60–65 8 C, the lipase preparation constituting 10 % water. A 3-fold excess of free
fatty acids or ethyl esters was used, as based on number of molar equivalents of
esters present in the TG. The reactions are demonstrated in Scheme 1.
In both reactions the lipase displayed a significantly higher activity toward EPA
than DHA, and the interesterification reaction took place considerably faster than the
acidolysis reaction. Some hydrolysis side reaction was observed, especially for the
interesterification reaction, but this could be reduced considerably by lowering the
water content of the lipase (Haraldsson et al., 1993b).
Despite the 1,3-regiospecificity of the lipase, the mid-position became enriched to
an equal extent to the end-positions. Intramolecular nonenzyme-promoted acyl-mi-
gration processes (Kodali et al., 1990) were responsible for this, as was established
by investigating the fatty acid composition of individual positions of the acylglycer-
ols when the reactions proceeded (Haraldsson and Almarsson, 1991). This means
that, at equilibrium, the fatty acid composition of the TG was reflected by a
weighted average of the initial composition of the cod liver oil TG and the concen-
trates. This is exactly what was wanted, since the aim was to enrich the fish oil to the
maximum levels. In order to allow that, prolonged reaction time was required be-
cause the acyl-migration processes were considerably slower than the lipase-pro-
moted processes and rate-limiting for the equilibrium. In order to obtain an equili-
brium for the interesterification, a 24-h reaction was needed, whereas the acidolysis
reaction required 48 h.
Scheme 2 shows a simplified proposal of the transesterification reactions. This is a
multi-component system involving more than 50 different fatty acids, TG, DG, MG
as well as PUFA either as free acids or ethyl esters. Each component has its own
distinguished relationship to the biocatalyst. The processes must be initiated by li-
pase-promoted hydrolysis at the end-positions to produce 1(3),2-DG, which can
either undergo an acyl migration to form a more stable 1,3-DG or a much faster
lipase-catalyzed esterification to reform a PUFA-enriched TG. Once formed, the
1,3-DG can undergo a second hydrolysis to form 1(3)-MG, which in turn can under-
go PUFA acylation at the end-position to form a PUFA enriched 1,3-DG, followed by
a subsequent PUFA acyl migration to form a 1(3),2-DG with PUFA located at the
mid-position. This continues until an equilibrium has been reached with an identical
fatty acid composition of each constituent and individual positions of the acylgly-
174 10 Enrichment of Lipids with EPA and DHA by Lipase
Scheme 1. Lipozyme-catalyzed enrichment of cod liver oil with PUFA by acidolysis (R = H) or in-
teresterification (R = Et). PUFA* refers to equilibrium composition, but R1, R2 and R3 to the initial fatty
acid composition of individual positions of cod liver oil, sn-1, sn-2 and sn-3, respectively.