wherein said fatty acid esters
are formed by a process comprising contacting an oil from
Schizochytrium sp. with an alcohol in the presence of a base or
acid, wherein said fatty acid esters are formed from said alcohol
and fatty acid residues of at least one glyceride in said oil, and
said alcohol is R.sub.3--OH.
64. A process according to claim 63, wherein said fatty acid esters
are formed under a non-oxidizing atmosphere.
Description
FIELD OF THE INVENTION
[0001] This invention relates generally to a process for making an
enriched mixture of esters of polyunsaturated fatty acids from a
natural source containing a high concentration of glycerides
(particularly triglycerides) which comprise one or more
polyunsaturated fatty acid residues. In a particularly preferred
embodiment, this invention is directed to making an enriched
mixture of esters of polyunsaturated fatty acids (particularly
esters of docosahexaenoic acid) from an oil obtained from the
marine algae identified as Schizochytrium sp.
BACKGROUND OF THE INVENTION
[0002] Many polyunsaturated fatty acids are known to have
therapeutic and nutritional benefits. One such fatty acid is
docosahexaenoic acid ("DHA"). DHA is a 22-carbon, naturally
occurring, unbranched fatty acid comprising 6 carbon-carbon double
bonds (with the biologically active form containing all cis
carbon-carbon double bonds). DHA and many of its derivatives (e.g.,
esters comprising a DHA residue, particularly the ethyl ester of
DHA and triglycerides containing one or more DHA residues) have
been used, for example, to treat cardiovascular and inflammatory
diseases. They also have been added to infant milk to promote the
development of brain and retina functions. Use of DHA esters (as
opposed to the free DHA fatty acid) is often particularly
advantageous because such esters (especially the ethyl ester and
triglycerides) tend to have a palatable taste and tend to be easily
absorbed by animal digestive systems.
[0003] Sources of DHA and derivatives thereof include marine animal
oils, fish oils (e.g., menhaden oil, salmon oil, mackerel oil, cod
oil, herring oil, sardine oil, capelin oil, and tuna oil), marine
algae (e.g., Schizochytrium sp.), and human milk. Such sources,
however, normally contain a substantial amount of saturated fatty
acid residues (often as residues of triglyceride molecules) which
dilute the concentration of DHA residues in the oil. It is
therefore advantageous to reduce the concentration of undesirable
saturated fatty acid residues in the oil while increasing the
concentration of DHA or a derivative(s) thereof.
[0004] Numerous methods have been used alone or in combination to
isolate (or at least concentrate) and recover specific fatty acids
and their derivatives from various naturally occurring sources.
These processes include fractional crystallization at low
temperatures, molecular distillation, urea adduct crystallization,
extraction with metal salt solutions, super critical fluid
fractionation on countercurrent columns, and high performance
liquid chromatography.
[0005] In W. W. Christie, Lipid Analysis, pp. 147-49 (Pergamon
Press, 1976), a method is disclosed generally for using urea to
separate methyl esters of saturated fatty acids from a mixture
which also contains methyl esters of polyunsaturated fatty acids.
According to Christie, when urea is permitted to crystallize in the
presence of various long-chain aliphatic compounds, it forms
hexagonal crystals which incorporate the aliphatic compounds (a
urea crystal which incorporates an aliphatic compound is sometimes
referred to as a "urea complex"), thereby allowing the aliphatic
compounds to be easily separated from the solution via filtration.
Christie states generally that methyl esters of saturated fatty
acids form urea complexes more readily than methyl esters of
unsaturated fatty acids having the same length, and that methyl
esters of unsaturated fatty acids having trans double bonds form
urea complexes more readily than methyl esters of analogous fatty
acids having cis double bonds. Christie also reports using urea
crystallization to concentrate methyl esters of polyunsaturated
fatty acids from a mixture containing methyl esters of
polyunsaturated fatty acids and methyl esters of saturated fatty
acids.
[0006] Another reference directed to separating methyl esters of
fatty acids using urea crystallization is T. Nakahara, T. Yokochi,
T. Higashihara, S. Tanaka, T. Yaguchi, & D. Honda, "Production
of Docosahexaenoic and Docosapentaenoic Acids by Schizochytrium sp.
Isolated from Yap Islands," JAOCS, vol. 73, no. 11, pp. 1421-26
(1996). Nakahara et al. report making a mixture of methyl esters of
fatty acids by washing and drying Schizochytrium sp. cells, and
then methyl-esterifying the cells directly with methanol in the
presence of 10% HCl. Nakahara et al. report that 34.9% of the
resulting methyl esters contained DHA residues, and 8.7% contained
DPA residues. To concentrate these polyunsaturated fatty acid
methyl esters, Nakahara et al. report adding methanol and urea to
the mixture, heating the mixture to 60.degree. C. to dissolve the
urea, and then cooling the mixture to 10.degree. C. to crystallize
the urea. Nakahara et al. report that they were able to recover
73.3% of the DHA methyl esters and 17.7% of the DPA methyl esters
using this method.
[0007] The growing use of polyunsaturated fatty acids (particularly
DHA) and esters thereof in medical and nutritional applications has
created a further need for a cost-effective and reliable process
that may be used to prepare a composition comprising an enriched
concentration of polyunsaturated fatty acid compounds (and a
minimal concentration of saturated fatty acid compounds) from
sources (particularly naturally occuring sources) of glycerides
having at least one polyunsaturated fatty acid residue.
SUMMARY OF THE INVENTION
[0008] This invention provides for a novel and useful process for
making an enriched composition comprising polyunsaturated fatty
acid compounds (particularly compounds containing a DHA residue).
This process is particularly advantageous because it provides a
method for enrichment of polyunsaturated fatty acid esters (e.g.,
methyl and ethyl esters) despite the fact that: (1) the compounds
involved here are highly complex molecules (e.g., they contain
carbon chains having from 3 to 21 carbon atoms and up to 6 double
bonds), and (2) there often is only a subtle difference in
structure between polyunsaturated fatty acid compounds and many
saturated fatty acid compounds.
[0009] Briefly, therefore, this invention is directed to a process
for making a mixture comprising a polyunsaturated fatty acid ester
having the Formula (V): 2
[0010] In one embodiment, the process comprises transesterifying an
oil from Schizochytrium sp. with an alcohol in the presence of a
base to form a saturated fatty acid ester and the polyunsaturated
fatty acid ester (these fatty acid esters are formed from the
alcohol and fatty acid residues of at least one glyceride in the
oil). Urea is subsequently dissolved in a medium comprising the
fatty acid esters to form a medium comprising the fatty acid esters
and dissolved urea. This medium is then cooled or concentrated to
form (a) a precipitate comprising urea crystals and at least a
portion of the saturated fatty acid ester, which is trapped within
the urea crystals; and (b) a liquid fraction comprising at least
most of the polyunsaturated fatty acid ester. Afterward, the
precipitate is separated from the liquid fraction. Here, the
alcohol is R.sub.3--OH, R.sub.3 is a hydrocarbyl or a substituted
hydrocarbyl, and R.sub.4 is a straight chain hydrocarbyl comprising
21 carbon atoms and at least 2 carbon-carbon double bonds.
[0011] In another embodiment, the process comprises
transesterifying an oil from Schizochytrium sp. with an alcohol in
the presence of an acid to form a saturated fatty acid ester and
the polyunsaturated fatty acid ester (these fatty acid esters are
formed from the alcohol and fatty acid residues of at least one
glyceride in the oil). Urea is subsequently dissolved in a medium
comprising the fatty acid esters to form a medium comprising the
fatty acid esters and dissolved urea. This medium, in turn, is
cooled to a temperature of no less than about 15.degree. C. to form
(a) a precipitate comprising urea crystals and at least a portion
of the saturated fatty acid ester, which is trapped within the urea
crystals; and (b) a liquid fraction comprising at least most of the
polyunsaturated fatty acid ester. Afterward, the precipitate is
separated from the liquid fraction. Here, the alcohol is
R.sub.3--OH, R.sub.3 is a hydrocarbyl or a substituted hydrocarbyl,
and R.sub.4 is a straight chain hydrocarbyl comprising 21 carbon
atoms and at least 2 carbon-carbon double bonds.
[0012] In another embodiment, the process comprises
transesterifying an oil from Schizochytrium sp. with an alcohol in
the presence of an acid to form a reaction mixture comprising a
saturated fatty acid ester and the polyunsaturated fatty acid ester
(the fatty acid esters are formed from the alcohol and fatty acid
residues of at least one glyceride in the oil). At least most of
the polyunsaturated fatty acid ester is subsequently separated from
the reaction mixture to form a mixture comprising the
polyunsaturated fatty acid and a residual amount of the saturated
fatty acid ester. Urea is then dissolved in a medium comprising the
separated polyunsaturated fatty acid ester and residual saturated
fatty acid ester to form a medium comprising the separated
polyunsaturated fatty acid ester, residual saturated fatty acid
ester, and dissolved urea. This medium, in turn, is cooled or
concentrated to form (a) a precipitate comprising urea crystals and
at least a portion of the residual saturated fatty acid ester,
which is trapped within the urea crystals; and (b) a liquid
fraction comprising at least most of the separated polyunsaturated
fatty acid ester. Afterward, the precipitate is separated from the
liquid fraction. Here, the alcohol is R.sub.3--OH, R.sub.3 is a
hydrocarbyl or a substituted hydrocarbyl, and R.sub.4 is a straight
chain hydrocarbyl comprising 21 carbon atoms and at least 2
carbon-carbon double bonds.
[0013] In another embodiment, the process comprises
transesterifying an oil from Schizochytrium sp. with an alcohol in
the presence of an acid to form a saturated fatty acid ester and
the polyunsaturated fatty acid ester (these fatty acid esters are
formed from the alcohol and fatty acid residues of at least one
glyceride in the oil). Urea is subsequently dissolved in a medium
comprising the fatty acid esters to form a medium comprising the
fatty acid esters and dissolved urea. This medium, in turn, is
cooled to a temperature of no less than 10.degree. C. to form (a) a
precipitate comprising urea crystals and at least a portion of the
saturated fatty acid ester, which is trapped within the urea
crystals; and (b) a liquid fraction comprising at least most of the
polyunsaturated fatty acid ester. Afterward, the precipitate is
separated from the liquid fraction. Here, the alcohol is
R.sub.3--OH, R.sub.3 comprises at least 2 carbon atoms and is a
hydrocarbyl or substituted hydrocarbyl, and R.sub.4 is a straight
chain hydrocarbyl comprising 21 carbon atoms and at least 2
carbon-carbon double bonds.
[0014] In another embodiment, the process comprises forming the
polyunsaturated fatty acid ester and a saturated fatty acid ester
(these fatty acid esters are formed from at least one glyceride
obtained from Schizochytrium sp.). A solvent comprising the fatty
acid esters is subsequently cooled to a temperature of no less than
about -30.degree. C. and no greater than about 0.degree. C. to form
(a) a precipitate comprising at least a portion of the saturated
fatty acid ester, and (b) a liquid fraction comprising at least
most of the polyunsaturated fatty acid ester. Afterward, the
precipitate is separated from the liquid fraction. Here, the
saturated fatty acid ester has Formula (VI): 3
[0015] R.sub.3 is a hydrocarbyl or a substituted hydrocarbyl,
R.sub.4 is a straight chain hydrocarbyl comprising 21 carbon atoms
and at least 2 carbon-carbon double bonds, and R.sub.5 is a
hydrocarbyl comprising no double bonds.
[0016] Other features of this invention will be in part apparent
and in part pointed out hereinafter.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0017] In accordance with the present invention, a novel and useful
process has been developed for making a composition containing an
enriched concentration of polyunsaturated fatty acid esters
(particularly esters containing a DHA residue) from sources which
comprise a glyceride having at least one polyunsaturated fatty acid
residue. This process generally comprises 2 steps: (1)
transesterifying the starting material to form a polyunsaturated
fatty acid ester (particularly an ester of DHA) and a saturated
fatty acid ester; and (2) separating the polyunsaturated fatty acid
ester from at least a portion of the saturated fatty acid ester via
urea crystallization or winterization, thereby enriching the
concentration of the polyunsaturated fatty acid ester.
[0018] A. Starting Material
[0019] The starting materials that may be used in accordance with
this invention vary widely. Preferably, the starting material is a
naturally occurring source that comprises at least one glyceride
(most preferably a triglyceride) which comprises at least one
polyunsaturated fatty acid residue. In a particularly preferred
embodiment, at least about 10% (more preferably at least about 25%,
and most preferably at least about 30%) of the fatty acid residues
in the source are the desired polyunsaturated fatty acid residues.
Where, for example, the desired polyunsaturated fatty acid compound
is DHA or a derivative thereof, the source preferably is a marine
animal oil, fish oil (e.g., menhaden oil, salmon oil, mackerel oil,
cod oil, herring oil, sardine oil, capelin oil, and tuna oil),
marine algae, or human milk. In an especially preferred embodiment,
the DHA source is the oil from the marine algae identified as
Schizochytrium sp. This oil is commercially available, for example,
under the trade name SEAGOLD from Monsanto Co. (St. Louis,
Mo.).
[0020] As used herein, a "glyceride" is an ester of glycerol and at
least one fatty acid, wherein from 1 to 3 of the hydroxyl groups of
the glycerol have been replaced by a fatty acid residue. Where
multiple fatty acid residues are present, the fatty acid residues
may be the same or different.
[0021] In many suitable starting materials, the bulk of glycerides
are triglycerides. A "triglyceride" is an ester of three fatty acid
residues and glycerol, and has the general chemical formula:
CH.sub.2(OOCR.sup.1)CH(OOCR.sup.2)CH.sub.2(OOCR.sup.3), wherein
OOCR.sup.1, OOCR.sup.2, and OOCR.sup.3 are each fatty acid
residues. To illustrate, a triglyceride having two
4,7,10,13,16,19-22:6 residues (i.e., a DHA fatty acid residue
containing 22 carbon atoms and 6 carbon-carbon cis double bonds
between the 4th & 5th, 7th & 8th, 10th & 11th, 13th
& 14th, 16th & 17th, and 19th & 20th carbon atoms,
counting from the carbonyl group of the residue, the carbon of the
carbonyl group being the first carbon counted) and one palmitic
acid residue (i.e., a fatty acid residue comprising 16 carbon atoms
containing no carbon-carbon double bonds) may have the following
Formula 4
[0022] To illustrate further, a triglyceride having a
4,7,10,13,16,19-22:6 residue; a 4,7,10,13,16-22:5 residue (i.e., a
DPA fatty acid residue containing 22 carbon atoms and 5
carbon-carbon cis double bonds between the 4th & 5th, 7th &
8th, 10th & 11th, 13th & 14th, and 16th & 17th carbon
atoms, counting from the carbonyl group of the residue, the carbon
of the carbonyl group being the first carbon counted); and a
palmitic acid residue may have the following Formula (II): 5
[0023] As shown in Formulas (I) and (II), each fatty acid residue
may be either saturated (i.e., all the bonds between the carbon
atoms are single bonds) or unsaturated (i.e., there is at least one
carbon-carbon double or triple bond). Unsaturated fatty acid
residues are sometimes identified herein by an omega (".omega.")
number. This number denominates the position of the first double
bond, when counting from the terminal methyl group of the fatty
acid or fatty acid residue. For example, in Formulas (I) and (II),
the DHA residues are .omega.-3 fatty acid residues. The DPA residue
in Formula (II), on the other hand, is an .omega.-6 fatty acid
residue. Generally, polyunsaturated fatty acid residues having the
most beneficial medical and nutritional properties are .omega.-3
fatty acid residues.
[0024] B. Transesterification of the Glycerides in the Starting
Material to Form Separate Esters of the Fatty Acid Residues of the
Glycerides
[0025] The purpose of the transesterification step is to cleave the
fatty acid residues from the glycerol backbone of the glycerides in
the starting material and form separate esters of each of the
residues so that they can be isolated from each other. As used
herein, an ester of a fatty acid residue has the following Formula
(III): 6
[0026] wherein R.sub.1 is the straight hydrocarbyl chain of the
fatty acid residue, and R.sub.2 is a hydrocarbyl or substituted
hydrocarbyl. For example, the methyl ester of 4,7,10,13,16,19-22:6
having all cis carbon-carbon double bonds (i.e., the methyl ester
of DHA) has the following Formula (IV): 7
[0027] In a preferred embodiment, the glycerides are
transesterified to form alkyl esters of the fatty acid residues.
These alkyl esters preferably are lower alkyl esters (i.e., R.sub.2
in Formula (III) is a hydrocarbyl containing from 1 to 6 carbon
atoms). More preferably, the esters are methyl esters or ethyl
esters (i.e., R.sub.2 in Formula (III) is a hydrocarbyl containing
1 or 2 carbon atoms). Most preferably, the esters are ethyl esters
(i.e., R.sub.2 in Formula (III) is a hydrocarbyl containing 2
carbon atoms). Ethyl esters typically taste better and are less
toxic (e.g., when ethyl esters hydrolyze in the digestive tract to
form free fatty acids, they produce ethanol as a byproduct; on the
other hand, the hydrolysis of methyl esters produces methanol as a
byproduct, which is generally more toxic than ethanol).
[0028] In a particularly preferred embodiment of this invention,
the transesterification reaction is catalyzed by a base or an acid.
This reaction comprises contacting the starting material with an
alcohol in the presence of the base or acid, as the following
reaction illustrates for a triglyceride starting material: 8
[0029] wherein R.sub.2--OH is the alcohol; R.sub.2 is a hydrocarbyl
or a substituted hydrocarbyl; and R.sub.6, R.sub.7, and R.sub.8 are
the straight hydrocarbyl chains of the fatty acid residues. It is
presently believed that, during base-catalyzed transesterification,
the alcohol is de-protonated to form an oxide ion, which, in turn,
attacks the carbonyl groups on the triglyceride to form separate
R.sub.2-esters of each of the fatty acid residues of the
triglyceride. During acid-catalyzed transesterification, on the
other hand, the carbonyl oxygen atoms of the glyceride are
protonated and then subjected to nucleophilic attack by the alcohol
to form the separate R.sub.2-esters of each of the fatty acid
residues.
[0030] Because formation of lower alkyl esters is generally
preferred, the alcohol preferably is a lower alkyl alcohol
containing from 1 to 6 carbon atoms. More preferably, the alcohol
is methanol (which reacts with glycerides to form methyl esters of
the fatty acid residues) or ethanol (which reacts with glycerides
to form ethyl esters of the fatty acid residues). Most preferably,
the alcohol is ethanol.
[0031] Acid-catalyzed transesterification may be carried out, for
example, by incubating a triglyceride at from about 0 to about
150.degree. C. in a mixture containing the alcohol and an acid
(e.g., HCl), preferably under a non-oxidizing atmosphere and in the
absence of water. In one embodiment, the triglyceride/acid/alcohol
mixture is refluxed for at least about 2 hours. In another
embodiment, the triglyceride/acid/alcohol mixture is maintained at
from about 0 to about 50.degree. C. overnight. The alcohol
preferably has the formula R.sub.2--OH (wherein R.sub.2 is defined
above for Formula III), and is selected to form the desired fatty
acid ester. For example, methanol may be used to form methyl
esters, and ethanol may be used to form ethyl esters. Because
acid-catalyzed transesterification is typically reversible, the
alcohol preferably is present in a large excess so that the
reaction proceeds essentially to completion. Preferably, the
triglyceride concentration in the alcohol/acid mixture is from
about 0.1 to about 15% by weight, and most preferably about 3% by
weight. If the acid is HCl, the concentration of HCl in the
alcohol/HCl mixture preferably is from about 4 to about 15% by
weight, and most preferably about 10% by weight. Such a mixture may
be prepared by various methods known in the art, such as bubbling
dry gaseous hydrogen chloride into dry ethanol, or adding 1 ml of
acetylchloride to each 10 ml of alcohol (to form approximately 10%
by weight HCl in alcohol). Although HCl is most preferred, other
acids may alternatively be used. One such acid is or
H.sub.2SO.sub.4, which typically is used at a concentration of from
about 0.5 to about 5% by weight in the alcohol. It should be noted,
however, that because H.sub.2SO.sub.4 is a strong oxidizing agent,
it preferably is not used with long reflux times (i.e., greater
than about 6 hours), at high concentrations (i.e., greater than
about 5% by weight), or at high temperatures (i.e., greater than
150.degree. C.). Another example of a suitable acid is boron
trifluoride, which preferably is used at a concentration of from
about 1 to about 20% by weight in the alcohol. Boron trifluoride,
however, is less preferred than HCl because boron trifluoride has a
greater tendency to produce undesirable byproducts.
[0032] A triglyceride alternatively may be transesterified by, for
example, base-catalyzed transesterification wherein the
triglyceride is transesterified by an alcohol in the presence of a
basic catalyst. In this instance, the base may be, for example,
sodium methoxide, potassium methoxide, elemental sodium, sodium
hydroxide, or potassium hydroxide. Preferably, the volumetric ratio
of triglyceride to the base/alcohol mixture is at least about 1:1,
and most preferably about 1:2. The concentration of the base in the
alcohol preferably is from about 0.1 to about 2 M. In one
embodiment, the base-catalyzed transesterification reaction is
conducted at room temperature (i.e., at a temperature of from about
20 to about 25.degree. C.) for from about 6 to about 20 hours. In
another embodiment, the base-catalyzed transesterification reaction
is conducted at a temperature greater than room temperature. In
this embodiment, the glyceride/alcohol/catalyst solution preferably
is heated to a temperature of at least about 40.degree. C., more
preferably from about 70 to about 150.degree. C., and most
preferably at about 100.degree. C. In a particularly preferred
embodiment, the solution is heated using a reflux condenser so that
the reaction mixture may be heated to temperatures above the
boiling point of one or more components in the mixture without
losing the components into the vapor phase (i.e., when the
components vaporize, they rise into the reflux condenser which has
a cooler temperature, thereby causing the vapor to condense into a
liquid and flow back into the liquid mixture).
[0033] During the transesterification reaction, the reacting
mixture preferably is placed under a non-oxidizing atmosphere, such
as an atmosphere consisting essentially of a noble gas, N.sub.2, or
a combination thereof. Use of such an atmosphere is particularly
preferred if the transesterification reaction is conducted over a
period of time exceeding about 10 minutes. An atmosphere consisting
essentially of N.sub.2 is most preferred due to the relatively low
cost of N.sub.2. Placing the reacting mixture under a non-oxidizing
atmosphere is advantageous because oxidizing atmospheres tend to
cause the carbon-carbon double bonds of the polyunsaturated fatty
acid residues to oxidize to form, for example, aldehydes and
epoxides. Such oxidized residues are undesirable, in part, because
they tend to make the fatty acid moieties less palatable. An
antioxidant (e.g., ascorbyl palmitate or propyl gallate) may also
be added to the reacting mixture to prevent auto-oxidation, and is
particularly preferred where a non-oxidizing atmosphere is not
used.
[0034] The transesterification may be conducted in a mixture
comprising an organic solvent. This solvent may vary widely, but
preferably is capable of solubilizing the glyceride that comprises
the polyunsaturated fatty acid residue. Where the starting material
comprises more than one glyceride, the organic solvent preferably
is capable of solubilizing all the glycerides. Examples of often
suitable solvents include dichloromethane, acetonitrile, ethyl
acetate, and diethyl ether. Dichloromethane is presently most
preferred.
[0035] Following the transesterification reaction, the esters
preferably are separated from the reaction mixture by adding water.
Often, these esters (which are organic) rise to the top of the
reaction mixture and may simply be skimmed from the remaining
reaction mixture. This is particularly true in large-scale,
industrial applications.
[0036] Alternatively, liquid-liquid solvent extraction may be used
to separate the esters from the remaining reaction mixture. This
extraction may vary widely. In one embodiment, for example, the
extraction generally begins by adding water to the mixture, and
then extracting the esters with a non-polar solvent. The amount of
water added to the reaction mixture may vary widely, and most
typically is about 1:1. If the transesterification is
base-catalyzed, the water preferably comprises sufficient acid to
neutralize the mixture, or, even more preferably, to impart a
slightly acidic pH to the mixture. The acid used is not critical,
and may, for example, be hydrochloric acid, citric acid, or acetic
acid, with hydrochloric acid being most preferred. The ratio of
total volume of non-polar solvent to the volume of reaction mass
(including the water added) also may vary widely, and is most
preferably from about 1:3 to about 4:3. In a particularly preferred
embodiment, the mixture is extracted with multiple fractions
(preferably 3 fractions) of the non-polar organic solvent which are
subsequently combined after the individual extractions are
completed. Examples of suitable non-polar solvents include
petroleum ether, pentane, hexane, cyclohexane, or heptane, with
hexane and petroleum ether being the most preferred. The non-polar
solvent also may contain a small amount of slightly polar organic
solvent as well, such as diethyl ether. Use of such a slightly
polar component tends to improve the extraction of fatty acid
esters from the aqueous layer because such esters also are slightly
polar. If a slightly polar organic component is used, the
volumetric concentration of the slightly polar component to the
non-polar component preferably is no greater than about 20%, more
preferably no greater than about 10%, and most preferably from
about 5 to about 10%. The resulting extraction organic solvent
layer may be washed to remove, for example, any residual free acid
and/or residual water in the layer. Removal of residual free acid
preferably is achieved by washing the layer with an aqueous
solution containing a weak base, e.g., an aqueous solution
containing about 2% by weight of potassium bicarbonate
concentration. Removal of residual water may be achieved, for
example, by washing the layer with brine (i.e., a saturated salt
solution) and/or passing the layer over an anhydrous salt (e.g.,
sodium sulfate or magnesium sulfate). If the solvent is washed with
brine, the volumetric ratio of brine to solvent preferably is about
1:6.
[0037] Following the extraction, the fatty acid esters in the
non-polar solvent layer may be concentrated. In one embodiment of
this invention, the esters are concentrated by evaporating a
portion of the non-polar solvent.
[0038] C. Separating the Polyunsaturated Fatty Acid Ester(s) from
at Least a Portion of at Least One Saturated Fatty Acid Ester via
Urea Crystallization or Winterization
[0039] Transesterification of a naturally occurring starting
material typically produces other fatty acid esters in addition to
the desired polyunsaturated fatty acid ester(s). As noted earlier,
many such fatty esters (particularly saturated fatty esters) tend
to have unknown and/or adverse medical and nutritional properties.
It is therefore often advantageous to remove at least a portion of
the saturated fatty esters from the transesterification reaction
mixture to form an enriched mixture containing the desired
polyunsaturated fatty acid(s). This separation preferably is
performed using either urea crystallization or winterization.
[0040] 1. Urea Crystallization
[0041] When urea crystallizes in a solution containing
polyunsaturated fatty acid esters (e.g., esters of DHA) and
saturated fatty acid esters formed by the transesterification of a
glyceride source using the techniques discussed above, a
precipitate forms which comprises the urea and at least a portion
of the saturated fatty acid esters. This precipitate, however,
comprises a substantially lesser fraction of the polyunsaturated
fatty acid esters than the initial reaction mixture. The bulk of
the polyunsaturated fatty acid esters, instead, remain in solution
and can therefore be easily separated from the precipitated
saturated fatty acid esters.
[0042] The urea crystallization separation process comprises first
forming a solution comprising fatty acid esters and urea. The
amount of urea preferably is proportional to the total amount of
saturated fatty acids to be separated from the solution. When
separating fatty acid esters from the transesterification reaction
mixtures described above, the mass ratio of the mixture of fatty
acid esters to urea is typically about 1:2. The solution also
preferably comprises an organic solvent which can solubilize urea
and the desired polyunsaturated fatty acid ester, and more
preferably can solubilize urea and all the fatty acid esters in the
mixture. Examples of often suitable solvents include alkyl alcohols
having from 1 to 4 carbons, with methanol and ethanol being more
preferred, and ethanol being the most preferred. The volumetric
ratio of the mixture of fatty acid esters to solvent is preferably
about 1:10.
[0043] Essentially all the urea preferably is dissolved in the
solution. This may generally be achieved by heating the solution.
The solution, however, preferably is not heated to a temperature
above the boiling point of the organic solvent. Typically, the
solution preferably is heated to a temperature of about 60.degree.
C.
[0044] Once the urea is dissolved in the solution, the mixture
preferably is cooled to form a precipitate comprising urea.
Preferably, the solution is cooled to a temperature which is
greater than 10.degree. C., more preferably to a temperature which
is no less than about 15.degree. C., still more preferably to a
temperature which is no less than about 20.degree. C., and most
preferably to a temperature of from about 20 to about 25.degree. C.
Once the solution is cooled, it preferably is allowed to stand for
a period of time (typically no greater than about 20 hours) at the
cooling temperature with occasional stirring.
[0045] In another embodiment of this invention, after the solution
comprising fatty acid esters and dissolved urea is formed, a
precipitate comprising urea is formed by concentrating the
solution. The solution may be concentrated, for example, by
evaporating a portion of the solvent in the solution. The amount of
solvent removed preferably is sufficient to cause the urea
concentration in the solution to exceed the saturation
concentration.
[0046] During the urea crystallization separation process, the
solution preferably is kept in a non-oxidizing atmosphere, such as
an atmosphere consisting essentially of a noble gas, N.sub.2, or a
combination thereof, with an atmosphere consisting essentially of
N.sub.2 being most preferred. As noted above, use of such an
atmosphere aids in minimizing oxidation of carbon-carbon double
bonds of the polyunsaturated fatty acid esters.
[0047] After the precipitate comprising urea has formed, the
precipitate preferably is separated from the liquid fraction
enriched in polyunsaturated esters. This may be achieved, for
example, by filtration or centrifugation. In a particularly
preferred embodiment, the precipitate is subsequently washed with a
small quantity of the organic solvent (preferably saturated with
urea) to recover any residual unprecipitated desired
polyunsaturated fatty acid ester that remains with the precipitate.
This solvent, in turn, preferably is combined with the liquid
fraction.
[0048] The liquid fraction preferably is concentrated, combined
with water, and then the esters therein are preferably extracted
with a non-polar solvent from the resulting mixture. The liquid
fraction may be concentrated, for example, by evaporating a portion
of the solvent from the liquid fraction (the amount of solvent
evaporated, however, preferably is not so great as to cause further
urea to precipitate). The amount of water subsequently combined
with the resulting concentrated liquid fraction may vary widely.
Preferably, the volume ratio of water to concentrated liquid
fraction is about 2:1 (in a particularly preferred embodiment,
sufficient acid (preferably HCl) is also introduced to neutralize
the urea). The non-polar solvent that may be used to extract the
fatty acid esters from the resulting
concentrated-mother-liquor/water mixture may be, for example,
petroleum ether, pentane, hexane, cyclohexane, ethyl acetate, or
heptane, with hexane being the most preferred. The volumetric ratio
of the non-polar solvent to the concentrated-mother-liquor/water
mixture preferably is about 2:3.
[0049] In an especially preferred embodiment, the liquid fraction
is also extracted with a slightly polar organic solvent to maximize
recovery of the fatty acid esters (which, as noted above, are
slightly polar). Examples of suitable slightly polar solvents
include diethyl ether and ethyl acetate, with diethyl ether being
most preferred. Preferably, the volumetric ratio of slightly polar
solvent to the mother-liquor/water mixture is about 2:3. Following
the extraction with this slightly polar solvent, the solvent
preferably is combined with the non-polar solvent used in the
initial extraction.
[0050] After the extraction is complete, any residual water may be
removed from the extraction solvent by, for example, washing the
solvent with brine and/or passing the solvent over an anhydrous
salt (e.g., sodium sulfate). The solution then preferably is
concentrated by, for example, evaporating a portion of the
solvent.
[0051] 2. Winterization
[0052] It has been found in accordance with this invention that
winterization is a time-efficient alternative to urea
crystallization for enriching the concentration of a
polyunsaturated fatty acid ester in a fatty acid ester mixture
comprising the polyunsaturated fatty acid ester and saturated fatty
acid esters. Winterization comprises cooling a solution comprising
the polyunsaturated fatty acid ester and the saturated fatty acid
esters to a temperature which will cause at least a portion of the
saturated fatty acid esters to precipitate, while causing a
substantially less proportion of the desired polyunsaturated fatty
acid ester to precipitate.
[0053] Winterization is typically conducted in an organic solvent
that can solubilize the desired polyunsaturated fatty acid ester
and at least one saturated fatty acid ester in the fatty acid ester
mixture. Suitable solvents include, for example, methanol and
ethanol, with methanol being the most preferred. Preferably, the
volumetric ratio of the fatty acid ester mixture to the organic
solvent is about 1:12.
[0054] Preferably, after the fatty acid mixture is dissolved in the
organic solvent, the solution is cooled to a temperature which is
low enough to cause a precipitate to form which comprises at least
one saturated fatty acid ester. Preferably, however, the solution
is not cooled to a temperature so low that the amount of the
desired polyunsaturated fatty acid ester(s) (e.g., an ester of DHA
and/or DPA) in the precipitate exceeds about 30 wt. % of the amount
of desired polyunsaturated fatty acid ester(s) in the fatty acid
mixture before conducting the winterization. More preferably, the
solution is not cooled to a temperature so low that the amount of
the desired polyunsaturated fatty acid ester(s) in the precipitate
exceeds 25 wt. % of the amount of the desired polyunsaturated fatty
acid ester(s) in the fatty acid mixture before conducting the
winterization. And most preferably, the solution is not cooled to a
temperature so low that the amount of the desired polyunsaturated
fatty acid ester(s) in the precipitate exceeds 20 wt. % of the
amount of the desired polyunsaturated fatty acid ester(s) in the
fatty acid mixture before conducting the winterization. In a
particularly preferred embodiment of this invention, the solution
is cooled to a temperature which is no greater than about 0.degree.
C., more preferably from about -30 to about -10.degree. C., still
more preferably from about -25 to about -15.degree. C., and most
preferably about -20.degree. C. The solution preferably is
maintained at these temperatures for up to about 20 hours, and kept
under a non-oxidizing atmosphere to minimize the oxidation of the
carbon-carbon double bonds of the polyunsaturated fatty acid
esters.
[0055] After forming the precipitate, the solution preferably is
separated from the precipitate to form a liquid fraction enriched
in the desired polyunsaturated fatty acid ester(s). This may be
achieved, for example, by filtration or centrifugation. After the
liquid fraction is separated, it may be concentrated by, for
example, evaporating the solvent in a rotary evaporator.
Definitions
[0056] Unless otherwise stated, the following definitions should be
used:
[0057] The term "hydrocarbyl" is defined as a radical consisting
exclusively of carbon and hydrogen. The hydrocarbyl may be branched
or unbranched, may be saturated or unsaturated, and may contain one
or more rings. Suitable hydrocarbyl residues include alkyl,
alkenyl, alkynyl, and aryl residues. They also include alkyl,
alkenyl, alkynyl, and aryl residues substituted with other
aliphatic or cyclic hydrocarbyl groups, such as alkaryl, alkenaryl
and alkynaryl.
[0058] The term "substituted hydrocarbyl" is defined as a
hydrocarbyl wherein at least one hydrogen atom has been substituted
with an atom other than hydrogen. For example, the hydrogen atom
may be replaced by a halogen atom, such as a chlorine or fluorine
atom. The hydrogen atom alternatively may be substituted by an
oxygen atom to form, for example, a hydroxy group, an ether, an
ester, an anhydride, an aldehyde, a ketone, or a carboxylic acid.
The hydrogen atom also may be replaced by a nitrogen atom to form,
for example, an amide or a nitro functionality. To illustrate
further, the hydrogen atom may be replaced with a sulfur atom to
form, for example, --SO.sub.3H.sub.2.
[0059] With reference to the use of the word(s) "comprise" or
"comprises" or "comprising" in this entire specification (including
the claims below), Applicant notes that unless the context requires
otherwise, those words are used on the basis and clear
understanding that they are to be interpreted inclusively, rather
than exclusively, and that Applicant intends each of those words to
be so interpreted in construing this entire specification.
EXAMPLES
[0060] The following examples are simply intended to further
illustrate and explain the present invention. This invention,
therefore, should not be limited to any of the details in these
examples.
Example 1
Base-Catalyzed Transesterification
[0061] Approximately 100 g of oil extracted from Schizochytrium sp.
(SEAGOLD, Monsanto Co., St. Louis, Mo.) was dissolved in 100 ml of
dichloromethane and 200 ml of methanol. Then 1 g of elemental
sodium was added. The solution was refluxed for 10 min., and then
poured into 500 ml of water containing 16 ml of 12N HCl. The
aqueous layer was extracted 3 times with 400 ml of hexane. The 3
hexane layers were combined and washed with an aqueous solution
containing 2% potassium bicarbonate, and finally dried over
anhydrous sodium sulfate.
Example 2
Acid-Catalyzed Transesterification
[0062] A mixture of HCl and methanol was prepared by slowly adding
100 ml of acetylchloride to 1000 ml of methanol at 0.degree. C.
Approximately 30 g of oil extracted from Schizochytrium sp. was
then added to 1000 ml of this mixture. The mixture was then stirred
overnight under an N.sub.2 atmosphere. Approximately 800 ml of ice
cold (i.e., roughly 0.degree. C.) distilled water was then added to
the mixture, and the mixture was transferred to a separatory funnel
where it was extracted three times with 200 ml of an organic
solvent containing diethyl ether and petroleum ether. The
volumetric ratio of the diethyl ether to petroleum ether in the
solvent was approximately 1:9. The combined organic layer was then
washed with brine (i.e., a saturated solution of NaCl) and dried
over anhydrous sodium sulfate. The solvent in the organic layer was
then evaporated using a rotary evaporator.
[0063] The yield of the transesterified product was 30 g. Gas
chromatography of the product revealed that 9.5 wt. % consisted of
the methyl ester of 14-carbon saturated fatty acid, 28.2 wt. %
consisted of the methyl ester of 16-carbon saturated fatty acid,
14.3 wt. % consisted of the methyl ester of .omega.-6 DPA, and 36.2
wt. % consisted of the methyl ester of .omega.-3 DHA.
Example 3
Using Urea Crystallization to Increase Concentration of Methyl
Ester of DHA
[0064] Approximately 50 g of methyl esters prepared using the
technique of Example 2 were dissolved in 500 ml of methanol in a
flask. Afterward, 100 g of urea was added, and the mixture was
heated until essentially all the urea was dissolved. The flask was
flushed with N.sub.2 and sealed with aluminum foil, and then
allowed to cool to room temperature (i.e., from about 20 to about
25.degree. C.). The mixture was allowed to sit overnight with
occasional swirling. The next day, the material was filtered
through a Buchner funnel to remove the urea crystals. After washing
the crystals twice with 25 ml of methanol (saturated with urea),
the methanol was added to the filtrate. A portion of the methanol
in the filtrate was then evaporated from the filtrate using a
rotary evaporator until the filtrate had a volume of 150 ml.
Approximately 300 ml of an aqueous solution of 1% HCl was then
added to the filtrate. Afterward, this mixture was extracted with
300 ml of hexane and then with 300 ml of diethyl ether. The organic
layers were combined and then washed twice with 50 ml of water,
washed once with 50 ml of brine, and finally dried over anhydrous
sodium sulfate. The solvent was then removed under reduced
pressure.
[0065] Approximately 23 g of crude product was obtained. Gas
chromatography of the product revealed that 23.4 wt. % consisted of
the methyl ester of .omega.-6 DPA, 65.2 wt. % consisted of the
methyl ester of .omega.-3 DHA, 2.9 wt. % consisted of the methyl
ester of 14-carbon saturated fatty acid, and 1.5 wt. % consisted of
the methyl ester of 16-carbon saturated fatty acid.
Example 4
Using Winterization to Increase Concentration of Methyl Ester of
DHA
[0066] Approximately 15 g of methyl esters prepared using the
technique of Example 2 were dissolved in 175 ml of methanol in a
flask. The flask was flushed with N.sub.2 and
* * * * *