unaffected. This enables the preparation of concentrates of EPA plus DHA.Pseu-
domonaslipases belong to this category, as has been demonstrated by Haraldsson and
co-workers (Haraldsson et al., 1997). Two commercially availablePseudomonas
lipases were observed to afford a concentrate of approximately 50 % EPA+DHA
in very high recovery (80–90 %) and highly efficiently without a solvent, since
simply a 2-fold stoichiometric amount of ethanol was required. This demonstrates
that lipases can be used as a valid alternative to conventional physical methods such
as molecular distillation. One of the main advantages was the considerable reduction
of bulkiness of the process, since no organic solvent was required and the ethyl esters
produced were directly distilled off by short-path distillation from the residual acyl-
glycerol mixture of EPA and DHA (Scheme 8).
Geotrichum candidumlipase also belongs to this category of lipases not discri-
minating much between EPA and DHA. That lipase was used to concentrate EPA
together with DHA in tuna oil by hydrolysis reaction up to the 50 % levels, similarly
with high recoveries of EPA and DHA, by Shimada and co-workers (Shimada et al.,
1994; 1995). It is of interest that the residual acylglyceride mixture from the reaction
comprised TG to a large extent (85 %) which was related to selectivity of that lipase
favoring MG and DG rather than TG. This lipase displayed a somewhat lower ac-
tivity toward DHA than EPA, which is the usual behavior of lipases. An exception to
that behavior was observed from thePseudomonaslipases mentioned above, which
displayed higher activity toward DHA than EPA (Haraldsson et al., 1997) (see also
Chapter 8).
Lipases belonging to the third class displaying moderate to strong discrimination
between EPA and DHA, all in favor of EPA, includeCandida rugosalipase (formerly
namedCandida cylindracea),Rhizopus delemarlipase, and the fungal lipase from
Rhizomucor miehei. Hydrolysis of tuna oil with theCandida rugosalipase afforded
an acylglycerol mixture highly enriched with DHA, up toward the 50 % level (Hos-
hino et al., 1990; Tanaka et al., 1992; McNeill et al., 1996; Moore and McNeill,
1996). With that lipase there are indications that TG molecules in fish oil containing
DHA may be resistant to the lipase (Tanaka et al., 1992). Shimada and co-workers
have demonstrated that theRhizopus delemarlipase can be used to highly enrich free
acids from tuna oil with DHA by a direct esterification with a long-chain alcohol
(Shimada et al., 1997a,b). The residual free acids contained 73 % DHA in very
high recovery (84 %), and no solvent was required. A second esterification afforded
further purification of DHA up to almost 90 % in 71 % overall recovery.
Similar levels of DHA enrichment were obtained by Haraldsson and Kristinsson
from tuna oil free acids in a direct esterification reaction with ethanol in the absence
184 10 Enrichment of Lipids with EPA and DHA by Lipase
Scheme 8. Ethanolysis of fish oul by Pseudomonas lipase.