9 Protein Cross-linking in Food 233dithiothreitol) and protein sulfhydryls. It is thought
to proceed by transient breakage of the protein di-
sulfide bonds by the enzyme, and reaction of the
exposed active cysteine sulfhydryl groups with other
appropriate residues to reform native linkages
(Singh 1991).
PDI has been found in most vertebrate tissues and
in peas, cabbage, yeast, wheat, and meat (Singh
1991). It has been shown to catalyze the formation
of disulfide bonds in gluten proteins synthesized in
vitro. Early reports suggested that a high level of
activity corresponded to a low bread-making quality
(Grynberg et al. 1977). The use of oxidoreduction
enzymes, such as PDI, to improve product quality is
an area of interest to the baking industry (van Oort
2000, Watanabe et al. 1998) and the food industry in
general (Hjort 2000). The potential of enzymes such
as protein disulfide isomerase to catalyze their inter-
change has been extensively reviewed by Shewry
and Tatham (1997).
Sulfhydryl (or thiol) oxidase catalyzes the oxida-
tive formation of disulfide bonds from sulfhydryl
groups and oxygen and occurs in milk (Matheis and
Whitaker 1988, Singh 1991). Immobilized sulfhy-
dryl oxidase has been used to eliminate the “cooked”
flavor of ultra high temperature treated milk (Swais-
good 1980). It is not a well-studied enzyme, although
the enzyme from chicken egg white has received
recent attention in biology (Hoober 1999). Protein
disulfide reductase catalyzes a further sulfhydryl-
disulfide interchange reaction and has been found in
liver, pea, and yeast (Singh 1991).
Peroxidase, lipoxygenase, and catechol oxidase
occur in various plant foods and are implicated in
the deterioration of foods during processing and
storage. They have been shown to cross-link several
food proteins, including bovine serum albumin,
casein, -lactoglobulin, and soy, although the un-
controlled nature of these reactions casts doubt on
their potential for food improvement (Singh 1991).
Lipoxygenase, in soy flour, is used in the baking
industry to improve dough properties and baking
performance. It acts on unsaturated fatty acids,
yielding peroxy free radicals and starting a chain
reaction. The cross-linking action of lipoxygenase
has been attributed to both the free radical oxidation
of free thiol groups to form disulfide bonds and to
the generation of reactive cross-linking molecules
such as malondialdehyde (Matheis and Whitaker
1987).
All enzymes discussed so far have been overshad-
owed in recent years by the explosion in research on
the enzyme transglutaminase. Due largely to its abil-
ity to induce the gelation of protein solutions, trans-
glutaminase has been investigated for uses in a
diverse range of foods and food-related products.
The use of this enzyme has been the subject of a
series of recent reviews, covering both the scientific
and patent literature (Nielsen 1995, Zhu et al. 1995,
Motoki and Seguro 1998, Kuraishi et al. 2001).
These are briefly highlighted below.TRANSGLUTAMINASEThe potential of transglutaminase in food processing
was hailed for many years before a practical source
of the enzyme became widely available. The pro-
duction of a microbially derived enzyme by Ajino-
moto, Inc., proved pivotal in paving the way for
industrial applications (Motoki and Seguro 1998). In
addition, the transglutaminases that were discovered
in the early years were calcium ion dependent,
which imposed a barrier for their use in foods that
did not contain a sufficient level of calcium. The
commercial preparation is not calcium dependent,
and thus finds much wider applicability (Motoki and
Seguro 1998). The production of microbial transglu-
taminase, derived from Streptoverticillium mobara-
ense,is described by Zhu (1995), and methods with
which to purify and assay the enzyme are reviewed
by Wilhelm (1996). The commercial enzyme oper-
ates effectively over the pH range 4–9, from 0–50°C
(Motoki and Seguro 1998).
There is a seemingly endless list of foods in
which the use of transglutaminase has been success-
fully used (De Jong and Koppelman 2002): seafood,
surimi, meat, dairy, baked goods, sausages (as a po-
tential replacement for phosphates and other salts),
gelatin (Kuraishi et al. 2001), and noodles and pasta
(Larre et al. 1998, 2000). It is finding increasing use
in restructured products, such as those derived from
scallops and pork (Kuraishi et al. 1997). In all cases,
transglutaminase is reported to improve firmness,
elasticity, water-holding capacity, and heat stability
(Kuraishi et al. 2001). It also has potential to allevi-
ate the allergenicity of some proteins (Watanabe et
al. 1994). Dickinson (1997) reviewed the applica-
tion of transglutaminase to cross-link different kinds
of colloidal structures in food and enhance their
solid-like character in gelled and emulsified systems,