Food Lipids: Chemistry, Nutrition, and Biotechnology

are better because they can partition to the interface. More polar substrates tend to
stay in the water phase and interact less with the interface (76). The disadvantages
of reverse micelle systems are that lipase activity is decreased rapidly, and the system
can alter lipase specificity (73,76,77). Reverse micelles can also be used with immobilized
lipases, where the reverse micelle is formed around the support and immobilized
lipase. This method has been used with hexane to produce cocoa butter
equivalents (73). Although they have been used in experimental form to produce
triacylglycerols from diacylglycerols and oleic acid (78), as well as triacylglycerols
suitable for use as cocoa butter substitutes (74), reverse micelles are not used in
industrial enzymatic interesterification applications.
IV. IMMOBILIZATION
Immobilization of lipases has become increasingly popular for both hydrolysis and
synthesis reactions. The advantages of immobilized enzyme systems compared to
free enzyme systems include reusability, rapid termination of reactions, lowered cost,
controlled product formation, and ease of separation of the enzyme from the reactants
and products. In addition, immobilization of different lipases can affect their selectivity
and chemical and physical properties. Immobilization also provides the possibility
of achieving both purification of the lipase from an impure extract and immobilization
simultaneously, with minimal inactivation of the lipase (79). Methods
for immobilization of enzymes include chemical forms, such as covalent bonding,
and physical forms, such as adsorption and entrapment in a gel matrix or microcapsules
(7,80).
The easiest and most common type of immobilization used in interesterification
reactions is adsorption, which involves contacting an aqueous solution of the lipase
with an organic or inorganic surface-active adsorbent. The objective of immobilization
is to maximize the level of enzyme loading per unit volume of support. The
process of adsorption can be accomplished through ion exchange or through hydrophobic
or hydrophilic interactions and van der Waals interactions (81). After a short
period of mixing of the free enzyme and support, the immobilized enzyme is washed
to remove any free enzyme that is left, after which the product is dried (79). The
same adsorption process can be accomplished by precipitating an aqueous lipase
solution onto the support using acetone, ethanol, or methanol, then drying as previously
described (9,81). Although desorption can occur, most immobilized lipase
preparations are stable in aqueous solutions for several weeks. The preparations are
stable because as the lipase adsorbs to the support, it unfolds slightly, allowing
several points of interaction between the lipase and support. In order for desorption
to occur, simultaneous loss of interactions at all contact sites must occur, which is
unlikely (82).
The degree of immobilization depends on several conditions, including pH,
temperature, solvent type, ionic strength, and protein and adsorbent concentrations.
The choice of carrier is dependent on its mechanical strength, loading capacity, cost,
chemical durability, functionality, and hydrophobic or hydrophilic character (83). In
general, lipases retain the highest degree of activity when immobilized on hydrophobic
supports, where desorption of lipase from the support after immobilization is
negligible, and improved activity has been attributed to increased concentrations of
hydrophobic substrate at the interface (7,50). The disadvantages of using hydrophilic
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