Note: Descriptions are shown in the official language in which they were submitted.
CA 02693070 2010-01-14
-1-
SPECIFICATION
METHOD FOR PRODUCING EPA-ENRICHED OIL AND DHA-ENRICHED OIL
TECHNICAL FIELD
[0001] The present invention relates to a method for producing enriched oils
containing
high concentrations of eicosapentaenoic acid (EPA hereafter) and
docosahexaenoic acid
(DHA hereafter) respectively using a lipase reaction.
BACKGROUND ART
[0002] Eicosapentaenoic acid (EPA hereafter) and docosahexaenoic acid (DHA
hereafter)
are n-3 polyunsaturated fatty acids (PUFA hereafter) having a variety of
biological effects,
and are used as medical products, health food products, food product
materials, and the like.
EPA ethyl ester is used as a therapeutic agent for arteriosclerosis and
hyperlipidemia, and
beverages to which fish oil containing EPA and DHA has been added have been
approved as
a food for specified health uses. Furthermore, the demand for these fatty
acids as a
supplement is very high in Japan and other countries.
[0003] PUFAs have many double bonds and are therefore very unstable to
oxidation.
Consequently, that an enzyme reaction that proceeds under mild conditions at
room
temperature is very desirable for the steps involved in producing PUFA-
containing oils.
[0004] There are lipase products for industrial use that are obtained from
primarily
microorganisms and that have the property of hardly reacting with PUFAs. PUFA-
enriched
oils and fats can be produced using lipases having such a property by
emphasizing liberation
and removal of fatty acids having few carbons. For instance, a method is
disclosed whereby
DHA-enriched oils and fats are produced by hydrolysis of tuna oil using
Candida
cylindoracea lipase and then removal of the free fatty acids (Patent Reference
1).
[0005] It is known that water has an important effect on enzyme activation for
enzyme
reactions in an organic solvent (non-Patent Reference 1). It is reported that
when a PUFA is
concentrated from cod lever oil using alcoholysis, which is a reaction wherein
the fatty acids
are severed from glycerides by exposure to an alcohol, the addition of water
promotes the
CA 02693070 2010-01-14
-2-
lipase reaction (Non-Patent Reference 2). On the other hand, it is disclosed
that alcoholysis
of oils and fats proceeds under virtually anhydrous conditions with certain
lipases.
Nevertheless, the amount of lipase used must be very high at 10% the amount of
oil, and the
lipase must be immobilized in order to improve productivity (Patent Reference
2).
[0006] A method is cited in Patent Reference 3 wherein an alkali salt is used
when oils and
fats containing long-chain polyunsaturated fatty acids as constituent fatty
acids are
hydrolyzed by a lipase having positions 1 and 3 specificity.
Patent Reference 1 JP (Publication of Unexamined Patent) 58-165796
Patent Reference 2 JP (National Publication) 9-510091
Patent Reference 3 JP (Publication of Unexamined Patent) 3-108489
Non-Patent Reference 1 J. S. Dorclick, "Enzymatic catalysis in monophasic
organic
solvents," Enzyme Microb. Technol., 1989, 11, April, 194-211.
Non-Patent Reference 2 L. Zui and O.P Ward, "Lipase-catalyzed alcoholysis to
concentrate the n-3 polyunsaturated fatty acids of cod liver oil," Enzyme
Microb. Technol.,
1993, 15, July, 601-606.
DISCLOSURE OF THE INVENTION
PROBLEMS TO BE SOLVED BY THE INVENTION
[0007] Although the market already carries oil that has been enriched with
PUFA from fish
oil and the like using lipase having the above-mentioned property, there are
limits to the
degree of concentration, and it is either difficult to obtain a highly
concentrated product or a
large amount of enzyme is necessary. An object of the present invention is to
provide a
method for efficiently enriching both the EI'A and DHA respectively from PUFAs
contained
in starting oils.
MEANS FOR SOLVING THE PROBLEMS
[0008] As a result of conducting comprehensive research of reactions using
industrial
lipases, the inventors discovered that the lipase reaction efficiency can be
dramatically
improved, even when a small amount of lipase is used, by adding a small amount
of a
compound selected from, for instance, magiiesium oxide (MgO hereafter),
magnesium
CA 02693070 2010-01-14
-3-
hydroxide, calcium oxide, and calcium hydroxide. They further discovered that
this reaction
has sufficient substrate specificity and that it is appropriate for methods
whereby both the
EPA and DHA are highly concentrated respectively.
[0009] The present invention is primarily a production method characterized by
reacting a
lipase, having substrate specificity for fatty acids having 18 carbons or
less, with oils and fats
containing EPA and DHA as constituent fatty acids in the presence of at least
one selected
from the group consisting of magnesium oxide, magnesium hydroxide, calcium
oxide, and
calcium hydroxide as a reaction additive; severing the fatty acids having 18
carbons or less
from the glyceride and then separating the glyceride fraction; and further
reacting this
glyceride fraction with a lipase having substrate specificity for fatty acids
having 20 carbons
or less in the presence of at least one compound selected from the group
consisting of
magnesium oxide, magnesium hydroxide, calcium oxide, and calcium hydroxide to
simultaneously obtain EPA-enriched oil (as a lower alcohol ester) and DHA-
enriched oil (as
a glyceride fraction).
[0010] That is, the present invention is a method for producing EPA-enriched
oil and
DHA-enriched oil from oils and fats contaiiiing EPA and DHA as constituent
fatty acids, this
method comprising
a) a step wherein the oils and fats are subjected to alcoholysis, or
alcoholysis
accompanied by hydrolysis, by a lipase having substrate specificity for fatty
acids having 18
carbons or less and in the presence of an alc:ohol or hydrated alcohol and at
least one
compound selected from the group consisting of magnesium oxide, magnesium
hydroxide,
calcium oxide, and calcium hydroxide as a reaction additive, and an
eicosapentaenoic acid
and docosahexaenoic acid-enriched glyceri(le fraction is obtained from the
resulting reaction
mixture and
b) a step wherein the glyceride fraction is subjected to alcoholysis, or
alcoholysis
accompanied by hydrolysis, by a lipase having substrate specificity for fatty
acids having 20
carbons or less and in the presence of an alcohol or hydrated alcohol and at
least one
compound selected from the group consisting of magnesium oxide, magnesium
hydroxide,
CA 02693070 2010-01-14
-4-
calcium oxide, or calcium hydroxide as a reaction additive, and the EPA-
enriched ester
fraction and DHA-enriched glyceride fraction are separated from the resulting
reaction
mixture.
ADVANTAGES OF THE INVENTION
[0011] The present invention increases enzyme reactivity and realizes a
reaction that is
highly substrate specific in each step by adding a small amount of an
inexpensive reaction
additive such as magnesium oxide. As a result, EPA-enriched oil and DHA-
enriched oil can
be produced simultaneously at a high yield and inexpensively.
EMBODIMENT OF THE INVENTION
[0012] There are no special restrictions to the oils and fats used as starting
materials in the
present invention as long as they are oils containing EPA and DHA as fatty
acids formed by
the glycerides contained in these oils and fats, and examples are marine
product oils
including fish oil, microorganism oils, seaweed oils, and vegetable oils. When
used as the
starting material of the present invention, these can be clude oils (expressed
oils), or they can
be oils that have been subjected to some type of purification process. It is
preferred that the
starting materials used in the present invention have as high an EPA and DHA
content as
possible, and preferred lipids are sardine oil (for instance, 17% EPA and 12%
DHA), tuna oil
(for instance, 7% EPA and 25% DHA), bonito oil (for instance, 5% EPA and 24%
DHA),
and salmon oil (for instance, 9% EPA and 1.4% DHA).
[0013] The term "oils and fats" usually means the triglycerides of fatty
acids, but in the
present invention the term also includes other glycerides that lipases will
affect, such as
diglycerides, monoglycerides, and the like. The term "glyceride" in the
present invention is a
general term for the triglycerides, diglycerides, and monoglycerides of fatty
acids.
[0014] The phrase "EPA or DHA-enriched" in the present invention means that
the [amount
of EPA or DHA/total amount of fatty acids] after the reaction is greater than
the [amount of
EPA or DHA./total amount of fatty acids] in. the starting oils and fats, and
oil having a higher
[amount of EPA or DHA/total amount of fatty acids] when compared to the
starting oils and
fats is EPA-enriched oil or DHA-enriched oil.
CA 02693070 2010-01-14
-5-
[0015] The term "alcohol" in the present invention includes one or multiple
types of
alcohols.
[0016] The lipase reaction performed by the method of the present invention is
alcoholysis
wherein a fatty acid ester is produced from a glyceride. This lipase reaction
is performed in a
mixed solvent of alcohol and water, and can also be a reaction by which free
fatty acids and
fatty acid esters are produced. Examples of lower alcohol solvents used in the
present
invention are ethanol, methanol, 2-propanol, and butanol. Ethanol is
particularly preferred.
[0017] There are no special restrictions to the lipase used in step a) of the
present invention
as long as it has substrate specificity to fatty acids having 18 carbons or
less. Examples of
preferred lipases are lipase obtained from niicroorganisms belonging to
Alcaligenes sp.
(Lipase QLM, Lipase QLC, Lipase QLG, Lipase PL, all produced by Meito Sangyo
Co.,
Ltd.). Lipase QLM is particularly preferrecl. Lipase QLC, which is easily
obtained from
Meito Sangyo Co., Ltd., has the following characteristics and properties.
Description: Beige
powder; Activity: Approximately 60,000 U/g; Molecular weight: 31 kDa;
Isoelectric point:
4.9; Optimal pH: 7 to 9; and Optimal Temperature: 65 to 70 C. Moreover, lipase
QLC is
lipase QLM that has been immobilized on diatomaceous earth, and lipase QLG is
lipase
QLM that has been immobilized on granulated diatomaceous earth.
[0018] Although there are no special restrictions to the amount of lipase
used, it is preferred
that the amount of lipase in powder form in terms of oils and fats be 10
units/g or more,
preferably 30 units/g or more taking into consideration practicality based on
the reaction rate,
and it is preferred that the amount of immobilized lipase in terms of oils and
fats be 0.01%
(w/w) or more.
[0019] There are no special restrictions to the lipase used in step b) of the
present invention
as long as it has substrate specificity for fatty acids having 20 carbons or
less. An example of
a preferred lipase is lipase obtained from microorganisms belonging to
Thermomyces
lanuginosus (Lipozyme TL IM made by Novozymes). Lipozyme TL IM, which can be
obtained from Novozymes, has the following characteristics and properties.
Molecular
weight: 30 kDa; Isoelectric Point: 4.8, pH: 6 to 11, Optimal Temperature: 60
to 70 C;
CA 02693070 2010-01-14
-6-
Immobilization Carrier: Silica Gel; Grain Size: 300 to 1,000 m (primarily 500
to 900 m):
and Specific Gravity: 0.54 g/mL. Although there are no special restrictions to
the amount of
lipase used, it is preferred that the amount of lipase in powder form in terms
of glyceride
fraction be 10 units/g or more, preferably 30 units/g or more taking into
consideration
practicality based on the reaction rate, and it is preferred that the amount
of immobilized
lipase in terms of oils and fats be 0.01% (w,iw) or more.
[0020] Magnesium oxide, magnesium hydroxide, calcium oxide, and calcium
hydroxide
can be used as reaction additives, but magnesium oxide is particularly
preferred because it
has the most effective and can be used in food products. Additives in powder,
particle, or
granule form are easy to handle, and those sold for industrial use can be
used. There are no
particular restrictions to the amount of reaction additive added, but in step
a) the reaction
additive is used within a range of 0.01% (w/w) to 30% (w/w), more preferably a
range of
0.05% (w/w) to 5% (w/w), in terms of starting oils and fats. Moreover, in step
b) the reaction
additives are used within a range of 0.01% (w/w) to 30% (w/w), preferably
0.05% (w/w) to
5% (w/w), in terms of glyceride fraction of the starting materials.
[0021] There are no special restrictions to the reaction method as long as a
predetermined
amount of starting oils and fats, reaction additives, alcohol, and the like
can be mixed. The
reaction can be performed in accordance with conventional technological
knowledge
regarding reactions that use ordinary lipases. Generally, the reactants are
stirred such that
they are thoroughly mixed for a reaction time of 1 to 24 hours at a reaction
temperature at
which the enzyme is very active (for instance, 20 to 60 ). It is also possible
to use
immobilized enzyme packed in a column and the like in the reaction.
[0022] When the lipase reaction is an alcoholysis reaction, after the reaction
it is possible
to remove the reaction additive, enzyme, and the like by, for instance,
filtration or washing
with an aqueous solution.
[0023] Although there are no special restrictions to the method used to
separate the
glyceride fraction in steps a) and b), distillation, such as molecular
distillation or short- path
distillation, and separation methods that use various types of chromatography
can be used.
CA 02693070 2010-01-14
-7-
The purification method can be one that is iiormally used for purification of
oils and fats, and
various types of chromatography and steam distillation are examples.
[00241 As one of the embodiment of the present invention, the lipase reaction
can be
performed by adding a small amount of water to the reaction system during
either of steps a)
and b) or both of steps a) and b). When water is added, it is added in the
amount of 1% (v/v)
to 30% (v/v), further preferably 5% (v/v) to 20% (v/v), in terms of the amount
of lower
alcohol used. The amount of water contained in the oils and fats should be
taken into
consideration.
[0025] The amount of lower alcohol is, for instance, 0.2 to 5 equivalents,
more preferably
0.2 to 1.5 equivalents, in terms of fatty acids contained in the oils and fats
or glyceride
fraction in the reaction system.
[0026] The fatty acid lower alcohol esters and free fatty acids produced when
step a) is
performed in the presence of a hydrated lower alcohol can be removed by
distillation (such as
thin film distillation, molecular distillation, or short-path distillation), a
deacidification step
using an alkali, and the like.
[0027] The present invention is described below in specific terms using
working examples,
but the present invention is in no way limited to these working examples. It
should be noted
that the EPA and DHA content of the starting oil and glyceride fraction was
determined from
the gas chromatography area ratio after methyl esterification. Moreover,
methyl
esterification that was performed prior to gas chromatography analysis was
conducted in
accordance with the standard oils and fats testing method specified by the
Japan Oil
Chemists' Society (Japan Oil Chemists' Society Standard Methods for the
Analysis of Fats,
Oils and Related Materials (1), 1996, 2.4.1 Fatty acid Derivation Methods:
2.4.1.2-1996
Methyl esterification methods (boron trifluoride-methanol method)).
WORKING EXAMPLE 1
[0028] (1) lipase reaction of step a)
1.49 g of Lipase QLM (100 units/g), 20.7 g of water, 30 g (2.5% in terms of
oil) of
magnesium oxide (Junsei Chemical Co., Ltd., special grade reagent having a
purity of 99% or
CA 02693070 2010-01-14
-8-
higher, powder), and 207 mL of ethanol were added to 1.20 kg of purified
sardine oil (17.4%
EPA, 11.9% DHA, Nippon Suisan Kaisha, Ltd..) and the mixture was stirred for
16 hours at
40 C. After the reaction, the solid content was filtered and rinsed with 560
mL of 20%
sulfuric acid and 200 mL of brine. Then the water content and ethanol
remaining in the oil
layer were distilled off to obtain 1.21 kg of oil. A small amount of the
resulting oil was
sampled, the glyceride fraction was separated by preparative TLC and methyl
esterified, and
the fatty acid composition was analyzed by gas chromatography. The analysis
conditions are
described below (unless otherwise noted, analysis by preparative TLC and gas
chromatography was performed by the same method later).
[0029] TLC fractionation and methyl esterification conditions:
1 mL of hexane and 10 mL of saturated brine were added to 50 L of reaction
solution and hexane extraction was performed. 150,uL of the resulting hexane
layer were
applied to preparative TLC and developed using hexane:diethyl ether:acetic
acid (70:30:1,
volume ratio). After development, the glyceride fraction other than the ethyl
ester was
collected and, without further treatment of the fraction, methyl
esterification was performed
by methylation. In essence, 2 mL of a 1 N sodium methoxide/methanol solution
were added
and heated for one minute at 80 C. Then 2 mL of 1 N hydrochloric acid were
added and the
product was heated for one minute at 80 C in order to stop the reaction. Next,
0.5 mL of
hexane and 6 mL of saturated brine were added, the mixture was shaken and then
set aside,
and the hexane layer was analyzed by gas chromatography.
[0030] Gas chromatography conditions:
Capillary column: DB-WAX (J & W Scientific), Fused Silica Capillary Column,
0.25 mm I.D. x 30 m, 0.25,um film thickness
Carrier gas: helium
Detector: 250 C, FID
Injector: 250 C, split ratio: 100:1
Column temperature: 180 C - 3"C/min - 230 C (15 min)
Device: Hewlett Packard 6890
CA 02693070 2010-01-14
-9-
Moreover, after spotting a 5 wt% hexane solution (1,uL) on a silica gel rod,
the rod
was developed with hexane:diethyl ether:acetic acid (90:10:1, volume ratio)
and the lipid
composition was analyzed using TLC/FID. Tables 1 and 2 show the lipid
composition and
fatty acid composition of the starting sardine oil, the lipid composition
(area%) of the
resulting oil, and fatty acid composition (area%) of the glyceride fraction.
The EPA and
DHA were concentrated to 46.7% and 20.6%, respectively, in the glyceride
fraction by this
reaction. The yield of EPA and DHA was a high at 106.6% and 68.7%,
respectively. The
EPA and DHA yield (%) was calculated from the (ratio (%) of EPA (or DHA) in
the
glyceride fraction after the reaction x the glyceride content (%))/(ratio (%)
of EPA (or DHA)
in the oils and fats prior to the reaction).
[0031]
CA 02693070 2010-01-14
-10-
[Table 1]
Table 1 Fatty acid composition (area%)
Purified sardine oil After step a) reaction (glyceride fraction)
C14:0 6.3 2.3
C15:0 0.5 0.0
C16:0 14.9 2.8
C16:1 8.1 2.4
C16:2 1.6 1.7
C16:3 1.3 0.4
C16:4 1.8 0.4
C18:0 3.5 0.7
C18:1 13.4 4.5
C18:2n-6 1.2 0.3
C18:3n-3 0.7 0.0
C18:4n-3 2.5 0.5
C20:1 3.1 2.3
C20:4n-6 1.6 3.6
C20:4n-3 0.9 0.3
C20:5 (EPA) 17.4 46.7
C22:1 1.9 1.5
C22:4 0.7 0.9
C22:5n-3 2.4 5.8
C22:6 (DHA) 11.9 20.6
Others 4.6 2.2
[0032] [Table 2]
Table 2 Lipid composition (area%)
CA 02693070 2010-01-14
-11-
Purified sardine oil After step a) reaction
Ethyl ester 0.0 43.5
Glyceride fraction Triglycerides 100.0 0.0
Diglycerides 0.0 9.1
Monoglycerides 0.0 30.6
Free fatty acids 0.0 16.9
[0033] (2) Glyceride fraction separation
The ethyl ester and fatty acids were distilled off from the oils obtained in
Working
Example 1 using a thin layer distillation device. The thin layer distillation
device was
short-path distillation device KDL-5 (evaporation area of 0.048 m2) made by
UIC GmbH,
and 2-pass treatment was performed at 130 C, 1 x 10-3 mbar, and 0.60 L.Uh.
Tables 3 and 4
show the fatty acid composition (area%) and the lipid composition (area%) of
the residual oil
after distillation. It was confirmed that the ethyl ester and free fatty acids
had been distilled
off from the lipid composition and that EPA- and DHA-enriched glyceride
fraction had been
obtained.
[0034]
CA 02693070 2010-01-14
-12-
[Table 3]
Table 3 Fatty acid composition (area%)
After thin layer distillation (after two passes)
C14:0 1.9
C15:0 0.0
C16:0 2.8
C16:1 2.9
C16:2 1.5
C16:3 0.5
C16:4 0.4
C18:0 0.9
C18:1 4.8
C18:2n-6 0.3
C18:3n-3 0.0
C18:4n-3 0.6
C20:1 2.4
C20:4n-6 3.9
C20:4n-3 0.4
C20:5 (EPA) 45.1
C22:1 1.7
C22:4 0.7
C22:5n-3 5.8
C22:6 (DHA) 20.5
Others 2.7
[0035]
CA 02693070 2010-01-14
-13-
[Table 4]
Table 4 Lipid composition (area%)
After thin layer distillation
After 1 pass After 2 passes
Ethyl ester 4.5 0.8
Triglycerides 0.0 0.0
Diglycerides 35.2 44.2
Monoglycerides 54.5 51.7
Free fatty acids 5.8 3.3
[0036] (3) Step b) lipase reaction and product fractions
L of water, 20 mg of Lipozynie TL IM (2.0% in terms of oil), 25 mg of
magnesium oxide (2.5% in terms of oil), and 173 L of ethanol were added to 1
g of the oil
(glyceride fraction) obtained in step b), and the mixture was stirred for 16
hours at 40 C.
Then the solid was filtered and washed with brine, and the lipid composition
of the resulting
oil was analyzed. The ethyl ester and glyceride fraction were separated by
preparative TLC
and the fatty acid composition was analyzed. The conditions for preparative
TLC were the
same as those for step a) of Working Example 1 and this time analysis was
performed on
both the ethyl ester and the glyceride fraction. Tables 5 and 6 show the fatty
acid
composition (area%) and the lipid composition after the reaction. The DHA was
concentrated in the glyceride fraction to 76.4% and the EPA was concentrated
in the ethyl
ester fraction to 52.1%, and DHA-enriched oil and EPA-enriched oil could be
simultaneously
obtained. The DHA yield from the glyceride fraction by this reaction was 71.6%
and the
EPA yield from the ethyl ester fraction was 74.9%. The DHA yield (%) was
calculated from
the (ratio (%) of DHA in the glyceride fraction after the reaction x the
glyceride content (%)
after the reaction)/(the ratio (%) of DHA in the glyceride fraction before the
reaction), and
the EPA yield was calculated from (the ratio (%) of EPA from the ethyl ester
after the
CA 02693070 2010-01-14
-14-
reaction x the ethyl ester content (%) after the reaction)/(the ratio (%) of
EPA before the
reaction ).
[0037] [Table 5]
Table 5 Fatty acid composition (area%)
After step b) reaction
Glyceride fraction Ethyl ester fraction
C14:0 1.1 1.7
C15:0 0.0 0.0
C16:0 0.8 2.3
C16:1 0.6 1.9
C16:2 3.3 0.8
C16:3 0.3 0.4
C16:4 0.5 0.5
C18:0 0.0 0.6
C18:1 0.6 4.0
C18:2n-6 0.0 0.0
C18:3n-3 0.0 0.0
C18:4n-3 0.8 0.7
C20:1 0.0 1.9
C20:4n-6 0.7 3.8
C20:4n-3 0.0 0.5
C20:5 (EPA) 10.2 52.1
C22:1 0.0 1.5
C22:4 2.7 0.6
C22:5n-3 1.2 7.1
C22:6 (DHA) 76.4 16.4
Others 0.9 3.1
CA 02693070 2010-01-14
-15-
[0038] [Table 6]
Table 6 Lipid composition (area%)
After step b) reaction
Ethyl ester 64.8
Triglycerides 0.0
Diglycerides 1.8
Monoglycerides 17.4
Free fatty acids 15.2
WORKING EXAMPLE 2
[0039] The amount of Lipozyme TL IM in step (3) of Working Example 1 was
changed to
mg (0.5% in terms of oil) and the reaction was performed under exactly the
same
conditions as in step (3) of Working Example 1. Tables 7 and 8 show the
results.
DHA-enriched oil and EPA-enriched oil were simultaneously obtained, even when
the
amount of lipase was reduced. The DHA yield of the glyceride fraction in the
reaction was
114.7% and the EPA yield in the ethyl ester fraction was 58.9%. (Yield results
exceeding
100% are due to the fact that this yield calculation method was simplified
method. That is,
the FID measurement used to calculate yield is high for fatty acids having a
high molecular
weight, such as DHA, because relative sensitivity increases with an increase
in molecular
weight. The same is true for the following working examples.)
[0040]
CA 02693070 2010-01-14
-16-
[Table 7]
Table 7 Fatty acid composition (area%)
After step b) reaction
Glyceride fraction Ethyl ester fraction
C14:0 1.2 2.4
C15:0 0.0 0.0
C16:0 0.7 3.5
C16:1 0.6 2.5
C16:2 2.0 1.0
C16:3 0.6 0.3
C16:4 0.9 0.9
C18:0 0.0 5.1
C18:1 0.6 0.3
C18:2n-6 0.0 0.0
C18:3n-3 0.0 0.0
C18:4n-3 1.3 0.4
C20:1 0.0 1.8
C20:4n-6 1.8 4.0
C20:4n-3 0.4 0.6
C20:5 (EPA) 24.8 54.8
C22:1 0.0 1.9
C22:4 2.1 0.5
C22:5n-3 2.0 8.3
C22:6 (DHA) 58.8 7.2
Others 2.4 4.6
[0041]
CA 02693070 2010-01-14
-17-
[Table 8]
Table 8 Lipid composition (area%)
After step b)'reaction
Ethyl ester 48.5
Triglycerides 0.0
Diglycerides 6.5
Monoglycerides 33.5
Free fatty acids 10.2
WORKING EXAMPLE 3
[0042] DHA-enriched oil and EPA-enriched oil were prepared using winterized
and
purified EPA rich sardine oil (29.0% EPA, 12.5% DHA, Nippon Suisan Kaisha,
Ltd..) as the
starting lipid. The step a) reaction involve(i using 1.2 kg of purified
sardine oil, 1.49 g of
Lipase QLM, 20.7 mL of water, 60 g of magnesium oxide, and 207 mL of ethanol,
reacting
the mixture for 16 hours at 40 C, and performing the same treatment as in
Working Example
1. Then 2-pass thin layer distillation was performed under the same conditions
as in Working
Example 1 to obtain a glyceride fraction. The step b) reaction involved using
1 g of the oil
obtained from step a), 10,uL of water, 20 nig of Lipozyme TL IM, 25 mg of
magnesium
oxide, and 173,uL of ethanol, and reacting the mixture for 16 hours at 40 C.
The ethyl ester
and glyceride fractions were both fractionated by preparative TLC (the
conditions were the
same as in Working Example 1). Table 9 shows the fatty acid composition of
starting
purified sardine oil, after step a) reaction mixture, residual oil after 2-
pass thin layer
distillation, and fractions after step b) reaction. Table 10 shows the lipid
composition after the
step a) and step b) reactions. It was possible to simultaneously obtain high
concentrations by
a two-step reaction, with the EPA concentration being 73.9% and the DHA
concentration
being 50.8%. In the step a) reaction the EI'A yield was 95.8% and the DHA
yield was 53.7%,
and in the step b) reaction the EPA yield of the ethyl ester fraction was
62.6% and the DHA
CA 02693070 2010-01-14
-18-
yield of the glyceride fraction was 114.6%.
[0043] [Table 9]
Table 9 Fatty acid composition (area%)
Winterized and After step a) After thin layer After step b) reaction
purified EPA reaction distillation
rich sardine oil (glyceride (after 2 passes) Glyceride Ethyl ester
fraction) fraction fraction
C14:0 5.3 1.1. 0.7 1.3 0.7
C15:0 0.0 0.0 0.1 0.0 0.0
C16:0 7.5 0.8 0.7 0.7 0.8
C16:1 8.6 1.4 1.3 0.8 1.1
C16:2 1.9 1.1 0.9 1.6 0.5
C16:3 2.3 0.4 0.3 0.6 0.5
C16:4 4.0 0.5 0.5 1.1 0.0
C18:0 1.9 0.0 0.1 0.0 1.6
C18:1 8.7 1.6 1.5 0.5 0.0
C18:2n-6 1.1 0.0 0.2 0.0 0.0
C18:3n-3 0.9 0.0 0.1 0.0 0.0
C18:4n-3 4.4 0.`i 0.6 1.4 0.6
C20:1 0.9 0.3 0.3 0.0 0.3
C20:4n-6 1.6 3.7 3.5 1.8 4.1
C20:4n-3 1.3 0.3 0.3 0.3 0.4
C20:5 (EPA) 29.0 65.4 62.0 33.4 73.9
C22:1 0.0 0.0 0.7 0.0 0.0
C22:4 2.1 0.`i 0.2 1.4 0.3
C22:5n-3 3.0 5.5 5.4 2.0 6.9
C22:6 (DHA) 12.5 15.8 14.8 50.8 7.6
Others 3.1 1.3 5.7 2.4 1.0
CA 02693070 2010-01-14
-19-
[0044] [Table 10]
Table 10 Lipid composition (area%)
After step a) reaction After step b) reaction
Ethyl ester 36.2 52.5
Triglycerides 0.0 0.0
Diglycerides 6.8 3.0
Monoglycerides 35.7 30.4
Free fatty acids 22.3 14.1
[0045] Conditions for executing the present invention were studied as shown
below in
Reference Examples 1 through 13
REFERENCE EXAMPLE 1
[0046] 1.65 mg of Lipase QLM (Alcaligenes sp., Meito Sangyo Co., Ltd) (100
units/g),
17 L of water, MgO (Junsei Chemical Co., Ltd., special grade reagent, purity
of 99% or
higher) (0.25% (w/w) or 2.5% (w/w) in terrns of oil), and 170 L of ethanol
(0.75 equivalents
in terms of fatty acids) were added to 1 g of purified sardine oil (28.2% of
EPA, 12.5% of
DHA, Nippon Suisan Kaisha, Ltd..), and stirred for 16.hours at 40 C. After the
reaction, the
solid content was filtered and the filtrate was extracted with hexane. The
glyceride fraction
was separated by preparative TLC using the following method.
[0047] The resulting glyceride fraction was methyl esterified and the fatty
acid composition
was analyzed by gas chromatography. The conditions for preparative TLC, methyl
esterification, and gas chromatography analysis were the same as in Working
Example 1.
[0048] 3.3 mg of Lipase PS (Burkholderias cepacia, Amano Enzymes) (100
units/g) were
reacted under the same conditions.
[0049] By way of comparison, ethanolysis with the each lipase was performed
under the
same above-mentioned conditions with the exception that no water or MgO was
added, only
water was added, or only MgO was added at 0.25% (w/w).
CA 02693070 2010-01-14
-20-
[0050] The lipid composition of the glyceride fraction was analyzed using
TLC/FID
(latroscan TH-10, Mitsubishi Kagaku Yatron Corporation) by spotting a 5 wt%
hexane
solution (1 uL) on a silica gel rod and then developing the rod using
hexane:diethyl
ether:acetic acid (90:10:1, volume ratio). The glyceride and ester peak area
ratios were
obtained from the resulting charts, and the glyceride yield was calculated
based on these
ratios. The EPA and DHA yields were calculated from (the PUFA ratio (%) of the
glyceride
after reaction x the glyceride content (%))/(the PUFA ratio (%) before the
reaction). Table
11 shows the results of the EPA and DHA area%, the EPA and DHA fatty acid
yield, and
glyceride yield. Table 12 shows the results of the comparative examples.
[0051] When the results in Table 11 are compared with the comparative example
results in
Table 12, it is clear that the addition of water and MgO has an effect on EPA
and DHA
concentration even with the same amount of lipase. Moreover, it is clear that
the EPA and
DH:A become more concentrated as the amount of MgO added increases.
Furthermore, the
EPA and DHA yields are very high and fatty acid selectivity of this reaction
is maintained.
[0052] [Table 11]
Table 11
Starting Lipase QLM Lipase QLM Lipase PS Lipase PS
purified 0.25% MgO 2.5% MgO 0.25% MgO 2.5% MgO
sardine oil + water + water + water + water
EPA area% 28.8 50.6 61.5 52.2 59.7
DHA area% 12.5 16.3 16.2 15.6 17.9
EPA yield (%) 98.5 97.5 93.2 91.7
DHA yield (%) 90.4 91.7 83.6 70.2
Glyceride yield (%) 67.8 50.6 60.5 49.1
[0053]
CA 02693070 2010-01-14
-21-
[Table 12]
Table 12
Lipase Lipase Lipase Lipase PS Lipase PS Lipase PS
QLM QLM QLM + water + MgO
+ water + Mg0
EPA area% 36.1 43.0 41.7 30.7 45.0 30.1
DHA area% 15.2 17.5 16.4 12.9 17.1 12.3
EPA yield (%) 99.5 94.8 98.5 99.6 85.9 99.6
DHA yield (%) 99.2 89.0 90.0 98.6 75.2 97.5
Glyceride yield (%) 83.4 74.6 69.8 96.2 55.0 98.0
REFERENCE EXAMPLE 2
[0054] Using as the starting material sardine oil (15.7% EPA, 8.99% DHA,
Nippon Suisan
Kaisha, Ltd..) having lower EPA and DHA contents than the sardine oil used in
Reference
Example 1, ethanolysis was performed for 16 hours at 40 C under the same
conditions as in
Reference Example 1 after adding 1.65 mg of Lipase QLM (100 units/g), 17 L of
water,
2.5% (w/w) MgO, and 170 L of ethanol to 1 g of oils and fats. Table 13 shows
the area%
and yield of EPA and DHA and glyceride yield.
[0055] [Table 13]
Table 13
Lipase QLM 2.5% Mg0 + water
EPA area% 43.5
DHA area% 17.3
EPA yield (%) 95.5
DHA yield (%) 80.5
Glyceride yield (%) 41.9
REFERENCE EXAMPLE 3
CA 02693070 2010-01-14
-22-
[0056] 2 mg of Lipozyme TL IM (Thermomyces lanuginosus, Novozymes) (0.1% (w/w)
in
terms of oil), 34,u L of water, MgO (0.25% (w/w) or 2.5% (w/w)), and 340 L of
ethanol
were added to 2 g of purified tuna oil (6.75% of EPA, 24.3% of DHA, Nippon
Suisan Kaisha,
Ltd..), and the mixture was stirred for 16 hours at 40 C. By way of
comparison, ethanolysis
was performed under the same above-mentioned conditions with the exception
that no water
or MgO was added, only water was added, or 0.25% (w/w) of MgO only was added.
After
the reaction, the solid content was filtered, the glyceride fraction was
separated by
preparative TLC, methyl esterification was performed, and the fatty acid
composition was
analyzed. Table 14 shows the EPA and DHA fatty acids yield and glyceride
yield, and
Table 15 shows the EPA and DHA area%, fatty acid yield, and glyceride yield of
the
comparative example.
[0057] When Lipozyme TL IM was used, the degree of concentration of the DHA
concentration increased and ethanolysis of the EPA proceeded. The degree of
enrichment of
the DHA concentration improved with an increase in the amount of MgO added.
Even
though the same amount of enzyme was used in the comparative example, the DHA
was
hardly concentrated.
[0058] [Table 14]
Table 14
Purified tuna oil Lipozyme TL IM Lipozyme TL IM
0.25% MgO + water 2.5% MgO + water
EPA area% 6.8 9.4 8.4
DHA area% 24.3 48.2 68.7
EPA yield (%) 69.6 37.0
DHA yield (%) 99.1 83.5
Glyceride yield (%) 50.0 29.6
[0059]
CA 02693070 2010-01-14
-23-
[Table 15]
Table 15
Lipozyme TL IM Lipozyme TL IM Lipozyme TL IM
+ water + Mg0
EPA area% 7=2 7.2 7.1
DI-IA area% 26.2 26.5 25.5
EPA yield (%) 99.2 99.0 99.5
DHA yield (%) 99.6 99.8 99.5
Glyceride yield (%) 97.2 93.5 98.0
REFERENCE EXAMPLE 4
[0060] In order to investigate the effects of reaction additives other than
MgO, nine reaction
additives were added at 1% (w/w) in terms of starting oil and reacted under
the same reaction
conditions as in Reference Example 1. That is, 1.65 mg of Lipase QLM
(Alcaligenes sp.,
Meito Sangyo Co., Ltd) (100 units/g), 17 L of water, the nine types of
reaction additives
shown in Table 16 at 1% (w/w) in terms of oil, and 170,uL of ethanol (0.75
equivalents in
terms of fatty acids) were added to 1 g of purified sardine oil (28.2% EPA,
12.5% DHA,
Nippon Suisan Kaisha, Ltd..), and the mixture was stirred for 16 hours at 40
C. Once the
reaction was over, the solid content was filtered, the glyceride fraction was
separated by
preparative TLC, methyl esterification was performed, and the fatty acid
composition was
determined. Table 16 shows the EPA area% of the glyceride fraction. It is
clear that in
addition to MgO, magnesium hydroxide, calcium oxide, and calcium hydroxide
have an
EPA-enriching effect.
[0061]
CA 02693070 2010-01-14
-24-
[Table 16]
Table 16
EPA Manufacturer Grade Purity min%
area%
Magnesium oxide 56.3 Junsei Chemical Co., Special grade 99
I.td.
Magnesium 54.5 Wako Pure Chemical First grade 97
hydroxide Industries, Ltd.
Magnesium 44.7 Nacalai Tesque, Inc. Special grade MgCO3 60 to
carbonate (basic) 55%
MgO 40 to 45%
Magnesium 30.7 Wako Pure Chemical Special grade 98
chloride Industries, Ltd.
Calcium oxide 46.9 Wako Pure Chemical Special grade 99.9
Industries, Ltd.
Calcium hydroxide 46.6 Nacalai Tesque Inc. Special grade 95
Calcium chloride 29.6 Nacalai Tesque Inc. Special grade 98.5
Calcium nitrate 30.1 Nacalai Tesque Inc. Special grade 99.5
Sodium carbonate 29.9 Wako Pure Chemical Special grade 99.5
Industries, Ltd.
Potassium 36.2 Nacalai Tesque, Inc. Special grade 99.7
bicarbonate
REFERENCE EXAMPLE 5
[0062] Production of EPA-enriched oils and fats by Lipase QLM
0.83 g of Lipase QLM (Alcaligenes sp, Meito Sangyo Co., Ltd.), 17 g of water,
2.5 g
of MgO, and 173 mL of ethanol were added to 1 kg of purified sardine oil
(28.2% EPA,
12.5% DHA, Nippon Suisan Kaisha, Ltd..), and the mixture was stirred for 16
hours at 40 C.
After centrifugation, the solid content was removed, the ethanol was distilled
off, and 1.06 kg
were obtained. The product was washed with dilute sulfuric acid, rinsed with
warm water,
the ester and fatty acids were distilled off using a thin layer distillation
device, and 583 g of
CA 02693070 2010-01-14
- 25 -
EPA-enriched oil were obtained as the glyceride fraction. The fatty acid
composition was
measured to be 48.3% of EPA and 17.3% of DHA.
REFERENCE EXAMPLE 6
[0063] Production of DHA-enriched oils and fats by Lipozyme TL IM
1 g of Lipozyme TL IM (Thermomyces lanuginosus, Novozymes), 17 g of water,
g of MgO, and 173 mL of ethanol were acided to 1 g of purified tuna oil (6.75%
EPA and
24.3% DHA) and stirred for 16 hours at 40"C. After filtering the solid
content, the ethanol
was distilled off and 1.07 kg were obtained. After rinsing with phosphoric
acid, the product
was rinsed with warm water, the ester and fatty acids were distilled off by a
molecular
distillation device, and 416 g of DHA-enriched oil were obtained as glyceride
fraction. The
fatty acid composition was measured to be '9.4% of EPA and 52.8% of DHA.
REFERENCE EXAMPLE 7
[0064] Study of the amount of MgO added
Alcoholysis was performed under the same conditions as in Reference Example 1,
that is, 1.65 mg of Lipase QLM (100 units/g), 17,uL of water, MgO (0 to 10%
(w/w) in terms
of oil)), and 170 L of ethanol (0.75 equivalent in terms of fatty acids) were
added to 1 g of
purified sardine oil (28.2% EPA and 12.5% DHA, Nippon Suisan Kaisha, Ltd..)
and the
mixture was stirred for 16 hours at 40 C.
[0065] Table 17 shows the results. The reaction was promoted and the EPA was
concentrated in proportion to an increase in the amount of MgO added.
[0066]
CA 02693070 2010-01-14
-26-
[Table 17]
Table 17
Amount of MgO added EPA area% DHA area%
0 43.6 17.1
0.05% 46.7 16.1
0.1% 47.2 16.2
0.25% 50.6 16.3
1% 56.3 17.0
2.5% 61.5 16.2
5% 66.8 15.1
10% 67.6 15.4
REFERENCE EXAMPLE 8
[0067] Study of amount of ethanol
0.83 mg of Lipase QLM (50 units,/g), 17,uL of water, MgO (0.25% (w/w) in terms
of oil), and ethanol at 0.5 to 1.5 equivalents in terms of fatty acids were
added to 1 g of
purified sardine oil (28.2%EPA, 12.5% DHA, Nippon Suisan Kaisha, Ltd..) and
alcoholysis
was performed by stirring for 16 hours at 40 C.
[0068] Table 18 shows the results. It is clear that the preferred amount of
ethanol is 0.5 to
1.5 equivalents in terms of fatty acids.
[0069] [Table 18]
Table 18
Ethanol (equivalents in terms of fatty acids) 0.5 0.67 0.75 1 1.5
EPA area% 43.1 46.38 46.3 46.5 40.2
DHA area% 16.7 15.77 17.0 17.4 16.3
REFERENCE EXAMPLE 9
CA 02693070 2010-01-14
-27-
[0070] Study of amount of lipase used
to 50 units/g of Lipase QLM, 17 L of water, MgO (0.25 to 1% (w/w) in terms of
oil), and 0.75 equivalent of ethanol in terms of fatty acids were added to 1 g
of purified
sardine oil (28.2% EPA, 12.5% DHA, Nippon Suisan Kaisha, Ltd..), and
alcoholysis was
performed by stirring for 16 hours at 40 C.
[0071] Table 19 shows the results. It is clear that the preferred amount of
lipase is
25 units/g or more. Moreover, it was confirmed that, even with the same amount
of lipase,
by increasing the amount of MgO the reactivity could be increased.
[0072] [Table 19]
Table 19
QLM (units/g) 10 25 30 50 50
MgO (%) 2.5 2.5 1 0.25 2.5
EPA area% 32.1 37.17 48.2 47.8 63.9
DHA area% 13.1 15.4 17.2 17.2 17.2
EPA yield (%) 98.99 99.2 98.4 98.5 96.5
REFERENCE EXAMPLE 10
[0073] Study of reaction time
1.65 mg of Lipase QLM (100 units/g), 17 L of water, MgO (0.25% (w/w) in terms
of oil), and one equivalent of ethanol in terms of fatty acids were added to 1
g of purified
sardine oil (28.2% EPA, 12.5% DHA, Nippon Suisan Kaisha, Ltd..), and
alcoholysis was
performed by stirring for 0 to 24 hours at 40 C.
[0074] Table 20 shows the results.
[0075]
CA 02693070 2010-01-14
-28-
[Table 20]
Table 20
Reaction time 0 1 2 4 6 7 16 24
EPA area% 28.8 39.6 42.7 44.0 44.5 46.8 50.6 53.4
DHA area% 12.0 15.8 15.6 16.4 15.7 17.0 16.3 16.0
[0076] [Comparative Example]
Lipase reaction in which MgO and water were not used
Lipase QLM (100 to 1,000 units/g) and ethanol (one equivalent in terms of
fatty
acids) were added to 1 g of purified sardine oil (28.2% EPA, 12.5% DHA, Nippon
Suisan
Kaisha, Ltd..) and alcoholysis was performed by stirring for 16 hours at 40 C.
[0077] [Table 21]
Table 21
QLM (units/g) 100 250 500 750 1,000
EPA area% 36.1 41.4 45.9 46.7 46.3
DHA area% 15.2 36.1 16.2 17.92 17.97
EPA yield (%) 85.1 76.1 69.1 72.2 71.6
REFERENCE EXAMPLE 11
[0078] Application to Coho salmon extracted oil
2.0 mg (0.2%) of Lipozyme TL IM (Thermomyces lanuginosus, Novozymes), 10 L
of water, MgO (Junsei Chemical Co., Ltd., special grade, purity of 99% or
higher) (0.5%
(w/w) or 2.5% (w/w) in terms of oil), and 1'70 L of ethanol (0.75 equivalent
in terms of fatty
acids) were added to 1 g of Coho salmon extracted oil (9.8% EPA, 14.0% DHA),
and the
mixture was stirred for 16 hours at 40 C. After the reaction, the solid
content was filtered,
the glyceride fraction was separated by preparative TLC, methyl esterification
was performed,
and the fatty acid composition was analyzed by gas chromatography. The same
conditions as
CA 02693070 2010-01-14
-29-
in Working Example 1 were used for preparative TLC, methyl esterification, and
gas
chromatography analysis.
[0079] Moreover, by way of comparison, ethanolysis was performed under the
above-mentioned conditions with the exception that no water or MgO was added.
[0080] Table 22 shows the results of the glyceride fraction EPA and DHA area%,
the EPA
and DHA fatty acid yield, and the glyceride yield. Table 23 shows the results
of the
comparative example.
[0081] [Table 22]
Table 22
Starting Coho 0.2% Lipozyme 0.2% Lipozyme
salmon extracted oil TL IM TL IM
0.5% MgO + Water 2.5% MgO + Water
EPA area% 9.8 14.0 14.0
DHA area% 14.0 30.7 45.3
EPA + DHA area% 23.8 44.7 59.3
EPA yield (%) 69.6 29.1
DHA yield (%) 107.0 66.3
Glyceride yield (%) 48.8 20.5
[0082]
CA 02693070 2010-01-14
-30-
[Table 23]
Table 23
0.2% Lipozyme TL IM
Without MgO nor water
EPA area% 10.33
DHA area% 15.15
EPA + DHA area% 25.48
EPA yield (%) 98.9
DHA yield (%) 96.9
Glyceride yield (%) 95.28
REFERENCE EXAMPLE 12
[0083] Application to Pollack extracted oil
1.65 mg of Lipase QLM (100 units/g), 17 L of water, 2.5% (w/w) MgO, and
170 L of ethanol were added to 1 g of Pollack extracted oil (12.3% EPA, 7.9%
DHA) as the
starting oils and fats and ethanolysis was performed for 16 hours at 40 C.
Moreover,
similarly, water and MgO were added with 5 mg (0.5%) of Lipozyme TL IM and
ethanolysis
was performed. Table 24 shows the results of the area% and yield of EPA and
DHA and
glyceride yield. When Lipase QLM was used EPA was concentrated, and when
Lipozyme
TL IM was used DHA was concentrated. The EPA and DHA were concentrated such
that
their combined area% was at least twice that of the starting material.
[0084] By way of comparison, Table 25 shows the results of performing
ethanolysis under
the above-mentioned conditions with the exception that no MgO or water was
added.
[0085]
CA 02693070 2010-01-14
-31-
[Table 24]
Table 24
Starting pollack Lipase QLM 0.5% Lipozyme
extracted oil 100 units/g TL IM
2.5% MgO + Water 2.5% MgO + Water
EPA area% 12.3 30.9 14.0
DHA area% 7.9 12.9 38.3
EPA + DHA area% 20.2 43.8 49.4
EPA yield (%) 103.7 18.3
DHA yield (%) 73.7 78.1
Glyceride yield (%) 45.1 16.2
[0086] [Table 25]
Table 25
Lipase QLM 100 units/g 0.5% Lipozyme TL IM
Without MgO nor water Without MgO nor water
EPA area% 15.8 18.6
DHA area% 9.9 16.0
EPA + DHA area% 25.7 34.5
EPA yield (%) 101.0 64.3
DHA yield (%) 97.8 64.1
Glyceride yield (%) 78.6 79.4
REFERENCE EXAMPLE 13
[0087] Application to Sunfish liver oil
1.65 mg of Lipase QLM (100 units/g), 17,uL of water, 2.5% (w/w) MgO, and
170,uL of ethanol were added to 1 g of Sunfish liver oil (5.1% arachidonic
acid (AA), 4.2%
EPA, 7.7% docosapentaenoic acid (DPA), and 10.5% DHA) as the starting oils and
fats, and
CA 02693070 2010-01-14
-32-
ethanolysis was performed for 16 hours at 40 C. Moreover, similarly, water and
MgO were
added with 5 mg of Lipozyme TL IM (0.5%) and ethanolysis was performed. Table
26
shows the area% and yield of AA, EPA, DPA, and DHA and glyceride yield. In
contrast to
the fact that AA, EPA, DPA and DHA were concentrated when Lipase QLM was used,
only
DHA was concentrated when Lipozyme TL IM was used.
[0088] By way of comparison, Table 27 shows the results of performing
ethanolysis under
the above-mentioned conditions with the exception that no MgO or water was
added.
[0089] [Table 26]
Table 26
Starting Lipase QLM 100 units/g 0.5% Lipozyme TL IM
sunfish liver 2.5% MgO + Water 2.5% MgO + Water
oil
AA area% 5.1 12.9 2.8
EPA area% 4.2 10.7 2.6
DPA area% 7.7 17.9 3.6
DHA area% 10.5 17.5 59.8
AA + EPA + DPA 27.6 59.0 68.8
+ DHA area%
AA yield (%) 95.7 8.6
EPA yield (%) 96.8 9.9
DPA yield (%) 98.0 7.5
DHA yield (%) 81.1 90.5
Glyceride yield (%) 44.8 15.9
[0090]
CA 02693070 2010-01-14
-33-
[Table 27]
Table 27
Lipase QLM 100 units/g 0.5% Lipozyme TL IM
Without MgO nor water Without MgO nor water
AA area% 6.5 5.6
EPA area% 5.3 4.5
DPA area% 10.4 8.7
DHA area% 13.5 12.8
AA + EPA + DPA + DHA area% 35.7 31.7
AA yield (%) 98.5 90.3
EPA yield (%) 99.1 89.2
DPA yield (%) 104.9 93.1
DHA yield (%) 100.1 100.0
Glyceride yield (%) 67.9 82.3
