ducts, and exhibit catalytic action under mild conditions. Enzymatic reactions have
another advantage for the synthesis of sTAG containing PUFA because PUFAs are
very unstable. They are prone to be easily isomerized, oxidized and polymerized.
These properties necessitate the use of as mild conditions as possible, especially
oxygen-free conditions.
A monograph dealing with sTAG was published two years ago, which covers most
important aspects of sTAG such as technology, metabolism, medical and clinical
uses as well as of specific food applications (Christophe, 1998). In this chapter,
based on these perspectives, recent advances on the lipase-catalyzed synthesis of
sTAG containing PUFA will be summarized, with a special emphasis of both mon-
itoring the reaction and increasing the yield that we have recently studied (Han et al.,
1999a,b; Iwasaki et al., 1999; Rosu et al., 1999a,b; Han and Yamane, 1999; Iwasaki
and Yamane, 2000).
9.2 Monitoring the reaction
For the production of a targeted sTAG, it is essential to know which types of TAG are
formed, and how many FAs are incorporated at a specific hydroxyl position of gly-
cerol. When the intention is to synthesize two types of diacid sTAG containing
PUFA, i.e. ABA and AAB (or BAA) types (Table 1) where A¼MCFA and B¼
PUFA the ABA and AAB (or BAA) type sTAGs must be separated and determined
by a suitable analytical method. Moreover, when the intention is to produce pure
(EPA)C 8 C 8 (a AAB-type sTAG) by lipase-catalyzed transesterification between
eicosapentaenoic acid ethyl ester (EPAEE) and tricaprylin by the following Equa-
tion 1,
EPAEE + TricaprylinR(EPA)C 8 C 8 +C 8 EE (1)
a number of chemical species may appear during the reaction, including the two
substrates, the targeted sTAG, its positional isomers [C 8 (EPA)C 8 ] and inevitable
byproduct (C 8 EE). TAG containing two or three moles of EPA that may occur
by further replacement of EPAEE, and hydrolytic byproducts that may appear
from any esters. Table 2 lists in the first row almost all possible lipid species
that may appear in the course of the reaction according to Equation (1). It is highly
preferable to detect these species as well as the targeted sTAG. However, separation
of more than 11 chemical species in one analysis is not an easy task, and requires an
advanced analytical technique.
Several methods have been reported for the determination of the positional dis-
tribution of acyl groups in TAG, including enzymatic hydrolysis (Luddy et al., 1963;
Foglia et al., 1995), or chemical degradation using Grignard reagent (Becker et al.,
1993; Ando et al., 1996), followed by analysis of mono- and di-acylglycerol (MAG
and DAG) products by chromatographic techniques,^13 C-NMR (Gunstone, 1991;
Bergana and Lee, 1996), and silver-ion liquid chromatography (Dobson et al., 1995).
150 9 Lipase-Catalyzed Synthesis of Structured Triacylglycerols
