(GLA) and this technology has been used to enrich this biologically important fatty
acid from evening primrose or borage oil (Rahmatullah et al., 1994; Huang et al.,
1997).
Two approaches have been widely studied; hydrolysis of fish oil triglycerides (or
simple esters), and (trans)esterification of fish oil fatty acids. Hydrolysis of fish oil
triglycerides leads to partial glycerides enriched in EPA and DHA. The lipase from
Candida rugosa(formerly classified asC. cylindracea) has been shown to enrich
EPA and DHA from sardine oil, cod liver oil (Hoshino et al., 1990) and tuna oil
(Tanaka et al., 1992). Lipase fromGeotrichum candidumhas also been used (Shi-
mada et al., 1994), although the overall yields were lower unless a second step in-
volving removal of free fatty acids and a second hydrolysis was employed (see also
Chapter 8). As a result of their inability to hydrolyze fatty acids present at thesn-2
position of a triglyceride, 1,3-regioselective lipases are less widely used, although
Rhizomucor mieheilipase has been reported to enrich LCPUFA from anchovy oil
(Uston et al., 1997). The overall concentration of LCPUFA in the glyceride fraction
was raised from 27 % to 40 %. Selected data for the enrichment of LCPUFA are
shown in Table 2.
Although lipases from a number of sources have been shown to be effective in
these enrichment processes, the lipase fromCandida rugosais particularly interest-
ing as it has been shown to have relatively lower activity against DHA as compared
to EPA (Tanaka et al., 1992; McNeill et al., 1996). This observation has been
exploited to generate LCPUFA fractions enriched in these fatty acids. Using Chilean
fish oil the DHA:EPA ratio was raised to approximately 5 : 1 from 1 : 1 in the starting
oil (McNeill et al., 1996). If required the products of the enrichment process can be
re-esterified using immobilizedRhizomucor mieheilipase to generate triacylglycer-
ols enriched in these fatty acids (Moore and McNeill, 1996). The reaction scheme is
outlined in Figure 1.
Typically, a solution ofC. rugosalipase was added to fish oil at a ratio between
0.5 : 1 and 1 : 1. The enzymatic hydrolysis was allowed to proceed at 25 8 C under a
nitrogen blanket until the required level of free fatty acids was obtained. The reaction
was stopped by heating the mixture to 90 8 C. The aqueous phase was then allowed to
separate and the oil phase removed and dried under vacuum. The free fatty acids
were then separated from the glycerides by distillation.
1.2 Free fatty acids 7
Table 2.Enrichment of EPA and DHA from fish oils by lipase-catalyzed hydrolysis.
Oil Lipase Start, wt% Enriched, wt% Reference
EPA DHA EPA DHA
Chilean fish C. rugosa 16 12 10 36 McNeill et al., 1996
Tuna C. rugosa(two-step) 8 30 7 58 Shimada et al., 1994
Tuna G. candidum(two-step) 8 30 11 47 Shimada et al., 1994
Tuna C. rugosa 6 25 4 53 Tanaka et al., 1992
Cod liver A. niger 9 20 12 38 Hoshino et al., 1990
Refined sardine C. rugosa 15 10 10 37 Hoshino et al., 1990
