U.S. patent application number 12/163555 was filed with the patent office on 2009-01-22 for production and purification of esters of polyunsaturated fatty acids.
This patent application is currently assigned to MARTEK BIOSCIENCES CORPORATION. Invention is credited to Jung Lee, Neil Leininger, Chris Luigart, Krishna Raman.
Application Number | 20090023808 12/163555 |
Document ID | / |
Family ID | 40226489 |
Filed Date | 2009-01-22 |
United States Patent
Application |
20090023808 |
Kind Code |
A1 |
Raman; Krishna ; et
al. |
January 22, 2009 |
Production and Purification of Esters of Polyunsaturated Fatty
Acids
Abstract
The present invention includes methods for producing and
purifying esters of polyunsaturated fatty acids that include
reacting a composition having triglycerides with polyunsaturated
fatty acid residues in the presence of an alcohol and a base to
produce an ester of a polyunsaturated fatty acid from the
triglycerides. The composition can be a polyunsaturated fatty
acid-containing composition that has not been conventionally
processed. The reacted composition can be further processed by
distillation.
Inventors: |
Raman; Krishna; (Wilmington,
DE) ; Lee; Jung; (McLean, VA) ; Leininger;
Neil; (Winchester, KY) ; Luigart; Chris;
(Lexington, KY) |
Correspondence
Address: |
SHERIDAN ROSS PC
1560 BROADWAY, SUITE 1200
DENVER
CO
80202
US
|
Assignee: |
MARTEK BIOSCIENCES
CORPORATION
Columbia
MD
|
Family ID: |
40226489 |
Appl. No.: |
12/163555 |
Filed: |
June 27, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60947284 |
Jun 29, 2007 |
|
|
|
Current U.S.
Class: |
514/549 ;
554/167; 554/9 |
Current CPC
Class: |
C07C 67/08 20130101;
C07C 67/03 20130101; C07C 69/533 20130101; A61P 25/28 20180101;
A23L 33/12 20160801; A61P 3/06 20180101; C07C 69/533 20130101; C07C
67/08 20130101; C07C 67/03 20130101; C11C 3/003 20130101; A23K
20/158 20160501; A21D 2/16 20130101; C07C 69/587 20130101; C07C
67/60 20130101 |
Class at
Publication: |
514/549 ;
554/167; 554/9 |
International
Class: |
A61K 31/23 20060101
A61K031/23; A61P 25/28 20060101 A61P025/28; C11C 1/00 20060101
C11C001/00 |
Claims
1. A method for purifying a composition comprising triglycerides
having polyunsaturated fatty acid residues comprising: a) reacting
the composition in the presence of an alcohol and a base to produce
an ester of a polyunsaturated fatty acid from the triglycerides;
and b) distilling the composition to recover a fraction comprising
the ester of the polyunsaturated fatty acid.
2. The method of claim 1, wherein the step of reacting the
composition in the presence of an alcohol and a base is performed
at a temperature from about 60.degree. C. to about 120.degree.
C.
3. The method of claim 1, wherein the step of reacting the
composition in the presence of an alcohol and a base is performed
for a time from about 2 hours to about 12 hours.
4. The method of claim 1, wherein the composition comprising
triglycerides having polyunsaturated fatty acid residues has not
been subjected to one or more treatments selected from the group
consisting of refining, desolventization, deodorization,
winterization, chill filtration, and bleaching.
5. The method of claim 1, wherein the composition comprising
triglycerides having polyunsaturated fatty acid residues has not
been subjected to refining, desolventization, deodorization,
winterization, chill filtration, and bleaching.
6. The method of claim 1, wherein the composition comprising
triglycerides having polyunsaturated fatty acid residues is from a
source selected from the group consisting of a plant, a
microorganism, an animal, and mixtures of the foregoing.
7. The method of claim 6, wherein the source is a microorganism
selected from the group consisting of algae, bacteria, fungi and
protists.
8. The method of claim 6, wherein the source is selected from the
group consisting of plants selected from the group consisting of
soybean, corn, rice, safflower, sunflower, canola, flax, peanut,
mustard, rapeseed, chickpea, cotton, lentil, white clover, olive,
palm, borage, evening primrose, linseed and tobacco and mixtures
thereof.
9. The method of claim 6, wherein the source is selected from the
group consisting of a genetically modified plant and a genetically
modified microorganism, wherein the genetic modification comprises
the introduction of polyketide synthase genes.
10. The method of claim 6, wherein the source is a microorganism
selected from the group consisting of Thraustochytriales,
dinoflagellates, and Mortierella.
11. The method of claim 10, wherein the microorganism is
Thraustochytriales.
12. The method of claim 11, wherein the microorganism is
Schizochytrium.
13. The method of claim 11, wherein the microorganism is
Thraustochytrium.
14. The method of claim 10, wherein the microorganism is a
dinoflagellate of the genus Crypthecodinium.
15. The method of claim 6, wherein the source is an animal selected
from aquatic animals.
16. The method of claim 1, wherein the polyunsaturated fatty acid
is a polyunsaturated fatty acid having a chain length of at least
18 carbons.
17. The method of claim 1, wherein the polyunsaturated fatty acid
is a polyunsaturated fatty acid selected from the group consisting
of docosahexaenoic acid, docosapentaenoic acid, arachidonic acid,
eicosapentaenoic acid, stearidonic acid, linolenic acid, alpha
linolenic acid, gamma linolenic acid, conjugated linolenic acid and
mixtures thereof.
18. The method of claim 17, wherein the polyunsaturated fatty acid
is docosahexaenoic acid.
19. The method of claim 17, wherein the polyunsaturated fatty acid
is arachadonic acid.
20. The method of claim 1, wherein the base is a base of the
formula RO-M, wherein M is a monovalent cation and RO is an
alkoxide of a C.sub.1-6 alkyl alcohol.
21. The method of claim 1, wherein the base is sodium ethoxide.
22. The method of claim 1, wherein the alcohol is a C.sub.1-6 alkyl
alcohol.
23. The method of claim 1, wherein the alcohol is ethanol and the
ester is an ethyl ester of the polyunsaturated fatty acid.
24. The method of claim 1, wherein the step of distilling the
composition to recover a fraction comprising the ester of the
polyunsaturated fatty acid is performed under vacuum.
25. The method of claim 24, wherein the step of distilling the
composition to recover a fraction comprising the ester of the
polyunsaturated fatty acid is performed at a temperature of less
than about 170.degree. C.
26. The method of claim 1, wherein the fraction recovered comprises
at least about 50 wt. % ester of the polyunsaturated fatty
acid.
27. The method of claim 1, wherein the fraction recovered comprises
at least about 75 wt. % ester of the polyunsaturated fatty
acid.
28. The method of claim 1, wherein the fraction recovered comprises
at least about 90 wt. % ester of the polyunsaturated fatty
acid.
29. The method of claim 1, wherein the fraction recovered comprises
at least about 95 wt. % ester of the polyunsaturated fatty
acid.
30. The method of claim 1, wherein the step of reacting the
composition in the presence of an alcohol and a base produces an
ester of a polyunsaturated fatty acid from the triglycerides by
direct transesterification.
31. The method of claim 1, further comprising: a) combining the
fraction comprising the ester of the polyunsaturated fatty acid
with urea in a medium; b) cooling or concentrating the medium to
form a urea-containing precipitate and a liquid fraction; and c)
separating the precipitate from the liquid fraction.
32. The method of claim 31, wherein the medium further comprises an
organic solvent that can solubilize the ester of the
polyunsaturated fatty acid.
33. The method of claim 32, wherein the organic solvent comprises
an alkyl alcohol comprising from 1 to 4 carbon atoms.
34. The method of claim 33, wherein the organic solvent comprises
ethanol.
35. The method of claim 31, wherein the medium is cooled to a
temperature of from about 0.degree. C. to about 25.degree. C. to
form the urea-containing precipitate.
36. The method of claim 31, wherein at least a portion of the
urea-containing precipitate is formed under a non-oxidizing
atmosphere.
37. A method for producing an ester of a polyunsaturated fatty acid
from a composition comprising triglycerides having polyunsaturated
fatty acid residues comprising: a) transesterifying the composition
in the presence of an alcohol and a base to produce an ester of the
polyunsaturated fatty acid from the triglycerides; and b)
distilling the composition to recover a fraction comprising the
ester of the polyunsaturated fatty acid.
38. (canceled)
39. A method for preparing a composition comprising an ester of a
polyunsaturated fatty acid comprising: reacting a composition
comprising triglycerides having polyunsaturated fatty acid residues
in the presence of an alcohol and a base to produce an ester of a
polyunsaturated fatty acid from the triglycerides, wherein the
composition comprising triglycerides having polyunsaturated fatty
acid residues has not been subjected to one or more treatments
selected from the group consisting of refining, desolventization,
deodorization, winterization, chill filtration, and bleaching.
40-67. (canceled)
68. A composition comprising at least about 90 wt. % ethyl ester of
docosahexaenoic acid, wherein the composition further comprises at
least about 0.1 wt. % of 4,7,10,13,16,19,22,25 octacosaoctaenoic
acid (C28:8) or an ester thereof.
69. The composition of claim 68, wherein the composition comprises
at least about 0.5 wt. % of 4,7,10,13,16,19,22,25 octacosaoctaenoic
acid (C28:8) or an ester thereof.
70. The composition of claim 68, wherein the composition comprises
at least about 1.0 wt. % of 4,7,10,13,16,19,22,25 octacosaoctaenoic
acid (C28:8) or an ester thereof.
71. The composition of claim 68, wherein the composition comprises
at least about 1.2 wt. % of 4,7,10,13,16,19,22,25 octacosaoctaenoic
acid (C28:8) or an ester thereof.
72. The composition of claim 68, wherein the composition further
comprises at least about 0.1 wt. % of docosapentaenoic acid (n-3)
or an ester thereof.
73. The composition of claim 68, wherein the composition further
comprises at least about 0.3 wt. % of docosapentaenoic acid (n-3)
or an ester thereof.
74. The composition of claim 68, wherein the composition further
comprises at least about 0.4 wt. % of docosapentaenoic acid (n-3)
or an ester thereof.
75. The composition of claim 68, wherein the composition further
comprises at least about 0.5 wt. % of docosapentaenoic acid (n-3)
or an ester thereof.
76. The composition of claim 68, wherein the composition comprises
at least about 92 wt. % ethyl ester of docosahexaenoic acid.
77. The composition of claim 68, wherein the composition comprises
at least about 95 wt. % ethyl ester of docosahexaenoic acid.
78. The composition of claim 68, wherein the composition further
comprises less than about 1 wt. % eicosapentaenoic acid or an ester
thereof.
79. The composition of claim 68, wherein the composition further
comprises less than about 0.5 wt. % eicosapentaenoic acid or an
ester thereof.
80. The composition of claim 68, wherein the composition further
comprises less than about 0.25 wt. % eicosapentaenoic acid or an
ester thereof.
81. A composition comprising at least about 90 wt. % ethyl ester of
docosahexaenoic acid, wherein the composition further comprises at
least about 0.1 wt. % of docosapentaenoic acid (n-3) or an ester
thereof.
82. The composition of claim 81, wherein the composition comprises
at least about 0.3 wt. % of docosapentaenoic acid (n-3) or an ester
thereof.
83. The composition of claim 81, wherein the composition comprises
at least about 0.4 wt. % of docosapentaenoic acid (n-3) or an ester
thereof.
84. The composition of claim 81, wherein the composition comprises
at least about 0.5 wt. % of docosapentaenoic acid (n-3) or an ester
thereof.
85. The composition of claim 81, wherein the composition further
comprises at least about 0.5 wt. % of 4,7,10,13,16,19,22,25
octacosaoctaenoic acid (C28:8) or an ester thereof.
86. The composition of claim 81, wherein the composition further
comprises at least about 0.75 wt. % of 4,7,10,13,16,19,22,25
octacosaoctaenoic acid (C28:8) or an ester thereof.
87. The composition of claim 81, wherein the composition further
comprises at least about 1.0 wt. % of 4,7,10,13,16,19,22,25
octacosaoctaenoic acid (C28:8) or an ester thereof.
88. The composition of claim 81, wherein the composition further
comprises at least about 1.2 wt. % of 4,7,10,13,16,19,22,25
octacosaoctaenoic acid (C28:8) or an ester thereof.
89. The composition of claim 81, wherein the composition comprises
at least about 92 wt. % ethyl ester of docosahexaenoic acid.
90. The composition of claim 81, wherein the composition comprises
at least about 95 wt. % ethyl ester of docosahexaenoic acid.
91. The composition of claim 81, wherein the composition further
comprises less than about 1 wt. % eicosapentaenoic acid or an ester
thereof.
92. The composition of claim 81, wherein the composition further
comprises less than about 0.5 wt. % eicosapentaenoic acid or an
ester thereof.
93. The composition of claim 81, wherein the composition further
comprises less than about 0.25 wt. % eicosapentaenoic acid or an
ester thereof.
94. A composition comprising at least about 90 wt. % ethyl ester of
docosahexaenoic acid, wherein the composition further comprises at
least one additional fatty acid or an ester thereof with a boiling
point of about 150-170.degree. C. at a pressure of 0.8 mm Hg.
95-98. (canceled)
99. A composition comprising at least about 90 wt. % of a
combination of ethyl ester of docosahexaenoic acid and ethyl ester
of docosapentaenoic acid (n-6).
100-109. (canceled)
110. A method of treating a subject with high levels of
triglycerides comprising administering a composition according to
claim 68, 81, 94, or 99 to the subject.
111. A method of treating a subject with a neurological disorder,
dementia or a pre-dementia related condition comprising
administering a composition according to claim 68, 81, 94, or 99 to
the subject.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn. 119(e) of U.S. Provisional Application No.
60/947,284, filed Jun. 29, 2007, the disclosure of which is
incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to methods for producing and purifying
esters of polyunsaturated fatty acids from triglyceride containing
compositions. The invention also relates to compositions comprising
polyunsaturated fatty acids.
BACKGROUND OF THE INVENTION
[0003] It is desirable to increase the dietary intake of many
beneficial nutrients. Particularly beneficial nutrients include
fatty acids such as omega-3 and omega-6 long chain polyunsaturated
fatty acids (LC-PUFAs) and esters thereof. Omega-3 PUFAs are
recognized as important dietary compounds for preventing
arteriosclerosis and coronary heart disease, for alleviating
inflammatory conditions and for retarding the growth of tumor
cells. Omega-6 PUFAs serve not only as structural lipids in the
human body, but also as precursors for a number of factors in
inflammation, such as prostaglandins and leukotrienes. Long chain
omega-3 and the omega-6 PUFAs represent important classes of
PUFAs.
[0004] There are two main series or families of LC-PUFAs, depending
on the position of the double bond closest to the methyl end of the
fatty acid: the omega-3 series contains a double bond at the third
carbon, while the omega-6 series has no double bond until the sixth
carbon. Thus, docosahexaenoic acid ("DHA") has a chain length of 22
carbons with 6 double bonds beginning with the third carbon from
the methyl end and is designated "22:6 n-3". Other important
omega-3 LC-PUFAs include eicosapentaenoic acid ("EPA"), which is
designated "20:5 n-3," and omega-3 docosapentaenoic acid ("DPA
n-3"), which is designated "22:5 n-3." Important omega-6 LC-PUFAs
include arachidonic acid ("ARA"), which is designated "20:4 n-6,"
and omega-6 docosapentaenoic acid ("DPA n-6"), which is designated
"22:5 n-6."
[0005] Because humans and many other animals cannot directly
synthesize omega-3 and omega-6 essential fatty acids, they must be
obtained in the diet. Traditional dietary sources of PUFAs include
vegetable oils, marine animal oils, fish oils and oilseeds. In
addition, oils produced by certain microorganisms have been found
to be rich in LC-PUFAs. The oils derived from each of these
sources, however, also contain substantial levels of saturated
fatty acids and other undesirable impurities.
[0006] Numerous methods have been used to isolate or purify PUFAs
and derivatives thereof from crude oils. Among these processes are
fractional crystallization at low temperatures, urea adduct
crystallization, extraction with metal salt solutions, super
critical fluid fractionation on countercurrent columns and high
performance liquid chromatography.
[0007] The increased use of PUFAs and esters thereof in the fields
of medicine and nutrition has created a commensurate need for PUFAs
that are concentrated and free of impurities. Previous efforts
directed to purifying PUFAs, however, have suffered from problems
such as high costs and decreased yields due, in part, to the use of
harsh reagents. Accordingly, there is a need for improved methods
of isolating and purifying PUFAs in a form that can be consumed and
utilized by humans and other animals.
SUMMARY OF THE INVENTION
[0008] The present invention provides a method for purifying a
composition comprising triglycerides having polyunsaturated fatty
acid residues comprising reacting the composition in the presence
of an alcohol and a base to produce an ester of a polyunsaturated
fatty acid from the triglycerides and distilling the composition to
recover a fraction comprising the ester of the polyunsaturated
fatty acid.
[0009] In some embodiments, the step of reacting the composition in
the presence of an alcohol and a base is performed at a temperature
from about 60.degree. C. to about 120.degree. C.
[0010] In some embodiments, the step of reacting the composition in
the presence of an alcohol and a base is performed for a time from
about 2 hours to about 12 hours.
[0011] In some embodiments, the composition comprising
triglycerides having polyunsaturated fatty acid residues has not
been subjected to one or more treatments selected from the group
consisting of refining, desolventization, deodorization,
winterization, chill filtration, and bleaching.
[0012] In some embodiments, the composition comprising
triglycerides having polyunsaturated fatty acid residues has not
been subjected to refining, desolventization, deodorization,
winterization, chill filtration, and bleaching.
[0013] In some embodiments, the composition comprising
triglycerides having polyunsaturated fatty acid residues is from a
source selected from the group consisting of a plant, a
microorganism, an animal, and mixtures of the foregoing.
[0014] In some embodiments, the source is a microorganism selected
from the group consisting of algae, bacteria, fungi and
protists.
[0015] In some embodiments, the source is selected from the group
consisting of plants selected from the group consisting of soybean,
corn, rice, safflower, sunflower, canola, flax, peanut, mustard,
rapeseed, chickpea, cotton, lentil, white clover, olive, palm,
borage, evening primrose, linseed and tobacco and mixtures
thereof.
[0016] In some embodiments, the source is selected from the group
consisting of a genetically modified plant and a genetically
modified microorganism, wherein the genetic modification comprises
the introduction of polyketide synthase genes.
[0017] In some embodiments, the source is a microorganism selected
from the group consisting of Thraustochytriales, dinoflagellates,
and Mortierella.
[0018] In some embodiments, the microorganism is
Thraustochytriales, Schizochytrium or Thraustochytrium.
[0019] In some embodiments, the microorganism is a dinoflagellate
of the genus Crypthecodinium.
[0020] In some embodiments, the source is an animal selected from
aquatic animals.
[0021] In some embodiments, the polyunsaturated fatty acid is a
polyunsaturated fatty acid having a chain length of at least 18
carbons.
[0022] In some embodiments, the polyunsaturated fatty acid is a
polyunsaturated fatty acid selected from the group consisting of
docosahexaenoic acid, docosapentaenoic acid, arachidonic acid,
eicosapentaenoic acid, stearidonic acid, linolenic acid, alpha
linolenic acid, gamma linolenic acid, conjugated linolenic acid and
mixtures thereof.
[0023] In some embodiments, the polyunsaturated fatty acid is
docosahexaenoic acid.
[0024] In some embodiments, the polyunsaturated fatty acid is
arachadonic acid.
[0025] In some embodiments, the base is a base of the formula RO-M,
wherein M is a monovalent cation and RO is an alkoxide of a
C.sub.1-6 alkyl alcohol.
[0026] In some embodiments, the base is sodium ethoxide.
[0027] In some embodiments, the alcohol is a C.sub.1-6 alkyl
alcohol.
[0028] In some embodiments, the alcohol is ethanol and the ester is
an ethyl ester of the polyunsaturated fatty acid.
[0029] In some embodiments, the step of distilling the composition
to recover a fraction comprising the ester of the polyunsaturated
fatty acid is performed under vacuum.
[0030] In some embodiments, the step of distilling the composition
to recover a fraction comprising the ester of the polyunsaturated
fatty acid is performed at a temperature of less than about
170.degree. C.
[0031] In some embodiments, the fraction recovered comprises at
least about 50 wt. %, 75 wt. %, 90 wt. %, or 95 wt. % ester of the
polyunsaturated fatty acid.
[0032] In some embodiments, the step of reacting the composition in
the presence of an alcohol and a base produces an ester of a
polyunsaturated fatty acid from the triglycerides by direct
transesterification.
[0033] In some embodiments, the method further comprises a)
combining the fraction comprising the ester of the polyunsaturated
fatty acid with urea in a medium; b) cooling or concentrating the
medium to form a urea-containing precipitate and a liquid fraction;
and c) separating the precipitate from the liquid fraction.
[0034] In some embodiments, the medium further comprises an organic
solvent that can solubilize the ester of the polyunsaturated fatty
acid.
[0035] In some embodiments, the organic solvent comprises an alkyl
alcohol comprising from 1 to 4 carbon atoms.
[0036] In some embodiments, the organic solvent comprises
ethanol.
[0037] In some embodiments, the medium is cooled to a temperature
of from about 0.degree. C. to about 25.degree. C. to form the
urea-containing precipitate.
[0038] In some embodiments, at least a portion of the
urea-containing precipitate is formed under a non-oxidizing
atmosphere.
[0039] The present invention also provides a method for producing
an ester of a polyunsaturated fatty acid from a composition
comprising triglycerides having polyunsaturated fatty acid residues
comprising transesterifying the composition in the presence of an
alcohol and a base to produce an ester of the polyunsaturated fatty
acid from the triglycerides and distilling the composition to
recover a fraction comprising the ester of the polyunsaturated
fatty acid.
[0040] The present invention further provides a method for
purifying a composition comprising triglycerides having
polyunsaturated fatty acid residues comprising reacting the
composition in the presence of an alcohol and a base to produce an
ester of the polyunsaturated fatty acid from the triglycerides and
separating a fraction comprising at least about 75% ester of the
polyunsaturated fatty acid.
[0041] In some embodiments, the step of separating comprises
distilling.
[0042] The present invention also provides a method for preparing a
composition comprising an ester of a polyunsaturated fatty acid
comprising reacting a composition comprising triglycerides having
polyunsaturated fatty acid residues in the presence of an alcohol
and a base to produce an ester of a polyunsaturated fatty acid from
the triglycerides, wherein the composition comprising triglycerides
having polyunsaturated fatty acid residues has not been subjected
to one or more treatments selected from the group consisting of
refining, desolventization, deodorization, winterization, chill
filtration, and bleaching.
[0043] In some embodiments, the step of reacting the composition in
the presence of an alcohol and a base is performed at a temperature
from about 60.degree. C. to about 120.degree. C.
[0044] In some embodiments, the step of reacting the composition in
the presence of an alcohol and a base is performed for a time from
about 2 hours to about 12 hours.
[0045] In some embodiments, the method further comprises a step of
distilling the composition to recover a fraction comprising the
ester of the polyunsaturated fatty acid.
[0046] In some embodiments, the composition comprising
triglycerides having polyunsaturated fatty acid residues is from a
source selected from the group consisting of a plant, a
microorganism, an animal, and mixtures of the foregoing.
[0047] In some embodiments, the source is a microorganism selected
from the group consisting of algae, bacteria, fungi and
protists.
[0048] In some embodiments, the source is selected from the group
consisting of plants selected from the group consisting of soybean,
corn, rice, safflower, sunflower, canola, flax, peanut, mustard,
rapeseed, chickpea, cotton, lentil, white clover, olive, palm,
borage, evening primrose, linseed and tobacco and mixtures
thereof.
[0049] In some embodiments, the source is selected from the group
consisting of a genetically modified plant and a genetically
modified microorganism, wherein the genetic modification comprises
the introduction of polyketide synthase genes.
[0050] In some embodiments, the source is a microorganism selected
from the group consisting of Thraustochytriales, dinoflagellates,
and Mortierella.
[0051] In some embodiments, the source is a microorganism selected
from the group consisting of Thraustochytriales, dinoflagellates,
and Mortierella.
[0052] In some embodiments, the microorganism is a dinoflagellate
of the genus Crypthecodinium.
[0053] In some embodiments, the source is an animal selected from
aquatic animals.
[0054] In some embodiments, the polyunsaturated fatty acid is a
polyunsaturated fatty acid having a chain length of at least 18
carbons.
[0055] In some embodiments, the polyunsaturated fatty acid is a
polyunsaturated fatty acid selected from the group consisting of
docosahexaenoic acid, docosapentaenoic acid, arachidonic acid,
eicosapentaenoic acid, stearidonic acid, linolenic acid, alpha
linolenic acid, gamma linolenic acid, conjugated linolenic acid and
mixtures thereof.
[0056] In some embodiments, the polyunsaturated fatty acid is
docosahexaenoic acid.
[0057] In some embodiments, the polyunsaturated fatty acid is
arachadonic acid.
[0058] In some embodiments, the base is a base of the formula RO-M,
wherein M is a monovalent cation and RO is an alkoxide of a
C.sub.1-6 alkyl alcohol.
[0059] In some embodiments, the base is sodium ethoxide.
[0060] In some embodiments, the alcohol is a C.sub.1-6 alkyl
alcohol.
[0061] In some embodiments, the alcohol is ethanol and the ester is
an ethyl ester of the polyunsaturated fatty acid.
[0062] In some embodiments, the step of distilling the composition
to recover a fraction comprising the ester of the polyunsaturated
fatty acid is performed under vacuum.
[0063] In some embodiments, the step of distilling the composition
to recover a fraction comprising the ester of the polyunsaturated
fatty acid is performed at a temperature of less than about
170.degree. C.
[0064] In some embodiments, the fraction recovered comprises at
least about 50 wt. %, 75 wt. %, 90 wt. %, or 95 wt. % ester of the
polyunsaturated fatty acid.
[0065] In some embodiments, the step of reacting the composition in
the presence of an alcohol and a base produces an ester of a
polyunsaturated fatty acid from the triglycerides by direct
transesterification.
[0066] The present invention also provides a composition comprising
at least about 90 wt. % ethyl ester of docosahexaenoic acid,
wherein the composition further comprises at least about 0.1 wt. %
of 4,7,10,13,16,19,22,25 octacosaoctaenoic acid (C28:8) or an ester
thereof.
[0067] In some embodiments, the composition comprises at least
about 0.5 wt. %, 1.0 wt. %, or 1.2 wt. % of 4,7,10,13,16,19,22,25
octacosaoctaenoic acid (C28:8) or an ester thereof.
[0068] In some embodiments, the composition further comprises at
least about 0.1 wt. %, 0.3 wt. %, 0.4 wt. %, or 0.5 wt. % of
docosapentaenoic acid (n-3) or an ester thereof.
[0069] In some embodiments, the composition comprises at least
about 92 wt. % or 95 wt. % ethyl ester of docosahexaenoic acid.
[0070] In some embodiments, the composition further comprises less
than about 1 wt. %, 0.5 wt. % or 0.25 wt. % eicosapentaenoic acid
or an ester thereof.
[0071] The present invention further provides a composition
comprising at least about 90 wt. % ethyl ester of docosahexaenoic
acid, wherein the composition further comprises at least about 0.1
wt. % of docosapentaenoic acid (n-3) or an ester thereof.
[0072] In some embodiments, the composition comprises at least
about 0.3 wt. %, 0.4 wt. %, or 0.5 wt. % of docosapentaenoic acid
(n-3) or an ester thereof.
[0073] In some embodiments, the composition further comprises at
least about 0.5 wt. %, 0.75 wt. %, 1.0 wt. %, or 1.2 wt. % of
4,7,10,13,16,19,22,25 octacosaoctaenoic acid (C28:8) or an ester
thereof.
[0074] In some embodiments, the composition comprises at least
about 92 wt. % or 95 wt. % ethyl ester of docosahexaenoic acid.
[0075] In some embodiments, the composition further comprises less
than about 1 wt. %, 0.5 wt. % or 0.25 wt. % eicosapentaenoic acid
or an ester thereof.
[0076] The present invention also provides a composition comprising
at least about 90 wt. % ethyl ester of docosahexaenoic acid,
wherein the composition further comprises at least one additional
fatty acid or an ester thereof with a boiling point of about
150-170.degree. C. at a pressure of 0.8 mm Hg.
[0077] The present invention further provides a composition
comprising at least about 70 wt. % ethyl ester of docosahexaenoic
acid and at least about 25 wt. % ethyl ester of docosapentaenoic
acid (n-6).
[0078] In some embodiments, the composition further comprises less
than about 4% of a saturated fatty acid or an ester thereof.
[0079] In some embodiments, the saturated fatty acid or an ester
thereof contains less than 20 carbons.
[0080] In some embodiments, the saturated fatty acid or an ester
thereof contains 14 or 16 carbons.
[0081] The present invention also provides a composition comprising
at least about 90 wt. % of a combination of ethyl ester of
docosahexaenoic acid and ethyl ester of docosapentaenoic acid
(n-6).
[0082] In some embodiments, the composition comprises at least
about 10 wt. % ethyl ester of docosahexaenoic acid and at least
about 10 wt. % ethyl ester of docosapentaenoic acid (n-6).
[0083] In some embodiments, the composition further comprises less
than about 4% of a saturated fatty acid or an ester thereof.
[0084] In some embodiments, the saturated fatty acid or an ester
thereof contains less than 20 carbons.
[0085] In some embodiments, the saturated fatty acid or an ester
thereof contains 14 or 16 carbons.
[0086] The present invention further provides a comprising at least
about 90 wt. % of a combination of ethyl ester of docosahexaenoic
acid and ethyl ester of docosapentaenoic acid (n-6), wherein the
composition further comprises at least one additional fatty acid or
an ester thereof with a boiling point of about 150-175.degree. C.
at a pressure of 0.5 mm Hg.
[0087] The present invention also provides a method for preparing a
composition comprising an ester of a polyunsaturated fatty acid
comprising reacting a composition comprising triglycerides having
polyunsaturated fatty acid residues in the presence of an alcohol
and a base to produce an ester of a polyunsaturated fatty acid from
the triglycerides, wherein the composition comprising triglycerides
having polyunsaturated fatty acid residues comprises at least one
characteristic selected from the group consisting of: at least
about 300 ppm phosphorus, at least about 0.4% free fatty acids, and
a peroxide value of at least about 0.2 meq/kg.
[0088] The present invention also provides a method for purifying a
composition comprising triglycerides having polyunsaturated fatty
acid residues, wherein the composition comprises at least one
characteristic selected from the group consisting of: at least
about 300 ppm phosphorus, at least about 0.4% free fatty acids, and
a peroxide value of at least about 0.2 meq/kg, comprising a)
reacting the composition in the presence of an alcohol and a base
to produce an ester of a polyunsaturated fatty acid from the
triglycerides; and b) distilling the composition to recover a
fraction comprising the ester of the polyunsaturated fatty
acid.
[0089] The present invention further provides a composition
comprising at least about 60 wt. % esters of arachidonic acid.
[0090] In some embodiments, the composition further comprises less
than about 10 wt. % eicosapentaenoic acid.
[0091] In some embodiments, the esters of arachidonic acid are
ethyl esters of arachidonic acid.
[0092] The present invention also provides a method of treating a
subject with high levels of triglycerides comprising administering
a composition according to claim 68, 81, 94, 95, 99 or 104 to the
subject.
[0093] The present invention further provides a method of treating
a subject with a neurological disorder, dementia or a pre-dementia
related condition comprising administering a composition according
to claim 68, 81, 94, 95, 99 or 104 to the subject.
DESCRIPTION OF THE INVENTION
[0094] The present invention provides novel methods for the
purification of compositions containing triglycerides having PUFA
residues. In various aspects, the invention includes reacting the
composition in the presence of an alcohol and a base to produce an
ester of a polyunsaturated fatty acid from the triglycerides. In
one embodiment, the invention advantageously and efficiently is
conducted on relatively crude oils that have not been subjected to
conventional processing methods that can include refining,
bleaching, deodorizing and winterization. In another embodiment,
the invention includes producing esters from triglycerides and then
distilling the resulting composition to recover a fraction
comprising the ester of the polyunsaturated fatty acid. In an
additional embodiment, the fraction comprising the ester of the
polyunsaturated fatty acid is further purified by urea
crystallization. The present invention allows the efficient and
cost effective production of esters of PUFAs directly from crude or
processed oils.
[0095] The starting material for the methods of the present
invention is a composition comprising triglycerides having PUFA
residues. The terms "oils" and "compositions comprising
triglycerides having PUFA residues" are used interchangeably
throughout this application. As used herein, a "triglyceride" is an
ester of three fatty acid residues and glycerol having a general
chemical formula of
CH.sub.2(OOCR.sup.1)CH(OOCR.sup.2)CH.sub.2(OOCR.sup.3), wherein
each of OOCR.sup.1, OOCR.sup.2, and OOCR.sup.3 represents a fatty
acid residue. Suitable triglycerides contain at least one PUFA. In
some embodiments, the PUFA has a chain length of at least 18
carbons. Such PUFAs are referred to herein as long chain PUFAs or
LC PUFAs. In some embodiments, the PUFA can be docosahexaenoic acid
C22:6 n-3 (DHA), omega-3 docosapentaenoic acid C22:5 n-3 (DPA),
omega-6 docosapentaenoic acid C22:5 n-6 (DPA), arachidonic acid
C20:4 n-6 (ARA), eicosapentaenoic acid C20:5 n-3 (EPA), stearidonic
acid (SDA), linolenic acid (LLA), alpha linolenic acid (ALA), gamma
linolenic acid (GLA), conjugated linolenic acid (CLA) or mixtures
thereof. The PUFAs can also be present in any of the common forms
found in natural lipids including but not limited to
triacylglycerols, diacylglycerols, monoacylglycerols,
phospholipids, free fatty acids, or in natural or synthetic
derivative forms of these fatty acids (e.g. calcium salts of fatty
acids, and the like). Reference to an oil or other composition
comprising triglycerides having PUFA residues, as used in the
present invention, can refer to either a composition comprising
triglycerides having only a single type of LC PUFA residue such as
DHA or a composition comprising triglycerides having a mixture of
more than one type of LC PUFA residues such as more than one of
DHA, EPA and ARA.
[0096] Compositions comprising triglycerides having PUFA residues
can be obtained from or derived from any suitable source, such as a
plant (including oilseeds), a microorganism, an animal, or mixtures
of the foregoing. The microorganisms can be algae, bacteria, fungi
or protists. Microbial sources and methods for growing
microorganisms comprising nutrients and/or PUFAs are known in the
art (Industrial Microbiology and Biotechnology, 2nd edition, 1999,
American Society for Microbiology). For example, the microorganisms
can be cultured in a fermentation medium in a fermentor. Oils
produced by microorganisms can be used in the methods and
compositions of the present invention. In some embodiments,
organisms include those selected from the group consisting of
golden algae (such as microorganisms of the kingdom Stramenopiles),
green algae, diatoms, dinoflagellates (such as microorganisms of
the order Dinophyceae including members of the genus
Crypthecodinium such as, for example, Crypthecodinium cohnii),
yeast, and fungi of the genera Mucor and Mortierella, including but
not limited to Mortierella alpina and Mortierella sect. schmuckeri.
Members of the microbial group Stramenopiles include microalgae and
algae-like microorganisms, including the following groups of
microorganisms: Hamatores, Proteromonads, Opalines, Develpayella,
Diplophrys, Labrinthulids, Thraustochytrids, Biosecids, Oomycetes,
Hypochytridiomycetes, Commation, Reticulosphaera, Pelagomonas,
Pelagococcus, Ollicola, Aureococcus, Parmales, Diatoms,
Xanthophytes, Phaeophytes (brown algae), Eustigmatophytes,
Raphidophytes, Synurids, Axodines (including Rhizochromulinaales,
Pedinellales, Dictyochales), Chrysomeridales, Sarcinochrysidales,
Hydrurales, Hibberdiales, and Chromulinales. The Thraustochytrids
include the genera Schizochytrium (species include aggregatum,
limnaceum, mangrovei, minutum, octosporum), Thraustochytrium
(species include arudimentale, aureum, benthicola, globosum,
kinnei, motivum, multirudimentale, pachydermum, proliferum, roseum,
striatum), Ulkenia (species include amoeboidea, kerguelensis,
minuta, profunda, radiate, sailens, sarkariana, schizochytrops,
visurgensis, yorkensis), Aplanochytrium (species include
haliotidis, kerguelensis, profunda, stocchinoi), Japonochytrium
(species include marinum), Althornia (species include crouchii),
and Elina (species include marisalba, sinorifica). The
Labrinthulids include the genera Labyrinthula (species include
algeriensis, coenocystis, chattonii, macrocystis, macrocystis
atlantica, macrocystis macrocystis, marina, minuta, roscoffensis,
valkanovii, vitellina, vitellina pacifica, vitellina vitellina,
zopfi), Labyrinthomyxa (species include marina), Labyrinthuloides
(species include haliotidis, yorkensis), Diplophrys (species
include archeri), Pyrrhosorus* (species include marinus),
Sorodiplophrys* (species include stercorea), Chlamydomyxa* (species
include labyrinthuloides, montana). (*=there is no current general
consensus on the exact taxonomic placement of these genera).
[0097] Suitable microorganisms include those capable of producing
lipids comprising omega-3 and/or omega-6 polyunsaturated fatty
acids, and in particular microorganisms that are capable of
producing oils containing DHA, DPA, EPA or ARA will be described.
More particularly, preferred microorganisms are algae, such as
Thraustochytrids of the order Thraustochytriales, including
Thraustochytrium (including Ulkenia) and Schizochytrium and
including Thraustochytriales which are disclosed in commonly
assigned U.S. Pat. Nos. 5,340,594 and 5,340,742, both issued to
Barclay, all of which are incorporated herein by reference in their
entirety. More preferably, the microorganisms are selected from the
group consisting of microorganisms having the identifying
characteristics of ATCC number 20888, ATCC number 20889, ATCC
number 20890, ATCC number 20891 and ATCC number 20892. Since there
is some disagreement among experts as to whether Ulkenia is a
separate genus from the genus Thraustochytrium, for the purposes of
this application, the genus Thraustochytrium will include Ulkenia.
Also preferred are strains of Mortierella schmuckeri (e.g.,
including ATCC 74371) and Mortierella alpina. Also preferred are
strains of Crypthecodinium cohnii, including microorganisms having
the identifying characteristics of ATCC Nos. 30021, 30334-30348,
30541-30543, 30555-30557, 30571, 30572, 30772-30775, 30812, 40750,
50050-50060, and 50297-50300. Oleaginous microorganisms are also
preferred. As used herein, "oleaginous microorganisms" are defined
as microorganisms capable of accumulating greater than 20% of the
dry weight of their cells in the form of lipids. Genetically
modified microorganisms that produce PUFA-containing oils are also
suitable for the present invention. These can include naturally
PUFA-producing microorganisms that have been genetically modified
as well as microorganisms that do not naturally produce PUFAs but
that have been genetically modified to do so.
[0098] Suitable organisms can be obtained from a number of
available sources, including by collection from the natural
environment. For example, the American Type Culture Collection
currently lists many publicly available strains of microorganisms
identified above. As used herein, any organism, or any specific
type of organism, includes wild strains, mutants, or recombinant
types. Growth conditions in which to culture or grow these
organisms are known in the art, and appropriate growth conditions
for at least some of these organisms are disclosed in, for example,
U.S. Pat. No. 5,130,242, U.S. Pat. No. 5,407,957, U.S. Pat. No.
5,397,591, U.S. Pat. No. 5,492,938, U.S. Pat. No. 5,711,983 and
U.S. Pat. No. 6,607,900, all of which are incorporated herein by
reference in their entirety. When microbial oils are used, the
microorganisms are cultured in an effective medium, herein defined
as any medium capable of promoting oil production. Preferably, the
effective medium also promotes rapid microbial growth. The
microorganisms can be cultured in conventional fermentation modes,
which include, but are not limited to, batch, fed-batch, and
continuous.
[0099] Another source of oils suitable for the compositions and
methods of the present invention includes a plant source, such as
oilseed plants. PUFA-producing plants, in alternate embodiments,
can include those genetically engineered to express genes that
produce PUFAs and those that produce PUFAs naturally. Such genes
can include genes encoding proteins involved in the classical fatty
acid synthase pathways, or genes encoding proteins involved in the
PUFA polyketide synthase (PKS) pathway. The genes and proteins
involved in the classical fatty acid synthase pathways, and
genetically modified organisms, such as plants, transformed with
such genes, are described, for example, in Napier and Sayanova,
Proceedings of the Nutrition Society (2005), 64:387-393; Robert et
al., Functional Plant Biology (2005) 32:473-479; or U.S. Patent
Application Publication 2004/0172682. The PUFA PKS pathway, genes
and proteins included in this pathway, and genetically modified
microorganisms and plants transformed with such genes for the
expression and production of PUFAs are described in detail in: U.S.
Pat. No. 6,140,486, U.S. Pat. No. 6,566,583; U.S. Patent
Application Publication No. 20020194641, U.S. Pat. No. 7,211,418,
U.S. Patent Application Publication No. 20050100995A1, U.S. Patent
Application Publication No. 20070089199, PCT Publication No. WO
05/097982, and U.S. Patent Application Publication No. 20050014231,
each of which is incorporated herein by reference in its
entirety.
[0100] Oilseed crops suitable for use in the present invention
include soybeans, corn, rice, safflower, sunflower, canola, flax,
peanut, mustard, rapeseed, chickpea, cotton, lentil, white clover,
olive, palm oil, borage, evening primrose, linseed, and tobacco
that have been genetically modified to produce a PUFA as described
above.
[0101] Genetic transformation techniques for microorganisms and
plants are well-known in the art. Transformation techniques for
microorganisms are well known in the art and are discussed, for
example, in Sambrook et al., 1989, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Labs Press. A general technique for
transformation of dinoflagellates, which can be adapted for use
with Crypthecodinium cohnii, is described in detail in Lohuis and
Miller, The Plant Journal (1998) 13(3): 427-435. A general
technique for genetic transformation of Thraustochytrids is
described in detail in U.S. Patent Application Publication No.
20030166207, published Sep. 4, 2003. Methods for the genetic
engineering of plants are also well known in the art. For instance,
numerous methods for plant transformation have been developed,
including biological and physical transformation protocols. See,
for example, Miki et al., "Procedures for Introducing Foreign DNA
into Plants" in Methods in Plant Molecular Biology and
Biotechnology, Glick, B. R. and Thompson, J. E. Eds. (CRC Press,
Inc., Boca Raton, 1993) pp. 67-88. In addition, vectors and in
vitro culture methods for plant cell or tissue transformation and
regeneration of plants are available. See, for example, Gruber et
al., "Vectors for Plant Transformation" in Methods in Plant
Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J.
E. Eds. (CRC Press, Inc., Boca Raton, 1993) pp. 89-119. See also,
Horsch et al., Science 227:1229 (1985); Kado, C. I., Crit. Rev.
Plant. Sci. 10:1 (1991); Moloney et al., Plant Cell Reports 8:238
(1989); U.S. Pat. No. 4,940,838; U.S. Pat. No. 5,464,763; Sanford
et al., Part. Sci. Technol. 5:27 (1987); Sanford, J. C., Trends
Biotech. 6:299 (1988); Sanford, J. C., Physiol. Plant 79:206
(1990); Klein et al., Biotechnology 10:268 (1992); Zhang et al.,
Bio/Technology 9:996 (1991); Deshayes et al., EMBO J., 4:2731
(1985); Christou et al., Proc Natl. Acad. Sci. USA 84:3962 (1987);
Hain et al., Mol. Gen. Genet. 199:161 (1985); Draper et al., Plant
Cell Physiol. 23:451 (1982); Donn et al., In Abstracts of VIIth
International Congress on Plant Cell and Tissue Culture IAPTC,
A2-38, p. 53 (1990); D'Halluin et al., Plant Cell 4:1495-1505
(1992) and Spencer et al., Plant Mol. Biol. 24:51-61 (1994).
[0102] When oilseed plants are the source of PUFA-containing oils,
the seeds can be harvested and processed to remove any impurities,
debris or indigestible portions from the harvested seeds.
Processing steps vary depending on the type of oilseed and are
known in the art. Processing steps can include threshing (such as,
for example, when soybean seeds are separated from the pods),
dehulling (removing the dry outer covering, or husk, of a fruit,
seed, or nut), drying, cleaning, grinding, milling and flaking.
After the seeds have been processed to remove any impurities,
debris or indigestible materials, they can be added to an aqueous
solution and then mixed to produce a slurry. In some embodiments,
milling, crushing or flaking is performed prior to mixing with
water. A slurry produced in this manner can be treated and
processed the same way as described for a microbial fermentation
broth.
[0103] Another biomass source of PUFA-containing oils suitable for
the compositions and methods of the present invention includes an
animal source. Examples of animal sources include aquatic animals
(e.g., fish, marine mammals, and crustaceans such as krill and
other euphausids) and animal tissues (e.g., brain, liver, eyes,
etc.) and animal products such as eggs or milk. Techniques for
recovery of PUFA-containing oils from such sources are known in the
art.
[0104] While in one embodiment of the invention the composition
comprising triglycerides having PUFA residues can be a crude oil
(discussed in more detail below), other such compositions useful in
the present invention can be recovered from their sources by any
suitable means known to those in the art. For example, oils can be
recovered by extraction with solvents such as chloroform, hexane,
methylene chloride, methanol and the like, or by supercritical
fluid extraction. Alternatively, the oils can be extracted using
extraction techniques, such as are described in U.S. Pat. No.
6,750,048 and PCT Patent Application Serial No. US01/01806, both
filed Jan. 19, 2001, and entitled "Solventless Extraction Process,"
both of which are incorporated herein by reference in their
entirety. Additional extraction and/or purification techniques are
taught in PCT Patent Application Serial No. PCT/IB01/00841 entitled
"Method for the Fractionation of Oil and Polar Lipid-Containing
Native Raw Materials" filed Apr. 12, 2001; PCT Patent Application
Serial No. PCT/IB01/00963 entitled "Method for the Fractionation of
Oil and Polar Lipid-Containing Native Raw Materials Using
Water-Soluble Organic Solvent and Centrifugation" filed Apr. 12,
2001; U.S. Provisional Patent Application Ser. No. 60/291,484
entitled "Production and Use of a Polar Lipid-Rich Fraction
Containing Stearidonic Acid and Gamma Linolenic Acid from Plant
Seeds and Microbes filed May 14, 2001; U.S. Provisional Patent
Application Ser. No. 60/290,899 entitled "Production and Use of a
Polar-Lipid Fraction Containing Omega-3 and/or Omega-6 Highly
Unsaturated Fatty Acids from Microbes, Genetically Modified Plant
Seeds and Marine Organisms" filed May 14, 2001; U.S. Pat. No.
6,399,803 entitled "Process for Separating a Triglyceride
Comprising a Docosahexaenoic Acid Residue from a Mixture of
Triglycerides" issued Jun. 4, 2002 filed Feb. 17, 2000; and PCT
Patent Application Serial No. US01/01010 entitled "Process for
Making an Enriched Mixture of Polyunsaturated Fatty Acid Esters"
filed Jan. 11, 2001; all of which are incorporated herein by
reference in their entirety. The extracted oils can be evaporated
under reduced pressure to produce a sample of concentrated oil
material. Processes for the enzyme treatment of biomass for the
recovery of lipids are disclosed in U.S. Provisional Patent
Application No. 60/377,550, entitled "HIGH-QUALITY LIPIDS AND
METHODS FOR PRODUCING BY ENZYMATIC LIBERATION FROM BIOMASS," filed
on May 3, 2002; PCT Patent Application Serial No. PCT/US03/14177
entitled "HIGH-QUALITY LIPIDS AND METHODS FOR PRODUCING BY
ENZYMATIC LIBERATION FROM BIOMASS," filed on May 5, 2003; copending
U.S. patent application Ser. No. 10/971,723, entitled "HIGH-QUALITY
LIPIDS AND METHODS FOR PRODUCING BY LIBERATION FROM BIOMASS," filed
on Oct. 22, 2004; EP Patent Publication 0 776 356 and U.S. Pat. No.
5,928,696, both entitled "Process for extracting native products
which are not water-soluble from native substance mixtures by
centrifugal force," the disclosures of which are hereby
incorporated by reference herein in their entirety.
[0105] In some embodiments, an oil obtained from a source described
above can serve as the starting material for the methods of the
present invention even when it has not been subjected to
conventional processing. Examples of such conventional processes
that may be avoided include refining (e.g., physical refining,
silica refining or caustic refining), desolventization,
deodorization, winterization, chill filtration, and/or bleaching.
Thus, in certain embodiments, the composition containing
triglycerides having PUFA residues has not been subjected to one or
more treatments selected from refining, desolventization,
deodorization, winterization, chill filtration, and bleaching and
in further embodiments, the composition has not been subjected to
any one of refining, desolventization, deodorization,
winterization, chill filtration, and bleaching.
[0106] In further aspects of the invention, the composition
comprising triglycerides having polyunsaturated fatty acid residues
may be an oil having characteristics of oils that have not been
subjected to conventional processing, such as refining,
desolventization, deodorization, winterization, chill filtration,
and bleaching. Thus, a suitable oil can have a chemical or physical
characteristic of an unprocessed oil. For example, the oil may
contain an undesirable component (e.g., an impurity) at a level
that is typically not present in a conventionally processed oil.
For example, the oil may contain from about 300 ppm phosphorous to
about 1000 ppm phosphorous. In some embodiments, the oil comprises
at least about 300 ppm phosphorous; at least about 400 ppm
phosphorous; at least about 500 ppm phosphorous; at least about 600
ppm phosphorous; at least about 650 ppm phosphorous; at least about
700 ppm phosphorous; at least about 750 ppm phosphorous; at least
about 800 ppm phosphorous; at least about 850 ppm phosphorous; at
least about 900 ppm phosphorous; at least about 950 ppm
phosphorous; or at least about 1000 ppm phosphorous. In another
aspect, the oil may contain free fatty acids in a range of from
about 0.4 wt. % to about 1.4 wt. %. In certain embodiments, the oil
comprises at least about 0.4 wt. % free fatty acids; at least about
0.6 wt. % free fatty acids; at least about 0.8 wt. % free fatty
acids; at least about 0.9 wt. % free fatty acids; at least about
1.0 wt. % free fatty acids; at least about 1.1 wt. % free fatty
acids; at least about 1.2 wt. % free fatty acids; at least about
1.3 wt. % free fatty acids; or at least about 1.4 wt. % free fatty
acids. In another aspect, the oil may contain a peroxide value
ranging from about 0.2 meq/kg to about 2.5 meq/kg. In some
embodiments, the oil comprises a peroxide value of at least about
0.2 meq/kg; a peroxide value of at least about 0.4 meq/kg; a
peroxide value of at least about 0.6 meq/kg; a peroxide value of at
least about 0.8 meq/kg; a peroxide value of at least about 1.0
meq/kg; a peroxide value of at least about 1.2 meq/kg; a peroxide
value of at least about 1.4 meq/kg; a peroxide value of at least
about 1.5 meq/kg; a peroxide value of at least about 1.6 meq/kg; a
peroxide value of at least about 1.7 meq/kg; a peroxide value of at
least about 1.8 meq/kg; a peroxide value of at least about 1.9
meq/kg; a peroxide value of at least about 2.0 meq/kg; a peroxide
value of at least about 2.1 meq/kg; a peroxide value of at least
about 2.2 meq/kg; a peroxide value of at least about 2.3 meq/kg; a
peroxide value of at least about 2.4 meq/kg; or a peroxide value of
at least about 2.5 meq/kg.
[0107] In some embodiments, the crude oil may be isolated from a
microorganism using standard techniques, without being subjected to
further refinement or purification. In such embodiments, the oil is
a microbial oil that has only been subjected to solvent extraction,
such as hexane extraction, isopropanol extraction, or the like.
[0108] In other embodiments, compositions comprising triglycerides
having polyunsaturated fatty acid residues, such as oils described
above, may be subjected to further processing steps, such as
refining, desolventization, deodorization, winterization, chill
filtration, and/or bleaching. Such "processed" oils include
microbial oils that have been subjected to solvent extraction and
one or more of these additional processing steps. In some
embodiments, oils are minimally processed. "Minimally processed"
oils include microbial oils that have been subjected to solvent
extraction and filtration. In certain embodiments, minimally
processed oils are further subjected to winterization.
[0109] Methods of the present invention involve reacting
compositions containing triglycerides having PUFA residues in the
presence of an alcohol and a base to produce esters of the PUFAs
from the triglycerides.
[0110] Alcohols suitable for use in the present invention include
any lower alkyl alcohol containing from 1 to 6 carbon atoms (i.e.,
a C.sub.1-6 alkyl alcohol). Without being bound by theory, it is
believed that the use of lower alkyl alcohols in the methods of the
present invention produces lower alkyl esters of the PUFAs. For
example, the use of ethanol produces ethyl esters. In certain
embodiments, the alcohol is methanol or ethanol. In these
embodiments, the PUFA esters produced are a methyl ester and an
ethyl ester of the PUFA, respectively. In processes of the present
invention, the alcohol typically comprises between about 25 wt. %
and about 50 wt. %; between about 30 wt. % and about 45 wt. %, or
between about 35 wt. % and about 40 wt. % of the mixture of the
composition, the alcohol and the base. In some embodiments, the
alcohol comprises about 38 wt. % of the mixture of the composition,
the alcohol and the base. In certain embodiments, the composition
and the base can be added to either pure ethanol or pure methanol.
In general, the amount of alcohol used may vary with the solubility
of the oil or composition containing triglycerides having PUFA
residues in the alcohol.
[0111] Any base known in the art to be suitable for use as a
reactant may be used in the present invention. Bases of the formula
RO-M, wherein M is a monovalent cation and RO is an alkoxide of a
C.sub.1-6 alkyl alcohol are particularly suited for the present
invention. Examples of suitable bases include elemental sodium,
sodium methoxide, sodium ethoxide, potassium methoxide, and
potassium ethoxide. In some embodiments, the base is sodium
ethoxide. In processes of the present invention, the base is
typically added in an amount of between about 0.5 and about 1.5
molar equivalents of triglycerides, between about 0.7 and about 1.4
molar equivalents of triglycerides, between about 0.9 and about 1.3
molar equivalents of triglycerides, or between about 1.0 and about
1.2 molar equivalents of triglycerides to the reaction step with
the composition and the alcohol. In certain embodiments, the base
is typically added in an amount of about 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 0.95, 1.0, 1.01, 1.02, 1.03, 1.04, 1.05, 1.10,
1.15, 1.2, 1.3, 1.4, or 1.5 molar equivalents of triglycerides to
the reaction step with the composition and the alcohol. In some
embodiments, the base is added in an amount of 1.04 molar
equivalents of triglycerides to the reaction step with the
composition and the alcohol.
[0112] The composition comprising triglycerides having
polyunsaturated fatty acid residues, the alcohol and the base are
reacted together at a temperature and for an amount of time that
allows the production of an ester between the fatty acid residues
and the alcohol. Suitable reaction times and temperatures may be
determined by one of skill in the art to produce an ester. Without
intending to be bound by theory, the PUFA residues are believed to
be cleaved from the glycerol backbone of the triglyceride and
esters of each PUFA residue are formed during the step of reacting.
In certain embodiments, the step of reacting the composition in the
presence of an alcohol and a base is performed at a temperature
from about 60.degree. C. to about 120.degree. C., from about
70.degree. C. to about 110.degree. C., from about 75.degree. C. to
about 100.degree. C., or from about 80.degree. C. to about
90.degree. C. In further embodiments, the step of reacting the
composition in the presence of an alcohol and a base is performed
at a temperature of about 75.degree. C., 80.degree. C., 85.degree.
C., 90.degree. C., or 95.degree. C. In some embodiments, the step
of reacting the composition in the presence of an alcohol and a
base is performed for a time from about 2 hours to about 12 hours,
from about 3 hours to about 11 hours, from about 4 hours to about
10 hours, from about 5 hours to about 9 hours, or from about 6
hours to about 8 hours. In certain embodiments, the step of
reacting the composition in the presence of an alcohol and a base
is performed for about 5.5, 6, 6.5, 7, 7.5, 8, or 8.5 hours
[0113] In one embodiment, the step of reacting the oil composition,
alcohol and base may be conducted by refluxing the components to
produce the PUFA esters. In additional embodiments, the step of
reacting the oil composition may be carried out at a temperature
that does not result in the refluxing of the reaction components.
For example, carrying out the step of reacting the oil composition
under pressures greater than atmospheric pressure can increase the
boiling point of the solvents present in the reaction mixture.
Under such conditions, the reaction can occur at a temperature at
which the solvents would boil at atmospheric pressure, but would
not result in the refluxing of the reaction components. In some
embodiments, the reaction is conducted at a pressure from about 5
to about 20 pounds per square inch (psi); from about 7 to about 15
psi; or from about 9 to about 12 psi. In certain embodiments, the
reaction is conducted at a pressure of about 7, 8, 9, 10, 11, or 12
psi. Reactions conducted under pressure may be carried out at the
reaction temperatures listed above. In some embodiments, reactions
conducted under pressure may be carried out at about 70.degree. C.,
75.degree. C., 80.degree. C., 85.degree. C., or 90.degree. C.
[0114] The reaction mixture comprising PUFA esters can be further
processed to obtain the PUFA esters from the mixture. For example,
the mixture may be cooled, diluted with water, and the aqueous
solution extracted with a solvent such as hexane to produce a
composition comprising PUFA esters. Techniques for washing and/or
extracting crude reaction mixtures are known in the art.
[0115] In one embodiment of the present invention, PUFA esters are
separated from the reaction mixture by distilling the composition
to recover a fraction comprising the ester of the polyunsaturated
fatty acid. In this manner, a targeted fraction of the reaction
mixture including PUFA esters of interest can be separated from the
reaction mixture and recovered.
[0116] In certain embodiments, the distillation is performed under
vacuum. Without being bound by theory, distillation under vacuum
allows the distillation to be accomplished at a lower temperature
than in the absence of a vacuum and thus may prevent the
degradation of the esters. Typical distillation temperatures range
from about 120.degree. C. to about 170.degree. C. In some
embodiments, the step of distilling is performed at a temperature
of less than about 180.degree. C., less than about 175.degree. C.,
less than about 170.degree. C., less than about 165.degree. C.,
less than about 160.degree. C., less than about 155.degree. C.,
less than about 150.degree. C., less than about 145.degree. C.,
less than about 140.degree. C., less than about 135.degree. C., or
less than about 130.degree. C. Typical pressures for vacuum
distillation range from about 0.1 mm Hg to about 10 mm Hg. In some
embodiments, the pressure for vacuum distillation is about 0.1,
0.5, 1, 1.5, 2, 2, 5, 3, 3.5, or 4 mm Hg.
[0117] The methods of the present invention may be used to produce
compositions that contain a high percentage of PUFA esters. For
example, such compositions can contain between about 50 wt. % and
about 100 wt. % of an ester of a PUFA, and in other embodiments,
the composition can comprise at least about 50 wt. %, at least
about 55 wt. %, at least about 60 wt. %, at least about 65 wt. %,
at least about 70 wt. %, at least about 75 wt. %, at least about 80
wt. %, at least about 85 wt. %, at least about 90 wt. %, at least
about 95 wt. %, at least about 99 wt. % of esters of a PUFA.
[0118] In some embodiments, the PUFA esters are subjected to a urea
crystallization step. When urea crystallizes in a solution
containing PUFA 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 that
comprises the urea and at least a portion of the saturated fatty
acid esters. This precipitate, however, comprises a substantially
lesser fraction of the PUFA esters than the initial reaction
mixture. The bulk of the PUFA esters instead remain in solution and
can therefore be easily separated from the precipitated saturated
fatty acid esters.
[0119] 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 that can solubilize urea
and the desired PUFA ester, and more preferably can solubilize urea
and all the fatty acid esters in the mixture. Examples of 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.
[0120] 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 is heated to a temperature of about 60.degree. C.,
65.degree. C., 70.degree. C., 75.degree. C. or 80.degree. C.
[0121] Once the urea is dissolved, the PUFA esters are added to the
solution. Upon addition, if solids remain, the mixture may be
heated until solids dissolve. The solution may be cooled to form a
precipitate comprising urea adducts of fatty acid esters. In
certain embodiments, the solution is cooled to a temperature that
is from about 0.degree. C. to about 25.degree. C., such as from
about 15.degree. C. to about 25.degree. C. In other embodiments,
the solution is cooled to a temperature of about 0.degree. C. about
5.degree. C. about 10.degree. C. about 15.degree. C., about
20.degree. C., about 25.degree. C., or from about 20.degree. C. to
about 25.degree. C. Once the solution is cooled, it may be allowed
to stand for a period of time (typically no greater than about 20
hours) at the cooling temperature with occasional stirring.
[0122] 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.
[0123] During the urea crystallization separation process, the
solution may be 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. Use of such an atmosphere may aid in
minimizing oxidation of carbon-carbon double bonds of the PUFA
esters.
[0124] After the precipitate comprising urea has formed, the
precipitate may be separated from the liquid fraction enriched in
PUFA esters. This may be achieved, for example, by filtration or
centrifugation. In one embodiment, the precipitate may be
subsequently washed with a small quantity of the organic solvent
(preferably saturated with urea) to recover any residual
unprecipitated desired PUFA ester that remains with the
precipitate. This solvent, in turn, may be combined with the liquid
fraction.
[0125] The liquid fraction may be concentrated, combined with
water, and then the esters therein may be 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 H.sub.2SO.sub.4) 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.
[0126] In other embodiments, the liquid fraction may also be
extracted with a slightly polar organic solvent to maximize
recovery of the fatty acid esters (which 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 may be
combined with the non-polar solvent used in the initial
extraction.
[0127] 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.
[0128] By way of example, the methods of the present invention may
be used to purify ethyl arachidonate (arachidonic acid ethyl ester)
from a crude Mortierella alpina oil. A crude oil obtained from
Mortierella alpina by hexane extraction (typically with an ARA
content of about 0.5 g/g oil) can be used directly without any
further processing, such as winterization and/or RBD processing.
150 mL of absolute ethanol can be added to 175 g (approximately 0.2
moles) of the crude oil in a one-liter flask under N.sub.2 at room
temperature. The mixture can be allowed to stir for 15 minutes to
obtain a homogeneous solution. 67 g of a 21% solution of NaOEt/EtOH
(approximately 1.04 molar equivalents) can be then added to the
solution, and the mixture can be allowed to reflux under N.sub.2
for about 10 hours. The progress of the reaction may be monitored
by gas chromatography (GC) and/or thin-layer chromatography
(TLC).
[0129] When the reaction is completed, approximately 75 mL of
ethanol can be removed by distillation, and the mixture can be
allowed to cool to room temperature under N.sub.2. 300 mL of hexane
can be added to the cooled mixture, and the mixture can be allowed
to stir for 15 minutes at room temperature. 300 mL of deionized
water can be then added to the mixture, and the mixture can be
allowed to stir for an additional 15 minutes. After removing and
saving the organic layer, the aqueous layer can be washed twice
with 300 mL portions of hexane. The combined organic layer can be
washed with 200 mL of a saturated NaCl solution. A GC analysis of
the organic layer may be used to determine the amount of ARA ethyl
ester present in the crude product. In some embodiments,
approximately 50% of the crude product is ARA ethyl ester, with the
remaining materials being predominantly lower molecular weight
ethyl esters. The crude product may then be subjected to vacuum
fractional distillation or other purification procedures. In
certain embodiments, a purity of greater than about 60% ARA ethyl
ester may be achieved following the fractional distillation of the
crude product.
[0130] Without being bound by theory, it is believed that the
methods of the present invention result in the direct
transesterification of triglycerides having PUFA residues to
produce esters of the PUFAs. Previous methods utilized long
reaction times, large amounts of reagents, and subjected the oils
to harsh conditions such as high temperatures and highly acidic
conditions. The methods disclosed herein thus provide a more
efficient and economical purification process that yields a pure
product. Furthermore, the methods disclosed herein may be applied
to crude oils as well as purified oils, resulting in an additional
increase in efficiency and cost savings.
[0131] Other embodiments of the present invention include
compositions produced by the methods described herein. As noted
above, such compositions can contain greater than about 50 wt. %,
greater than about 55 wt. %, etc. of esters of a PUFA. In such
embodiments, the compositions can contain at least about 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, or 99 wt. % of PUFA esters. In
other embodiments, the compositions may further comprise less than
about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.25 or 0.1 wt. %
eicosapentaenoic acid. The compositions of the invention may
include any PUFA esters as described above, namely, DHA, omega-3
DPA, omega-6 DPA, ARA, SDA, LLA, ALA, GLA, or CLA or combinations
thereof. In some embodiments, the compositions may comprise ethyl
esters. In certain embodiments, the composition comprises at least
about 89 wt. % DHA esters. In other embodiments, the composition
comprises at least about 89 wt. % of a combination of DHA and DPA
esters.
[0132] Compositions of the present invention also include
compositions that contain at least about 60, 65, 70, 75, 80, 85,
90, or 95 wt. % ARA esters. In some embodiments, the ARA esters may
be ethyl esters of ARA. In other embodiments, the compositions may
further comprise less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1,
0.5, 0.25 or 0.1 wt. % eicosapentaenoic acid.
[0133] The present invention also provides compositions comprising
at least about 90 wt. % ethyl ester of docosahexaenoic acid (DHA)
and at least about 0.1 wt. % of 4,7,10,13,16,19,22,25
octacosaoctaenoic acid (C28:8). These compositions may be produced
by the methods disclosed herein. In some embodiments, the amount of
ethyl ester of DHA in the compositions may be at least about 91,
92, 93, 94, 95, 96, 97, 98, or 99 wt. %. In certain embodiments,
the amount of C28:8 in the compositions may be at least about 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5
wt. %. The C28:8 may be present in triglyceride or ester form. For
example, the C28:8 may be present in ethyl ester form.
[0134] The present invention also provides compositions comprising
at least about 90 wt. % ethyl ester of docosahexaenoic acid (DHA)
and at least about 0.1 wt % of DPA (n-3). These compositions may be
produced by the methods disclosed herein. In some embodiments, the
amount of ethyl ester of DHA in the compositions may be at least
about 91, 92, 93, 94, 95, 96, 97, 98, or 99 wt. %. In certain
embodiments, the amount of DPA (n-3) in the compositions may be at
least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 wt.
% of DPA (n-3). The DPA (n-3) may be present in triglyceride or
ester form. For example, the DPA (n-3) may be present in ethyl
ester form.
[0135] In certain embodiments, the compositions comprise all three
of the ethyl ester of DHA, C28:8 and DPA (n-3) in the concentration
ranges specified above.
[0136] In further embodiments, the compositions may comprise less
than about 1.0, 0.9, 0.8. 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 wt.
% EPA in addition to the ethyl ester of DHA and C28:8. In one
embodiment, the compositions may comprise less than about 0.25 wt.
% EPA. The EPA may be present in triglyceride or ester form. For
example, the EPA may be present in ethyl ester form. In some
embodiments, the compositions may comprise 0 wt. % EPA.
[0137] The present invention also provides compositions comprising
at least about 90 wt. % ethyl ester of docosahexaenoic acid and at
least one additional fatty acid or an ester thereof. In some
embodiments, the amount of ethyl ester of DHA in the compositions
may be at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99 wt. %.
In certain embodiments, the additional fatty acid may have a
boiling point of about 150-170.degree. C. at a pressure of 0.8 mm
Hg.
[0138] The present invention further includes compositions
comprising at least about 70 wt. % ethyl ester of docosahexaenoic
acid (DHA) and at least about 25 wt. % ethyl ester of
docosapentaenoic acid (n-6).
[0139] Compositions of the present invention also include
compositions that comprises at least about 90 wt. % of a
combination of ethyl ester of docosahexaenoic acid and ethyl ester
of docosapentaenoic acid (n-6). In certain embodiments, the
compositions may comprise at least about 91, 92, 93, 94, 95, 96,
97, 98, or 99 wt. % of a combination of ethyl ester of
docosahexaenoic acid and ethyl ester of docosapentaenoic acid
(n-6). In some embodiments, the compositions may comprise at least
about 10 wt. % ethyl ester of docosahexaenoic acid and at least
about 10 wt. % ethyl ester of docosapentaenoic acid (n-6). In other
embodiments, the compositions may comprise at least about 15 or 20
wt. % ethyl ester of docosahexaenoic acid and at least about 15 or
20 wt. % ethyl ester of docosapentaenoic acid (n-6).
[0140] The present invention also provides compositions comprising
at least about 90 wt. % of a combination of ethyl ester of
docosahexaenoic acid and ethyl ester of docosapentaenoic acid
(n-6), and at least one additional fatty acid or an ester thereof.
In certain embodiments, the compositions may comprise at least
about 91, 92, 93, 94, 95, 96, 97, 98, or 99 wt. % of a combination
of ethyl ester of docosahexaenoic acid and ethyl ester of
docosapentaenoic acid (n-6). In some embodiments, the additional
fatty acid may have a boiling point of about 150-170.degree. C. at
a pressure of 0.8 mm Hg.
[0141] The DHA/DPA (n-6) compositions described above may further
comprise less than about 4% of a saturated fatty acid or an ester
thereof. In certain embodiments, the compositions may comprise less
than about 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.0% or 0.5% of a
saturated fatty acid or an ester thereof.
[0142] In some embodiments, the saturated fatty acid or an ester
thereof may contain less than 20 carbons, such as, for example, a
saturated fatty acid or an ester thereof that contains 19, 18, 17.
16, 15, 14, 13, 12, 11, 10, 9 or 8 carbons. In certain embodiments,
the saturated fatty acid or ester thereof may contain 14 or 16
carbons.
[0143] PUFA esters and compositions of the present invention (at
times referred to collectively as "PUFA esters") may be used in
pharmaceutical products. In some embodiments, the pharmaceutical
products may contain PUFA esters without an additional
pharmaceutically active agent. In other embodiments, the
pharmaceutical product may comprise a pharmaceutically active
agent. Examples of pharmaceutically active agents include statins,
anti-hypertensive agents, anti-diabetic agents, anti-dementia
agents, anti-depressants, anti-obesity agents, appetite
suppressants and agents to enhance memory and/or cognitive
function. The pharmaceutical products may further comprises any
pharmaceutically acceptable excipient, carriers, binders or other
formulation components known in the art.
[0144] PUFA esters produced by the methods of the present invention
and compositions of the present invention are suitable for use as
therapeutic and experimental agents. An embodiment of the present
invention comprises the production of PUFA esters for treatment of
PUFA-deficient infants. The PUFA esters can be included in a
parenteral formulation that can be administered to an infant
through parenteral routes to fortify the infant's supply of a PUFA.
Preferred parenteral routes include, but are not limited to,
subcutaneous, intradermal, intravenous, intramuscular and
intraperitoneal routes. A parenteral formulation can include PUFA
esters of the present invention and a carrier suitable for
parenteral delivery. As used herein, a "carrier" refers to any
substance suitable as a vehicle for delivering a molecule or
composition to a suitable in vivo site of action. Examples of such
carriers include, but are not limited to water, phosphate buffered
saline, Ringer's solution, dextrose solution, serum-containing
solutions, Hank's solution and other aqueous physiologically
balanced solutions. Suitable carriers also include oil-based
carriers, non-aqueous solutions, suspensions, and emulsions.
Examples include propylene glycol, polyethylene glycol, vegetable
oils such as olive oil, injectable organic esters such as ethyl
oleate, polyethoxylated castor oil (cremaphor), and others known in
the art. Acceptable protocols to administer PUFA esters in an
effective manner include individual dose size, number of doses,
frequency of dose administration, and mode of administration.
Determination of such protocols can be accomplished by those
skilled in the art depending upon a variety of variables, including
the weight of the infant and the extent of PUFA deficiency. Another
embodiment of the present invention comprises the production of
PUFA esters for treatment of adults, in particular pregnant
mothers. The product may be used for augmenting long chain PUFA
levels in milk of warm-blooded animals. Acceptable protocols for
administration of PUFA esters to adults includes parenteral feeding
techniques or encapsulating PUFA esters of the present invention in
a capsule, such as gelatin (i.e., digestible) capsule, for oral
administration and/or in a liquid diet formulation. A liquid diet
formulation can comprise a liquid composition containing nutrients
suitable for supplementing a diet or nutrients sufficient as a
complete diet.
[0145] PUFA esters produced by the methods of the present invention
and compositions of the present invention may also be used to treat
subjects (e.g., humans or animals) with high levels of
triglycerides, including subjects with triglyceridemia. For
example, subjects with fasting triglycerides of 150 mg/dL or above
may benefit from treatment with the PUFA esters of the present
invention, and, additionally, the elevation of post-parandial
triglyercides may be reduced by treatment with the PUFA esters of
the present invention. In some embodiments, individual PUFA esters
may be administered to a subject to treat high levels of
triglycerides. In certain embodiments, the PUFA ester may be DHA or
ARA. In other embodiments combinations of PUFA esters may be
administered to a subject to treat high levels of triglycerides. In
certain embodiments, the combination of PUFA esters may comprise
omega-3 and omega-6 PUFAS such as DHA and DPA n-6. In some
embodiments, the PUFA esters may comprise about 90% of a
composition administered to the subject. The PUFA esters may be
administered with other components and excipients, such as the
carriers described above. The PUFA esters may also be used to treat
subjects with diseases that can be associated with high levels of
triglycerides, such as cardiovascular disease or hypertension.
[0146] PUFA esters and compositions of the present invention may be
used to treat subjects with neurological disorders, dementia and
pre-dementia related conditions. These conditions include
Alzheimer's Disease, Vascular Dementia, Mixed Dementia, Dementia
with Lewy Bodies, as well as secondary dementias caused by drugs,
delirium, or depression.
[0147] Therapeutic compounds appropriate to use with the PUFA
esters and compositions of the present invention include any
therapeutic which can be used to protect an individual against any
of the conditions or diseases discussed herein, and may include a
protein, an amino acid, a drug, other natural products and a
carbohydrate. Such therapeutic compounds will be well known to
those of skill in the art for the particular disease or condition
being treated. Some preferred therapeutic compounds to combine with
a composition or formulation of the invention include, but are not
limited to: Tacrine (COGNEX); Donepezil (ARICEPT); Rivastigmine
(EXELON); Galantamine (REMINYL); Memantine (AKATINOL); Neotropin;
Nootropics; Alpha-tocopherol (vitamin E); Selegeline (ELDEPRYL);
non-steroidal anti-inflammatory agents (NSAIDS); Gingko biloba;
estrogen; .beta.-secretase inhibitors; vaccines, including lipid or
liposome-based vaccines, that dissolve plaques in the brain; B
complex vitamins; calcium channel blockers; HMG CoA reductase
inhibitors; statins and other anti-cholesterol drugs (e.g., ZOCOR
(simvastatin), LIPITOR (atorvastatin calcium), LESCOL
(fluvastatin), LOPID (gemfibrozil), or PRAVACHOL (pravastatin
sodium)); policosanols; fibrates; Clioquinol; (and other natural
products (e.g., curcumin, lignans, phytoestrogens, phytosterols;
niacin, and vitamin supplements).
[0148] Dosages and routes of administration are known in the art
and may be determined by those of skill in the art.
[0149] Although PUFA esters and compositions of the present
invention can be administered topically or as an injectable, the
most preferred route of administration is oral administration. The
PUFAs may be administered to individuals in the form of nutritional
supplements and/or foods and/or pharmaceutical formulations and/or
beverages. A preferred type of food is a medical food (e.g., a food
which is in a formulation to be consumed or administered externally
under the supervision of a physician and which is intended for the
specific dietary management of a disease or condition for which
distinctive nutritional requirements, based on recognized
scientific principles, are established by medical evaluation.) For
infants, the fatty acids are administered to infants as infant
formula, weaning foods, jarred baby foods, human milk fortifier
and/or infant cereals.
[0150] Any biologically acceptable dosage forms, and combinations
thereof, are contemplated by the inventive subject matter. Examples
of such dosage forms include, without limitation, chewable tablets,
quick dissolve tablets, effervescent tablets, reconstitutable
powders, elixirs, liquids, solutions, suspensions, emulsions,
tablets, multi-layer tablets, bi-layer tablets, capsules, soft
gelatin capsules, hard gelatin capsules, caplets, lozenges,
chewable lozenges, beads, powders, granules, particles,
microparticles, dispersible granules, cachets, douches,
suppositories, creams, topicals, inhalants, aerosol inhalants,
patches, particle inhalants, implants, depot implants, ingestibles,
injectables, infusions, health bars, confections, cereals, cereal
coatings, foods, nutritive foods, functional foods and combinations
thereof. The preparations of the above dosage forms are well known
to persons of ordinary skill in the art. Preferably, a food that is
enriched with the desired PUFA is selected from the group
including, but not limited to: baked goods and mixes; chewing gum;
breakfast cereals; cheese products; nuts and nut-based products;
gelatins, pudding, and fillings; frozen dairy products; milk
products; dairy product analogs; soft candy; soups and soup mixes;
snack foods; processed fruit juice; processed vegetable juice; fats
and oils; fish products; plant protein products; poultry products;
and meat products.
[0151] The present invention also includes a method of making any
of the above-described compositions of the invention, such as by
combining the components of the composition into any suitable
delivery form using any suitable method known in the art.
[0152] According to the present invention, the methods of the
present invention are suitable for use in an individual that is a
member of the Vertebrate class, Mammalia, including, without
limitation, primates, livestock and domestic pets (e.g., a
companion animal). Most typically, an individual will be a human
individual. The term "individual" can be interchanged with the term
"subject" or "patient" and refers to the subject of a protocol or
method according to the invention. Accordingly, an individual can
include a healthy, normal (non-diseased) individual, as well as an
individual who has or is at risk of developing pre-dementia or
dementia or a symptom or indicator thereof as described herein.
[0153] The PUFA esters produced by the methods of the present
invention may be used to produce PUFA salts. In some embodiments,
PUFA salts can be produced by reacting the PUFA esters of the
present invention in the presence of an alkaline metal base such as
an alkaline metal hydroxide (e.g., potassium hydroxide). The PUFA
salts formed from the PUFA esters of the present invention can be
used in a variety of applications, such as in foods, beverages, and
pharmaceuticals. In some embodiments, the PUFA salts produced using
the PUFA esters of the present invention are water-soluble and can
be used directly in foods, beverages, and pharmaceuticals.
[0154] PUFA esters produced by the methods of the present invention
can be used in any animal food material, particularly food
materials for humans, to create a food product having enhanced
concentrations of PUFAs. The amount of fatty acids naturally in
food products varies from one food product to another. A food
product of the present invention can have a normal amount of a PUFA
or a modified amount of a PUFA. In the former instance, a portion
of the naturally occurring lipids may be substituted by PUFA esters
of the present invention. In the latter instance, naturally
occurring lipids may be supplemented by PUFA esters of the present
invention.
[0155] PUFA esters may be added to foods for infants, such as
infant formula and baby food. According to the present invention,
an infant refers to infants and children less than about two years
old, including, in particular, premature infants. Certain PUFAs are
particularly important component of infant formula and baby food
because of the rapid growth of infants (i.e., doubling or tripling
in weight during the first year of life). An effective amount of
PUFA ester to supplement infant formula is an amount that
approximates the concentration of the PUFAs in human breast milk.
Preferred amounts of PUFA esters to add to infant formula or baby
food range from between about 0.1 to about 1.0% of total fatty
acids, more preferably from between about 0.1 to about 0.6% of
total fatty acids, and even more preferably about 0.4% of total
fatty acids.
[0156] Another aspect of the present invention includes a food
product comprising a food material combined with PUFA esters of the
present invention. PUFA esters may be added to a food material to
create a food product having enhanced concentrations of PUFAs. As
used herein, the term "food material" refers to any food type fed
to humans or non-human animals. Also within the scope of the
present invention is a method to make a food product comprising
adding PUFA esters produced by methods of the present invention to
a food material.
[0157] A suitable food material useful for the formation of a food
product of the present invention includes animal food. The term
"animal" means any organism belonging to the kingdom Animalia and
includes, without limitation, primates (e.g., humans and monkeys),
livestock and domestic pets. The term "food product" includes any
product to be fed to such animals. Preferred food materials to be
consumed by humans include infant formula and baby food. Preferred
food materials to be consumed by domestic pets include dog
foods.
[0158] PUFA esters produced by methods of the present invention can
be added to a wide range of products such as baked goods, vitamin
supplements, diet supplements, powdered drinks, etc. at various
stages of production. Numerous finished or semi-finished powdered
food products can be produced using the compositions of the present
invention.
[0159] A partial list of food products comprising the products of
the present invention includes doughs, batters, baked food items
including, for example, such items as cakes, cheesecakes, pies,
cupcakes, cookies, bars, breads, rolls, biscuits, muffins,
pastries, scones, and croutons; liquid food products, for example,
beverages, energy drinks, infant formula, liquid meals, fruit
juices, multivitamin syrups, meal replacers, medicinal foods, and
syrups; semi-solid food products such as baby food, yoghurt,
cheese, cereal, pancake mixes; food bars including energy bars;
processed meats; ice creams; frozen desserts; frozen yoghurts;
waffle mixes; salad dressings; and replacement egg mixes. Also
included are baked goods such as cookies, crackers, sweet goods,
snack cakes, pies, granola/snack bars, and toaster pastries; salted
snacks such as potato chips, corn chips, tortilla chips, extruded
snacks, popcorn, pretzels, potato crisps, and nuts; specialty
snacks such as dips, dried fruit snacks, meat snacks, pork rinds,
health food bars and rice/corn cakes; and confectionary snacks such
as candy.
[0160] The present invention, while disclosed in terms of specific
methods, products, and organisms, is intended to include all such
methods, products, and organisms obtainable and useful according to
the teachings disclosed herein, including all such substitutions,
modifications, and optimizations as would be available to those of
ordinary skill in the art. The following examples and test results
are provided for the purposes of illustration and are not intended
to limit the scope of the invention.
EXAMPLES
Example 1
[0161] This example illustrates a method of the present invention
for purifying ethyl docosahexaneoate (DHA ethyl ester) from
docosahexaneoic acid-containing single cell oil.
[0162] 150 mL of absolute ethanol (EtOH) was added to 175 g
(approximately 0.2 moles of triglyceride) of DHASCO.RTM.-T oil
(Martek Biosciences Corporation, Columbia, Md., having a DHA
content of 0.4 g/g oil) in a one-liter flask under nitrogen
(N.sub.2) at room temperature. DHASCO.RTM.-T oil is prepared from
the microalgae Crypthecodinium cohnii. The mixture was allowed to
stir for 15 minutes to obtain a homogeneous solution. 67 g of a 21%
solution of sodium ethoxide/ethanol (NaOEt/EtOH; approximately 1.04
molar equivalents of triglycerides) was then added to the solution
and the mixture was allowed to reflux under N.sub.2 for about 9
hours. The progress of the reaction was monitored by gas
chromatography (GC) and thin-layer chromatography (TLC). When the
reaction was completed, approximately 75 mL of EtOH was removed by
distillation. The reaction mixture was then allowed to cool to room
temperature under N.sub.2. 300 mL hexane was added to the cooled
reaction mixture, and the mixture was allowed to stir for 15
minutes at room temperature. 300 mL of deionized water was then
added to the mixture, and the mixture was allowed to stir for an
additional 15 minutes. After removing and saving the organic layer,
the aqueous layer was washed twice with 300 mL portions of hexane.
A dark brown aqueous layer was discarded. The combined organic
layers were then washed with 200 mL of a saturated NaCl solution. A
GC analysis of the organic layer indicated the presence of about
44.7% DHA ethyl ester; the remaining materials were predominantly
lower molecular weight ethyl esters (see Table 1).
[0163] The combined organic layer was concentrated under reduced
pressure. The crude concentrate was then subjected to vacuum
fractional distillation. The lower molecular weight ethyl esters
were collected at temperatures between 100-150.degree. C. and at a
pressure of 0.8 mm Hg. The major components of this fraction were
oleic, saturated C-14, and C-12 esters. The DHA ethyl ester was
collected at temperatures between 155-165.degree. C. and at a
pressure of 0.8 mm Hg. A GC analysis of the DHA ethyl ester
fraction showed a purity of about 91.3% DHA (see Table 1). From the
fractional distillation, 68 g (86% yield) of the DHA ethyl ester
was obtained as a light yellow oil.
TABLE-US-00001 TABLE 1 GC Analyses of DHASCO .RTM.-T Oil
Transesterification and Distillation Products DHA Ethyl
Ester-Containing Organic Layer After Fraction After Vacuum Sample
Transesterifaction Fractional Distillation % 22:6 (n-3) DHA 44.72
91.29 % 20:5 (n-3) EPA 0.00 0.00 % Additional 55.28 8.81
components
Example 2
[0164] This example illustrates a method of the present invention
for purifying ethyl docosahexaneoate (DHA ethyl ester) from a crude
Crypthecodinium cohnii oil.
[0165] A crude oil obtained from Crypthecodinium cohnii by hexane
extraction (DHA content of 0.5 g/g oil) was used directly without
any further processing, such as winterization and/or RBD
processing. 150 mL of absolute ethanol was added to 175 g
(approximately 0.2 moles of triglycerides) of the crude oil in a
one-liter flask under N.sub.2 at room temperature. The mixture was
allowed to stir for 15 minutes to obtain a homogeneous solution. 67
g of a 21% solution of NaOEt/EtOH (approximately 1.04 molar
equivalents of triglycerides) was then added to the solution, and
the mixture was allowed to reflux under N.sub.2 for about 10 hours.
The progress of the reaction was monitored by GC and TLC. When the
reaction was completed, approximately 75 mL of ethanol was removed
by distillation, and the mixture was allowed to cool to room
temperature under N.sub.2. 300 mL of hexane was added to the cooled
mixture, and the mixture was allowed to stir for 15 minutes at room
temperature. 300 mL of deionized water was then added to the
mixture, and the mixture was allowed to stir for an additional 15
minutes. After removing and saving the organic layer, the aqueous
layer was washed twice with 300 mL portions of hexane. The combined
organic layer was then washed with 200 mL of a saturated NaCl
solution. A GC analysis of the organic layer indicated the presence
of about 51% DHA ethyl ester; the remaining materials were
predominantly lower molecular weight ethyl esters (see Table
2).
[0166] The combined organic layer was concentrated under reduced
pressure. The crude concentrate was then subjected to vacuum
fractional distillation. The lower molecular weight ethyl esters
were collected at temperatures between 100-150.degree. C. and at a
pressure of 0.8 mm Hg. The major components of this fraction were
oleic, saturated C-14, and C-12 esters. The DHA ethyl ester was
collected at temperatures between 155-165.degree. C. and at a
pressure of 0.8 mm Hg. A GC analysis of the DHA ethyl ester
fraction showed a purity of about 92% DHA (see Table 2). From the
fractional distillation, 69 g (66% yield) of the DHA ethyl ester
was obtained as a light yellow oil.
TABLE-US-00002 TABLE 2 GC Analyses of Crude Crypthecodinium cohnii
Oil Transesterification and Distillation Products DHA Ethyl
Ester-Containing Organic Layer After Fraction After Vacuum Sample
Transesterifaction Fractional Distillation % 22:6 (n-3) DHA 51.25
91.80 % 20:5 (n-3) EPA 0.00 0.00 % Additional 48.75 8.20
components
Example 3
[0167] This example illustrates a method of the present invention
for purifying ethyl docosahexaenoate (as a DHA ethyl ester/DPA
ethyl ester mixture) from a crude Schizochytrium sp. oil.
[0168] A crude oil obtained from Schizochytrium sp. by hexane
extraction was used directly without any further processing, such
as winterization and/or RBD processing.
[0169] 150 mL of absolute ethanol was added to 175 g (approximately
0.2 moles of triglycerides) of the crude oil (DHA content 40%, DPA
content 15%) in a one-liter flask under N.sub.2 at room
temperature. The mixture was allowed to stir for 15 minutes to
obtain a homogeneous solution. 67 g of a 21% solution of NaOEt/EtOH
(approximately 1.04 molar equivalents of triglycerides) was then
added to the solution, and the mixture was allowed to reflux under
N.sub.2 for about 10 hours. The progress of the reaction was
monitored by GC and TLC. When the reaction was completed,
approximately 65 mL of ethanol was removed by distillation, and the
mixture was allowed to cool to room temperature under N.sub.2. 300
mL of hexane was added to the cooled mixture, and the mixture was
allowed to stir for 15 minutes at room temperature. 300 mL of
deionized water was then added to the mixture, and the mixture was
allowed to stir for an additional 15 minutes. After removing and
saving the organic layer, the aqueous layer was washed twice with
300 mL portions of hexane. The combined organic layer was washed
with 200 mL of a saturated NaCl solution. A GC analysis of the
organic indicated the presence of about 40% DHA ethyl ester and 15%
DPA ethyl ester; the remaining materials were predominantly lower
molecular weight ethyl esters (see Table 3).
[0170] The combined organic layer was concentrated under reduced
pressure. The crude concentrate was then subjected to vacuum
fractional distillation. The lower molecular weight ethyl esters
were collected at temperatures between 100-150.degree. C. and at a
pressure of 0.8 mm Hg. The major components of this fraction were
saturated C-14, and C-16 ethyl esters. The DHA ethyl ester/DPA
ethyl ester mixture was collected at temperatures between
155-170.degree. C. and at a pressure of about 0.5 mm Hg. A GC
analysis of the DHA/DPA ethyl ester fraction showed a combined
purity of about 93% (see Table 3). From the fractional
distillation, 85 g (85% yield) of the DHA/DPA ethyl ester was
obtained as a very light yellow oil.
TABLE-US-00003 TABLE 3 GC Analyses of Crude Schizochytrium sp. Oil
Transesterification and Distillation Products DHA/DPA Ethyl Ester-
Containing Fraction Organic Layer After After Vacuum Fractional
Sample Transesterifaction Distillation % 22:6 (n-3) DHA 40.07 67.31
% 22:5 (n-6) DPA 15.09 25.86 % 20:5 (n-3) EPA 1.21 0.32 %
Additional 43.63 6.5 components
Example 4
[0171] This example illustrates GC analyses of crude and purified
PUFA ethyl esters from Crypthecodinium cohnii oil and
Schizochytrium sp. oil.
[0172] A crude oil obtained from Schizochytrium sp. or
Crypthecodinium cohnii by hexane extraction was used directly
without any further processing, such as winterization and/or RBD
processing. The crude oils were then subjected to a
transesterification reaction as described above in Examples 2 and
3. The crude ethyl esters were then subjected to urea adduction as
described above, or to distillation as described in Examples 2 and
3. GC analyses were then performed on each sample along with a DPA
ethyl ester product or DHA ethyl ester product (Nu-Chek Prep, Inc.,
Elysian, Minn.). The results are presented below in Table 4
(Schizochytrium sp.) or Table 5 (Crypthecodinium cohnii). Two
analyses were performed on the crude and distilled ethyl esters
from the Crypthecodinium cohnii oil.
TABLE-US-00004 TABLE 4 GC Analyses of Schizochytrium sp. Ethyl
Ester Products Ethyl Esters Ethyl Esters after Urea after Crude
Adduction Distillation Ethyl of Crude of Crude Nu-Chek Fatty Acid
Esters Oil Oil DPA EE % C12:0 0.26 0.20 0.00 0.67 % C14:0 8.63 1.68
0.00 0.00 % C14:1 0.00 0.13 0.00 0.36 % C16:0 24.65 0.53 0.00 0.10
% C16:1 0.40 0.00 0.00 0.00 % C18:0 0.57 0.00 0.00 0.10 % C18:1
(n-9) 0.36 0.00 0.00 1.77 % C18:1 (n-7) 0.35 0.00 0.00 0.72 % C18:2
0.41 0.00 0.00 0.00 % C18:2 (n-6) 0.24 0.58 0.00 0.00 % C20:3 (n-6)
0.42 2.75 0.00 0.00 % C20:3 (n-3) 0.00 0.27 0.00 0.00 % C20:3 (n-6)
0.00 0.59 0.00 0.00 % C20:4 ARA 1.50 2.29 0.00 0.16 % C20:5 (n-3)
EPA 0.00 2.01 0.00 0.00 % C22:4 (n-6) 0.00 0.00 0.00 3.83 % C22:5
(n-6) DPA 15.89 22.78 26.46 87.15 % C22:5 (n-3) DPA 5.21 % C22:6
(n-3) DHA 40.65 57.67 71.83 0.00 % Additional 4.95 7.19 0.94 0.80
components
Example 5
[0173] This example illustrates a method of the present invention
for purifying ethyl docosahexaneoate (DHA ethyl ester) from a
mixture of fatty acid ethyl esters of docosahexaneoic
acid-containing single cell oil via urea crystallization.
[0174] 150 g crude mixture of fatty acid ethyl esters, obtained by
transesterification of docosahexaneoic acid-containing single cell
DHASCO.RTM.-T oil prepared from the microalgae Crypthecodinium
cohnii, was added to a solution of 262.5 g urea (1.75 wt. eq. of
esters) in 1050 mL methanol (7 vol eq of esters) at 70.degree. C.
under nitrogen. The resulting mixture of urea and esters was
continued to heat at 70.degree. C. under nitrogen for 1 hr. The
mixture was first allowed to cool to 20.degree. C. followed by
cooling to 0-4.degree. C. to complete the urea adduct
crystallization. The mixture was allowed to stand for additional 2
hours at 0-4.degree. C. The crystallized urea adduct was then
filtered at 0-4.degree. C.
[0175] The filtrate was diluted with 300 mL of water and the
mixture was acidified with dilute sulfuric acid to a pH of 1-2. The
acidified solution was extracted with 300 mL.times.3 of hexane. The
combined hexane extracts were washed with saturated NaCl solution.
The washed hexane solution was dried over anhydrous sodium sulfate
and concentrated in vacuo to obtain 70-75% of theoretical yield.
Typically, GC analysis showed purity of above obtained DHA Ethyl
ester around 90-96% (800-860 mg/g).
TABLE-US-00005 TABLE 5 GC Analyses of Crypthecodinium cohnii Ethyl
Ester Products Ethyl Esters Ethyl Ethyl after Urea Esters after
Crude Esters after Crude Adduction Distillation Ethyl Distillation
Ethyl of Crude of Crude Esters of Crude Oil Nu-Chek Fatty Acid
Esters Oil Oil #2 #2 DHA EE % C8:0 0.19 0.00 0.00 0.36 0.00 0.00 %
C10:0 0.95 0.00 0.00 1.86 0.00 0.00 % C12:0 4.04 0.00 0.00 7.18
0.00 0.00 % C13:0 0.00 0.09 0.00 0.00 0.00 0.00 % C14:0 13.77 0.00
0.00 19.99 0.00 0.00 % C14:1 0.17 0.00 0.00 0.19 0.00 0.00 % C16:0
11.08 0.00 0.10 16.15 0.00 0.10 % C16:1 2.83 0.00 0.00 2.24 0.00
0.00 % C18:0 0.21 0.00 0.00 0.48 0.00 0.00 % C18:1 (n-9) 9.77 0.00
0.00 10.27 0.00 0.00 % C18:1 (n-7) 0.00 0.00 0.00 0.00 0.18 0.00 %
C22:5 (n-3) 0.65 0.00 1.16 0.20 0.59 0.00 DPA % C22:6 (n-3) 55.64
93.00 89.67 41.26 96.73 99.95 DHA % C24:0 0.00 0.00 0.69 0.00 0.00
0.00 % C28:8 0.60 ** 1.40 0.25 1.20 0.00 % Additional 0.70 2.52
8.38 0.50 1.11 0.05 components ** Not Determined
[0176] The principles, preferred embodiments and modes of operation
of the present invention have been described in the foregoing
specification. The invention which is intended to be protected
herein should not, however, be construed as limited to the
particular forms disclosed, as these are to be regarded as
illustrative rather than restrictive. Variations and changes may be
made by those skilled in the art without departing from the spirit
of the present invention. Accordingly, the foregoing best mode of
carrying out the invention should be considered exemplary in nature
and not as limiting to the scope and spirit of the invention as set
forth in the appended claims.
* * * * *