Download The application of lipase for preparing various lipids enriched with

Rit Fiskideildar 16 (1999) 97-105
The application of lipase for preparing various lipids
enriched with omega-3 fatty acids
Gudmundur G. Haraldsson
Science Institute, University of Iceland
Dunhagi 3, IS-107 Reykjavik, Iceland
ABSTRACT
Immobilized lipases have been employed to generate various lipid classes, including triacylglycerols, phospholipids and non-polar ether lipids, highly enriched with eicosapentaenoic acid and
doscosahexaenoic acid. These are long-chain n-3 type polyunsaturated fatty acids, characteristic
of marine fat. Lipases are ideally suited as catalysts for transformations involving the highly labile
long-chain polyunsaturated fatty acids. The mildness they offer, most certainly protects them from
partial destruction of their natural all-cis n-3 framework by oxidation, double-bond migrations or
cis-trans isomerization during traditional chemical processes involving extremes of pH and high
temperature. The considerable reduction in bulkiness, related to the solvent-free conditions, renders the techniques described a high feasibility for industrialization.
Keywords: n-3 polyunsaturated fatty acids, fish oil, lipase, triacylglycerol, phospholipids, ether lipids.
INTRODUCTION
C14 to C24 of varying degree of unsaturation,
from saturated to polyunsaturated. Of the
polyunsaturated fatty acids (PUFA), EPA and
DHA usually account for between 5 and 15%
each, depending on type of fish species, with the
total n-3 content usually varying between 20 and
30%. As an example, cod liver oil has roughly
10% each of EPA and DHA, tuna oil is highly
enriched with DHA (approximately 5% EPA and
25% DHA) and sardine oil is enriched with EPA
(approximately 18% EPA and 12% DHA).
Herring oil and capelin oil have lower EPA and
DHA content, usually between 5 and 7 % each
of EPA and DHA. Seasonal variations and site
of catch influence the fatty acid composition of
fish and fish oil to a large extent within the same
The long-chain n-3 (or omega-3) type polyunsaturated fatty acids are characteristic of marine
fat (Ackman 1989). They are produced by phytoplankton and accumulate through the food web
in fish, which is unable to biosynthesise them.
The most ubiquitous of the n-3 fatty acids in fish
are cis-5,8,11,14,17-eicosapentaenoic acid (EPA)
and cis-4,7,10,13,16,19-docosahexaenoic acid
(DHA), which commonly occur in triacylglycerols
(TG; triglycerides) (Hölmer 1989) and phospholipids (PL) (Vaskovsky 1989). The chemical structure of EPA and DHA is shown in Figure 1. Fish
oil is virtually pure triacylglycerols, which
comprise more than fifty different fatty acids
(Ackman 1982). The chain length ranges from
Dedicated to Professor Unnsteinn Stefánsson in honour of his contributions to oceonography and education.
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DHA,
more
specific
methods are required, such
as the Corey´s chemical
modification, HPLC and,
more recently, kinetic resolution by lipase to separate
EPA and DHA (Haraldsson
et al. 1989).
Resynthesis of triacylglycerols or some other
natural lipid form, such as
ether lipids or phospholipids, is by no means
easy by conventional chemical methods. Such
traditional esterification methods involve
extremes of pH, high temperature and pressure
(Haraldsson 1989). Almost certainly, this will
partially destroy the all-cis n-3 framework of
EPA and DHA by oxidation, cis-trans isomerization, double-bond migrations or polymerization. In order to solve these problems lipases
have been introduced to processes involving the
highly labile n-3 polyunsaturated fatty acids
(Haraldsson and Hjaltason 1992). They offer
high efficiency and mildness and their application in organic media is now firmly established
(Haraldsson 1992).
The application of lipase for preparing various
lipid classes highly enriched or homogeneous
with EPA and/or DHA is described in this review,
which is largely confined to our work at the
University of Iceland. This includes triacylglycerols, phospholipids and ether lipids, which are
characteristic of shark liver oil. Furthermore, the
possibility of employing lipases to produce
monoester or free fatty acid concentrates of EPA
and DHA by kinetic resolution of fatty acids,
based on lipase fatty acid selectivity, will also
be discussed.
Figure 1. The structure of EPA and DHA.
species. In lean fish species such as cod, haddock
and pollack, the triacylglycerols are largely
confined to a large liver, whereas in fatty fish
species, such as herring and capelin, the triacylglycerols are confined to their oily flesh. Phospholipids are generally much more highly
enriched with EPA and DHA, 20 to 50% of each
depending on the type of phospholipids. The
total phospholipid content of fish usually
remains between 1 and 1.5% as based on total
fish wet weight, roughly an order of magnitude
lower than the triacylglycerols (Haraldsson et al.
1993).
The beneficial health effects of marine fat are
now well established and almost exclusively
attributed to the n-3 polyunsaturated fatty acids,
EPA and DHA in particular (Nelson 1991).
Consequently, there are strong demands for their
concentrates by the pharmaceutical industry as
well as the health food industry for use as food
supplements. Triacylglycerols, up to the level of
30 % EPA + DHA, can be prepared directly from
fish oils, without splitting the fat, by various
methods such as winterization, molecular distillation and solvent crystallization (Haraldsson et
al. 1989). Concentration beyond that level is
difficult and requires a cleavage of the fatty
acids off the acylglycerols, either as free fatty
acids or monoesters. This is a consequence of
the great combinations of the fatty acids in triacylglycerol oils, where they are more or less
randomly distributed, three and three together,
bound into the glycerol moiety. Several methods
or combination of methods are available for
concentrating them to the 50-80% level, including carbon dioxide supercritical fluid extraction,
urea complexation, molecular distillation and
kinetic resolution by lipase. In order to achieve
concentrates beyond the 90% level of EPA and
ENRICHMENT OF COD LIVER OIL
WITH EPA AND DHA
It is relatively easy to concentrate EPA and DHA
up to high levels as free fatty acids or ethyl
esters. The natural form of these fatty acids in
fish oil is triacylglycerols and the big challenge
was to provide natural triacylglycerols highly
enriched with EPA and DHA, far beyond the
30% level mentioned above. A highly successful solution to that problem was based on treat-
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ing cod liver oil as triacylglycerols with free fatty
acid or monoester concentrates of EPA and DHA in
the presence of lipase to
effect fatty acid exchange
between the natural triacylglycerols and the concentrates (Haraldsson et al.
1989).
A
1,3-regiospecific
lipase from the fungus
Mucor miehei, commercially available in an immobilized form by Novo
Nordisk in Denmark, was
employed to effect transesterification reactions of cod
liver oil with concentrates
of EPA and DHA. What 1,3-regiospecificity
means for a lipase, is to act with a high preference or exclusively at the primary alcoholic endpositions of the triacylglycerols. The cod liver
oil comprised approximately 9-10% each of EPA
and DHA, and triacylglycerols highly enriched
with n-3 polyunsaturated fatty acids were
accomplished, of high purity and in quantitative
yields. Interesterification and acidolysis reactions with ethyl ester and free fatty acid concentrates, respectively, both of 85% EPA + DHA
content, resulted in triacylglycerols containing
60-65% EPA + DHA and well over 70% total n3 polyunsaturated fatty acids. At that time, this
represented by far the highest EPA and DHA
enriched triacylglycerol product available. Both
reactions were conducted in the absence of any
solvent, using 10% dosage of lipase, as based on
the weight of fat, at 60-65°C. The reactions are
demonstrated in Scheme 1.
A three-fold excess of free fatty acids or ethyl
esters was used, as based on number of mol
equivalents of esters present in the triacylglycerols. Despite the 1,3-regiospecificity of the lipase,
the mid-position became enriched to an equal
extent as the end-positions. This means that at
an equilibrium the fatty acid composition of the
triacylglycerols was reflected by the weighted
average of the initial composition of the cod liver
oil triacylglycerols and the concentrates, as well
as the ratio between them. Intramolecular non-
Scheme 1
enzyme promoted acyl-migration processes were
responsible for this, as was established by investigating the fatty acid composition of individual
positions of the acylglycerols when the reactions
proceeded (Haraldsson and Almarsson 1991).
HOMOGENEOUS TRIACYLGLYCEROLS
OF EPA AND DHA
By the methodology described above, the EPA
and DHA fatty acid composition of the triacylglycerol product was determined by a weighted
average of the initial fatty acid composition of
the cod liver oil triacylglycerols and the composition of the n-3 concentrates. To avoid that limitation and obtain triacylglycerols of composition
identical to the concentrates being used, a procedure based on a direct esterification of free fatty
acids with glycerol was developed (Haraldsson
et al. 1995). This also opened the possibility of
synthesising triacylglycerols homogeneous with
either EPA or DHA, i.e. 100% EPA or DHA. This
had been considered a synthetic challenge and
our primary goal, the crown on the triacylglycerol issue. The structure of such homogeneous
triacylglycerols of EPA and DHA is depicted in
Figure 2.
A different lipase, the non-regiospecific yeast
lipase from Candida antarctica, also commercially available from Novo Nordisk in Denmark,
was highly efficient in generating triacylglycerols
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Figure 2. The structure of homogeneous triacylglycerols of EPA and DHA.
Scheme 2
containing 100% EPA and DHA. This was accomplished by a direct esterification of glycerol with
stoichiometric amount of pure EPA and DHA,
without any solvent, by stirring at 65°C under
vacuum, with a 10% dosage of the immobilized
lipase, as based on the weight of substrates. The
co-produced water was condensed into a liquid
nitrogen cooled trap during the progress of the
reaction, thus driving the reaction to completion.
This is demonstrated in Scheme 2 for EPA.
The resulting triacylglycerols, homogeneous
with either EPA or DHA, were afforded in nearly
quantitative yields of very high purity. High-field
1H and 13C NMR spectroscopy was found extremely valuable as a probe to monitor the progress of the reactions. It also enabled us to follow
the incorporation of EPA and DHA into glycerol
to form the various intermediary acylglycerols
participating in the direct esterification process,
the 1- and 2-monoacylglycerols (MG), 1,2- and
Scheme 3
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Figure 3. The structure of ether lipids homogeneous with EPA and DHA.
1,3-diacylglycerols (DG) and the triacylglycerols (TG). Again, it became evident, despite the
non-regio-specificity of the lipase, that acylmigration processes were playing an important
role during the process. The overall reaction
process is demonstrated in Scheme 3.
transesterification reactions with concentrates of
EPA and DHA, using Lipozyme in a manner
identical to the one previously described for cod
liver oil. Direct esterification of the 1-O-alkylsn-glycerols with EPA and DHA resulted in
homogeneous ether lipids, when using the
Candida antarctica lipase. These reactions are
demonstrated in Scheme 4 for EPA. It was interesting to notice that the ether lipids were apparently far less prone to acyl migrations as
compared to the triacylglycerols under the transesterification conditions.
ETHER LIPIDS HIGHLY ENRICHED
WITH EPA AND DHA
Non-polar glyceryl ether lipids of the 1-O-alkyl2,3-diacyl-sn-glycerol type are major constituents in liver oil of certain species of shark,
such as the Greenland shark (Somniosus microcephalus), making up to 30-60% of the oil (Sargent 1989). They have been claimed to display
various beneficial effects on human health (Mangold and Palthauf 1983). Ether lipids, highly enriched with EPA and DHA, were prepared by the
lipase catalysis procedures already described in
order to possibly combine the claimed beneficial
effects of both fish oil and the ether lipids
(Haraldsson and Thorarensen 1994). Such ether
lipids homogeneous with EPA and DHA are
shown in Figure 3.
The ether lipids were isolated in a pure state,
as was established by high-field NMR, from
shark liver oil concentrates using preparative
HPLC. There are three major fatty alcohol constituents present in the ether moiety, C16:0,
C18:0 and C18:1, the last one being the most
abundant, with EPA and DHA making up only
0.4 and 2.5% of the fatty acid composition of the
ether lipids acyl counterparts, respectively. They
were converted into pure 1-O-alkyl-sn-glycerols
by sodium methoxide catalyzed methanolysis.
Ether lipids highly enriched with either EPA
or DHA or both EPA and DHA were obtained by
PHOSPHOLIPIDS HIGLY ENRICHED
WITH EPA AND DHA
Phospholipids are major constituents of cell
membranes and play essential roles in biochemistry and physiology of the cell functions (Mead
et al. 1986). Phospholipids in fish and marine
species are highly enriched with the long-chain
n-3 type polyunsaturated fatty acids. 40-50%
content of EPA and DHA is not uncommon in
some phospholipid classes in fish (Haraldsson et
al. 1993). The n-3 polyunsaturated fatty acids
presumably play significant roles in adjusting the
membrane integrity and functions at the lower
temperatures, by adding to the membrane fluidity
and mobility as a result of their higher unsaturation. Among the phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE)
are by far the most abundant in fish flesh, especially the former one, which commonly makes
up 50-60% of the total phospholipid content. The
composition of individual phospholipid classes
is remarkably similar among fish species as is
the characteristic fatty acid composition of each
class (Haraldsson et al. 1993).
101
Scheme 4
Lecithins of plant and vegetable origin are popular as health supplements. They are highly enriched with n-6 fatty acids, which characterise
vegetable oil in a similar manner as do the n-3
fatty acid fish oils. Purified fish lecithins, which
are phospholipids highly enriched with n-3
polyunsaturated fatty acids, on the other hand,
are not available on the market at all. Unlike
plant or vegetable lecithins, they are by no means
readily obtainable, since tedious extraction procedures from fish are required (Haraldsson et al.
1993). We therefore decided to attempt the preparation of such phospolipids, highly enriched
with EPA and DHA, from the more readily available plant or animal lecithins (Haraldsson and
Thorarensen 1995). Figure 4 shows the structure
of phospatidylcholine (PC) homogeneous with
EPA and DHA.
Pure phosphatidylcholine was obtained from
egg yolk after purification by preparative HPLC
and was treated under the acidolysis reaction conditions described above for cod liver oil, using
the Mucor miehei lipase and pure EPA. As might
be anticipated, the rate of the reaction involving
the phospholipids, possessing the zwitterionic
head group, was much lower as compared to the
natural triacylglycerol substrates. Large quantities of lipase were therefore required, which
again resulted in high extent of hydrolysis sidereaction. Optimal reaction conditions offered a
mixture of phospholipids of approximately 40%
each of the desired phosphatidylcholine and
lysophosphatidylcholine (LPC) and 20%of glycerol phosphatidylcholine (GPC). In LPC one of
the acyl moiety has been hydrolysed and in glycerol phosphatidylcholine both acyl groups have
been hydrolysed (see Scheme 5). When pure EPA
was used, both the PC and the LPC fractions
were highly enriched with EPA, especially the
LPC fraction, 58 and 69%, respectively. This is
by far the highest enrichment of n-3 polyunsaturated fatty acids into phospholipids reported in
the literature.
More detailed information about the nature of
Figure 4. The structure of phosphatidylcholine homogeneous with EPA and DHA.
102
Scheme 5
the acidolysis reaction was afforded by 31P NMR
spectroscopy analysis (Haraldsson and Thorarensen 1995). The results revealed the complex role
played by water in terms of compromising the
lipase activity, hydrolysis side-reactions, reaction rate and extent of incorporation under the
non-aqueous reaction conditions. The rather
complicated reaction process is demonstrated in
Scheme 5. As before, acyl migrations were
believed to play important roles in involving the
mid-position in the overall process.
Recently, we described a highly efficient process to concentrate EPA together with DHA by
lipase-catalyzed ethanolysis of fish oil (Haraldsson et al. 1997; Breivik et al. 1997). Pseudomonas
lipases were observed to convert the bulk of the
unwanted saturated and monounsaturated fatty
acids of the fish oil triacylglycerols into ethyl
esters. The n-3 polyunsaturated fatty acids including both EPA and DHA, on the other hand,
remained attached to the residual acylglycerols,
mainly as mono- and diacylglycerols, but also
triacylglycerols, depending upon the extent of
conversion. This is demonstrated in Scheme 6.
No solvents were required and only stoichiometric amounts of ethanol were required. By that
method it became easy to obtain concentration
levels of 50% EPA + DHA in the residual acylglycerol mixture with very high EPA and DHA
recovery in sardine oil comprising 15% EPA and
10% DHA. It was interesting to observe that the
Pseudomonas lipases displayed preference for
DHA as compared to EPA, which is most unusual (Haraldsson et al. 1997).
In a further development of the work we became interested in discriminating between EPA
EPA AND DHA CONCENTRATES
FROM FISH OILS BY LIPASE
The use of lipases as catalysts for producing
monoester or free fatty acid concentrates of EPA
and DHA from fish oil, as an alternative to conventional chemical procedures, has also been
investigated. Such work is based on a wide range
of fatty acid selectivity among the various commercially available lipases. Many lipases do not
tolerate the long-chain n-3 fatty acids very well
as substrates. Lipases that do so usually display
preference for EPA as compared to DHA.
103
Scheme 6
and DHA in fish oil instead of concentrating both
EPA and DHA by lipase promoted kinetic resolution. Different requirements for the lipase
selectivity were therefore made and a lipase
strongly discriminating between EPA and DHA
was needed. In order to avoid complications related to regioselectivity of the lipase and nonhomogeneous distribution of EPA and DHA into
various positions of the triacylglycerols (Christie
1986) a different approach was required. That
approach was based on a direct esterification of
free fatty acids from fish oil, which is demonstrated in Scheme 7.
The immobilized Mucor miehei lipase was
observed to convert the bulk of the fatty acids
present, including EPA, into ethyl esters, leaving
the more reluctant DHA unaffected in the residual free fatty acids (Haraldsson and Kristinsson 1998). Free fatty acids from various fish oils
were treated under the direct esterification reaction conditions, using stoichiometric amounts of
ethanol at room temperature, in the absence of a
solvent. This modification of the lipase catalysed
separation of EPA and DHA was much more efficient and much faster than the corresponding
ethanolysis reaction involving fish oil triacylglycerols. As an example, when tuna oil free
fatty acids of 6% EPA and 23% DHA composition were directly esterified with ethanol, the
residual free fatty acids comprised 74% DHA
and only 3% EPA. The recovery of both DHA
into the residual free fatty acid fraction and EPA
into the ethyl ester product remained very high,
83 and 87%, respectively. Based on these results,
there are reasons to believe that in terms of separating EPA and DHA, lipase can be used as a
powerful alternative to traditional chemical techniques by a two-step or a multi-step approach.
ACKNOWLEDGEMENT
Lysi hf. in Reykjavik Iceland, Novo Nordisk AS
in Bagsvaerd Denmark, Norsk Hydro AS in
Porsgrunn Norway, Pronova Biocare AS in
Sandefjord Norway, The
Icelandic
Government
Science Fund and The
Scheme 7
University of Iceland Research Fund are acknowledged for financial support.
Freygarður
Þorsteinsson,
Páll A. Höskuldsson, Snorri
Þ. Sigurðsson, Örn Almars-
104
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son, Birgir Örn Guðmundsson, Björn Kristinsson, Atli Thorarensen, Guðmundur G. Guðmundsson, Ragnheiður Sigurðardóttir, Sigmundur Guðbjarnason, Baldur Hjaltason, Tomas
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