9 Protein Cross-linking in Food 231the cross-linking and aggregation of protein mole-
cules is an important mechanism for engineering
food structures with desirable mechanical properties
(Dickinson 1997). The cross-linking of food pro-
teins can influence many properties of food, includ-
ing texture, viscosity, solubility, emulsification, and
gelling properties (Motoki and Kumazawa 2000,
Kuraishi et al. 2000). Many traditional food textures
are derived from a protein gel, including those of yo-
gurt, cheese, sausage, tofu, and surimi. Cross-link-
ing provides an opportunity to create gel structures
from protein solutions, dispersions, colloidal sys-
tems, protein-coated emulsion droplets, or protein-
coated gas bubbles, and create new types of food or
improve the properties of traditional ones (Dick-
inson 1997). In addition, the judicious choice of
starting proteins for cross-linking can produce food
proteins of higher nutritional quality through cross-
linking of different proteins containing complemen-
tary amino acids (Kuraishi et al. 2000).
CHEMICALMETHODS
An increasing understanding of the chemistry of
protein cross-linking opens up opportunities to con-
trol these processes during food processing. Many
commercial cross-linking agents are available, for ex-
ample, from Pierce (2001). These are usually double-
headed reagents developed from molecules that
derivatize the side chains of proteins (Matheis and
Whitaker 1987, Feeney and Whitaker 1988) and
generally exploit the lysine and/or cysteine residues
of proteins in a specific manner. Doubt has recently
been cast on the accuracy with which reactivity of
these reagents can be predicted (Green 2001), but
they remain widely used for biochemical and bio-
technological applications.
Unfortunately, these reagents are expensive and
not often approved for food use, so their use has not
been widely explored (Singh 1991). They do, how-
ever, prove useful for “proof of principle” studies to
measure the possible effects of introducing spe-
cific new cross-links into food. If an improvement in
functional properties is seen after treatment with a
commercial cross-linking agent, then further re-
search effort is merited to find a food-approved, cost
effective means by which to introduce such cross-
links on a commercial scale. Such proof of principle
studies include the use of glutaraldehyde to demon-
strate the potential effects of controlled Maillard
cross-linking on the texture of wheat-based foods
(Gerrard et al. 2002a,b,c). The cross-linking of hen
egg white lysozyme with a double-headed reagent
has also been used to show that the protein is ren-
dered more stable to heat and enzyme digestion,
with the foaming and emulsifying capacity reduced
(Matheis and Whitaker 1987, Feeney and Whitaker
1988). Similarly, milk proteins cross-linked with
formaldehyde showed greater heat stability (Singh
1991).
Food preparation for consumption often involves
heating, which can result in a deterioration of the
functional properties of these proteins (Bouhallab et
al. 1999, Morgan et al. 1999a, Shepherd et al. 2000).
In an effort to protect them from denaturation, par-
ticularly in the milk processing industry, some have
harnessed the Maillard reaction to produce more
stable proteins following incubation with monosac-
charide (Aoki et al. 1999, 2001; Bouhallab et al.
1999; Chevalier et al. 2002; Handa and Kuroda
1999; Matsudomi et al. 2002; Morgan et al. 1999a;
Shepherd et al. 2000). Although increases in protein
stability at high temperatures and improved emulsi-
fying activity have been observed (Aoki et al. 1999,
Bouhallab et al. 1999, Shepherd et al. 2000), dimer-
ization and oligomerization of these proteins has
also been noted (French et al. 2002, Aoki et al. 1999,
Bouhallab et al. 1999, Chevalier et al. 2002, French
et al. 2002, Morgan et al. 1999a, Pellegrino et al.
1999). In -lactoglobulin, it has been suggested that
oligomerization is initiated by glycation of a -
lactoglobulin monomer, resulting in a conformation-
al change in this protein that engenders a propensity
to form stable covalent homodimers (Morgan et al.
1999b). From this point, it is thought that polymer-
ization occurs via hydrophobic interactions between
unfolded homodimers and modified monomers (Mor-
gan et al. 1999b). Experiments undertaken by
Bouhallab et al. showed that this final polymeriza-
tion process was not due to intermolecular cross-
linking via the Maillard reaction, but did confirm
that increased solubility of the protein at high tem-
peratures (65–90°C) was associated with polymer-
ization (Bouhallab et al. 1999), presumably invok-
ing disulfide linkages. Some recent data, however,
suggest that Maillard cross-links may after all be
important in the polymerization process (Chevalier
et al. 2002). Interestingly, Pellegrino et al. have ob-
served that an increase in pentosidine formation
coincided with heat-induced covalent aggregation of