234 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking
controlling rheology and stability. These applica-
tions are particularly appealing in view of the fact
that cross-linking by transglutaminase is thought to
protect nutritionally valuable lysine residues in food
from various deteriorative reactions (Seguro et al.
1996). Furthermore, the use of transglutaminase po-
tentially allows production of food proteins of high-
er nutritional quality, through cross-linking of dif-
ferent proteins containing complementary amino
acids (Zhu et al. 1995).
The use of transglutaminase in the dairy industry
has been explored extensively. The enzyme has been
trialed in many cheeses, from Gouda to Quark, and
the use of transglutaminase in ice cream is reported
to yield a product that is less icy and more easily
scooped (Kuraishi et al. 2001). Milk proteins form
emulsion gels, which are stabilized by cross-linking,
opening new opportunities for protein-based spreads,
desserts, and dressings (Dickinson and Yamamoto
1996). The use of transglutaminase has been ex-
plored in an effort to improve the functionality of
whey proteins (Truong et al. 2004); for example,
recent developments include the use of transglutam-
inase to incorporate whey protein into cheese (Coz-
zolino et al. 2003).
Soy products have also benefited from the intro-
duction of transglutaminase, with the enzyme pro-
viding manufacturers with a greater degree of tex-
ture control. The enzyme is reported to enhance the
quality of tofu made from old crops, giving a prod-
uct with increased water-holding capacity, a good
consistency, a silky and firmer texture, and a texture
that is more robust in the face of temperature change
(Kuraishi et al. 2001, Soeda 2003). Transglutam-
inase has also been used to incorporate soy protein
into new products, such as chicken sausages
(Muguruma et al. 2003).
New foods are being created using transglutami-
nase; for example, imitation shark fin for the South-
east Asian market has been generated by cross-
linking gelatin and collagen (Zhu et al. 1995).
Cross-linked proteins have also been tested as fat
substitutes in products such as salami and yogurt
(Nielson 1995), and the use of transglutaminase-
cross-linked protein films as edible films has been
patented (Nielson 1995).
Not surprisingly, the rate of cross-linking by trans-
glutaminase depends on the particular structure of
the protein acting as substrate. Most efficient cross-
linking occurs in proteins that contain a glutamine
residue in a flexible region of the protein, or within a
reverse turn (Dickinson 1997). Casein is a very good
substrate, but globular proteins such as ovalbumin
and -lactoglobulin are poor substrates (Dickinson
1997). Denaturation of proteins increases their reac-
tivity, as does chemical modification by disruption
of disulfide bonds, or by adsorption at an oil-water
interface (Dickinson 1997). Many of the reported
substrates of transglutaminase have actually been
acetylated and/or denatured with reagents such as
dithiothreitol under regimes that are not food ap-
proved (Nielson 1995). More work is needed to find
ways to modify certain proteins in a food-allowed
manner, to render them amenable to cross-linking
by transglutaminase in a commercial setting.
While the applications of transglutaminase have
been extensively reported in the scientific and patent
literature, the precise mode of action of the enzyme
in any one food-processing situation remains re-
latively unexplored. The specificity of the enzyme
suggests that in mixtures of food proteins, certain
proteins will react more efficiently than others, and
there is value in understanding precisely which pro-
tein modifications exert the most desirable effects.
Additionally, transglutaminase has more than one
activity: as well as cross-linking, the enzyme may
catalyze the incorporation of free amines into pro-
teins by attachment to a glutamine residue. Further-
more, in the absence of free amine, water becomes
the acyl acceptor, and the -carboxamide groups are
deamidated to glutamic acid residues (Ando et al.
1989). The extent of these side reactions in foods
and the consequences of any deamidation to the
functionality of food proteins have yet to be fully
explored.
Perhaps the most advanced understanding of the
specific molecular effects of transglutaminase in a
food product is seen in yogurt, where the treated
product has been analyzed by gel electrophoresis,
and specific functional effects have been correlated
to the loss of -casein, with the -casein remaining.
The specificity of the reaction was found to alter
according to the exact transglutaminase source
(Kuraishi et al. 2001). The specific effects of transg-
lutaminase in baked goods (Gerrard et al. 1998,
2000) have also been analyzed at a molecular level.
In particular, the enzyme produced a dramatic
increase in the volume of croissants and puff pas-
tries, with desirable flakiness and crumb texture
(Gerrard et al. 2001). These effects were later corre-