331
2008), and production of invert sugar syrup with invertase (Rebroš et al. 2007).
Flavourzyme, (a fungal protease/peptidase complex) entrapped in calcium alginate
(Anjani et al. 2007), k-carragenan, gellan, and higher melting-fat fraction of milk fat
(Kailasapathy and Lam 2005), was effectively used in cheese ripening, in order to
speed up the process, while avoiding the problems associated with the use of free
enzyme. These include deficient enzyme distribution, reduced yield and poorquality cheese, partly ascribed to excessive proteolysis and whey contamination
(Anjani et al. 2007). Calcium alginate beads were also used to immobilize glucose
isomerase (Tumturk et al. 2008) and α-amylase for starch hydrolysis to whey
(Rajagopalan and Krishnan 2008).
In a particularly favored technique immobilization of enzymes in liposomes,
known as dehydration-rehydration vesicles (DRVs), small (diameters usually below
50 nm) unilamellar vesicles (SUVs) is prepared in distilled water and mixed with an
aqueous solution of the enzyme to be encapsulated. The resulting vesicle suspension is then dehydrated under freeze drying or equivalent method. Upon rehydration, the resulting DRVs are multilamellar and larger (from 200 nm to a little above
1000 nm) than the original SUVs, and can capture solute molecules (Walde and
Ichikawa 2001;Grosová et al. 2008). Recent work in this particular application has
used lactase as enzyme model and has focused on the optimization and characterization of the liposome-based immobilized system (Rodríguez-Nogales and López
2006). Cocktails of enzymes, namely, Flavourzyme, bacterial proteases and Palatase
M (a commercial lipase preparation), were immobilized in liposomes and successfully used to speed up cheddar cheese ripening (Kheadr et al. 2003). Encapsulation
in lipid vesicles has been proved a mild method, providing high protection against
proteolysis.
Binding to a solid carrier, where enzyme-support interaction can be of covalent,
ionic, or physical nature. Curiously, the first reported application of enzyme immobilization was of invertase onto activated charcoal (Nelson and Griffin 1916).
Another example is the immobilization of pectinase in egg shell for the preparation
of low-methoxyl pectin. The immobilized biocatalyst could be reused for 32 times
at 30 °C, and it was used in a fluidized-bed reactor, operated at an optimum flow rate
of 5 mL h
−1
and 35 °C (Nighojkar et al. 1995). Other examples are the surface
immobilizations of α-amylase on alumina (Reshmi et al. 2006) and in zirconia
(Reshmi et al. 2007).
Carrier-free macroparticles, where a bifunctional reagent (namely, glutaraldehyde), is used to cross-link enzyme aggregates (CLEAs) or crystals (CLECs), leading to a biocatalyst displaying highly concentrated enzyme activity, high stability
and low production costs(Sheldon 2007; Roy and Abraham 2004). The use of
CLEAs is favored given the lower complexity of the process. This approach is
recent, as compared with entrapment and binding to a solid carrier, and there are
still relatively few examples of its application to enzymes used in the area of food
processing (Fernandes 2010) such as the immobilization of lactase for the hydrolysis of lactose, where, under similar operational conditions as for the free enzyme,
the CLEA yielded 78% monosaccharides in 12 h as compared to 3.9% of the free
form (Gaur et al. 2006).
Exogenous Enzymes
2008), and production of invert sugar syrup with invertase (Rebroš et al. 2007).
Flavourzyme, (a fungal protease/peptidase complex) entrapped in calcium alginate
(Anjani et al. 2007), k-carragenan, gellan, and higher melting-fat fraction of milk fat
(Kailasapathy and Lam 2005), was effectively used in cheese ripening, in order to
speed up the process, while avoiding the problems associated with the use of free
enzyme. These include deficient enzyme distribution, reduced yield and poorquality cheese, partly ascribed to excessive proteolysis and whey contamination
(Anjani et al. 2007). Calcium alginate beads were also used to immobilize glucose
isomerase (Tumturk et al. 2008) and α-amylase for starch hydrolysis to whey
(Rajagopalan and Krishnan 2008).
In a particularly favored technique immobilization of enzymes in liposomes,
known as dehydration-rehydration vesicles (DRVs), small (diameters usually below
50 nm) unilamellar vesicles (SUVs) is prepared in distilled water and mixed with an
aqueous solution of the enzyme to be encapsulated. The resulting vesicle suspension is then dehydrated under freeze drying or equivalent method. Upon rehydration, the resulting DRVs are multilamellar and larger (from 200 nm to a little above
1000 nm) than the original SUVs, and can capture solute molecules (Walde and
Ichikawa 2001;Grosová et al. 2008). Recent work in this particular application has
used lactase as enzyme model and has focused on the optimization and characterization of the liposome-based immobilized system (Rodríguez-Nogales and López
2006). Cocktails of enzymes, namely, Flavourzyme, bacterial proteases and Palatase
M (a commercial lipase preparation), were immobilized in liposomes and successfully used to speed up cheddar cheese ripening (Kheadr et al. 2003). Encapsulation
in lipid vesicles has been proved a mild method, providing high protection against
proteolysis.
Binding to a solid carrier, where enzyme-support interaction can be of covalent,
ionic, or physical nature. Curiously, the first reported application of enzyme immobilization was of invertase onto activated charcoal (Nelson and Griffin 1916).
Another example is the immobilization of pectinase in egg shell for the preparation
of low-methoxyl pectin. The immobilized biocatalyst could be reused for 32 times
at 30 °C, and it was used in a fluidized-bed reactor, operated at an optimum flow rate
of 5 mL h
−1
and 35 °C (Nighojkar et al. 1995). Other examples are the surface
immobilizations of α-amylase on alumina (Reshmi et al. 2006) and in zirconia
(Reshmi et al. 2007).
Carrier-free macroparticles, where a bifunctional reagent (namely, glutaraldehyde), is used to cross-link enzyme aggregates (CLEAs) or crystals (CLECs), leading to a biocatalyst displaying highly concentrated enzyme activity, high stability
and low production costs(Sheldon 2007; Roy and Abraham 2004). The use of
CLEAs is favored given the lower complexity of the process. This approach is
recent, as compared with entrapment and binding to a solid carrier, and there are
still relatively few examples of its application to enzymes used in the area of food
processing (Fernandes 2010) such as the immobilization of lactase for the hydrolysis of lactose, where, under similar operational conditions as for the free enzyme,
the CLEA yielded 78% monosaccharides in 12 h as compared to 3.9% of the free
form (Gaur et al. 2006).
Exogenous Enzymes