sence of this isomer (Pariza and Hargraves, 1985) and, subsequently, additional bio-
logical activities have been identified. These include prevention of body fat deposi-
tion during growth of some animal species, decreasing atherosclerosis and boosting
immune function (Nicolosi et al., 1997; Park et al., 1997; Belury, 1995; Miller et al.,
1994).
CLA in ruminant animals is believed to be generated by rumen bacteria during the
digestive process followed by incorporation into milk and body fat. However, it can
also be directly produced from linoleic acid or high-linoleate triglycerides such as
safflower oil. Under carefully controlled alkaline conditions the product comprises
of two main isomers;c-9,t-11 CLA andt-10,c-12 CLA, in approximately equal
amounts (Ip et al., 1991). It is believed that not all CLA isomers have equivalent
biological effects, and consequently there is now a great deal of interest in evaluat-
ing the individual isomers. As the properties of these isomers are similar, they are
difficult to separate by traditional physical or chemical means. However, the use of
lipases has proved effective in separating CLA isomers. Lipases obtained fromGeo-
trichum candidumhave been shown to be particularly useful (McNeill et al., 1999;
Haas et al., 1999; Chen and Sih, 1998), especially the B isoform (Haas et al., 1999),
although the use of lipase fromAspergillus nigerhas also been reported (Chen and
Sih, 1998).G. candidumB lipase has a very unusual selectivity as it shows an ex-
tremely high preference for fatty acids containing acis-D9 bond (see above). This
enzyme has been used in both hydrolysis and esterification reactions to generate
products containing>90 % of thec-9,t-11 isomer (based on total CLA content)
(see Table 3). The hydrolysis reactions have typically used CLA methyl esters as
substrate, whereas the esterification reactions are based on the use of primary ali-
phatic alcohols (e.g., methanol, ethanol, octanol, dodecanol).Geotrichum candidum
lipase has been used for the selective esterification of CLA with dodecanol on the
multi-kilo pilot plant scale (McNeill et al., 1999).
The enrichment process is shown schematically in Figure 2, and yielded CLA
fractions containing 92 %c-9,t-11 isomer and 81 %t-10,c-12 isomer based on
total CLA content. If required the CLA fractions can be incorporated into triglycer-
ides by interesterification using a non-selective lipase such as that obtained from
Rhizomucor miehei.
Plant lipases with unusual selectivities
Compared with microbial or mammalian lipases, only limited information has been
published on the fatty acid selectivity of plant lipases (Mukherjee and Hills, 1994).
However, the data that are available suggest that some lipases derived from germi-
1.2 Free fatty acids 9
Table 3.Enrichment ofc-9,t-11 CLA isomer by hydrolysis of mixed CLA methyl esters (adapted from
Haas et al., 1999).
Hydrolysis time (h) CLA 9,11 in ester fraction (%) CLA 9,11 in free fatty acid fraction (%)
G. candidum(GC-4)G. candidumB G. candidum(GC-4) G. candidumB0343400
4 17 8 77 94