BLBS102-c10 BLBS102-Simpson March 21, 2012 13:7 Trim: 276mm X 219mm Printer Name: Yet to Come
10 Protein Cross-linking in Food – Structure, Applications, Implications for Health and Food Safety 217example recent developments include the use of transglutami-
nase to incorporate whey protein into cheese (Cozzolino et al.
2003, Pereira et al. 2009) and yoghurt (Ozer et al. 2007, Gauche
et al. 2009).
Soy products have also benefited from the introduction of
transglutaminase, with the enzyme providing manufacturers
with a greater degree of texture control. The enzyme is reported
to enhance the quality of tofu made from old crops, giving a
product with increased water-holding capacity, a good consis-
tency, a silky and firmer texture and one that is more robust in the
face of temperature change (Kuraishi et al. 2001, Soeda 2003).
Transglutaminase has also been used to incorporate soy protein
into new products, such as chicken sausages (Muguruma et al.
2003). More recent studies have found that transglutaminase
can potentially reduce the extent of the Maillard reaction and
cross-links (Gan et al. 2009). Soy protein isolate that had been
heated in the presence of transglutaminase to initiate theε-(γ-
glutamyl)lysine bonds formed were then heated in the presence
of ribose to initiate Maillard reaction. It was found that theε-(γ-
glutamyl)lysine bonds formed during incubation of soy protein
isolate with transglutaminase reduced the number of free amino
groups able to take part in the following Maillard reaction with
ribose. Consequently, the use of transglutaminase is most likely
to be most beneficial in products that contain reducing sugars,
where it is advantageous for the Maillard reaction to be limited.
New foods are being created using transglutaminase, for ex-
ample, 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 substi-
tutes in products such as salami and yoghurt (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 transglutaminase
depends on the particular structure of the protein acting as sub-
strate. 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 andβ-lactoglobulin are
poor substrates (Dickinson 1997). Denaturation of proteins in-
creases their reactivity, as does chemical modification by disrup-
tion of disulfide bonds or by adsorption at an oil–water interface
(Dickinson 1997). Many of the reported substrates of transglu-
taminase have actually been acetylated and/or denatured with
reagents such as dithiothreitol under regimes that are not food
approved (Nielson 1995). More work needs to be done to find
ways to modify certain proteins in a food-allowed manner in
order to render them amenable to cross-linking by transglutam-
inase in a commercial setting.
Whilst the applications of transglutaminase have been exten-
sively reported in the scientific and patent literature, the precise
mode of action of the enzyme in any one food processing situa-
tion remains relatively 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 under-
standing precisely which protein modifications exert the most
desirable effects. Additionally, transglutaminase has more than
one activity: as well as cross-linking, the enzyme may catalysethe incorporation of free amines into proteins by attachment to
a glutamine residue. Furthermore, in the absence of free amine,
water becomes the acyl acceptor and theγ-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, has yet
to be fully explored. However, there have been suggestions that
in the case of wheat proteins, these side reactions may lead to
unwanted consequences, specifically associated with the celiac
response (Gerrard and Sutton 2005, Leszczynska et al. 2006,
Cabrera-Chavez et al. 2008), and the use in wheat products is
not advised until further research has been done to investigate
this possibility.
Perhaps the most advanced understanding of the specific
molecular effects of transglutaminase in a food product is seen
in yoghurt, where the treated product has been analysed by gel
electrophoresis and specific functional effects correlated to the
loss ofβ-casein, with theα-casein remaining. The specificity
of the reaction was found to alter according to the exact trans-
glutaminase source (Kuraishi et al. 2001). The specific effects
of transglutaminase in baked goods (Gerrard et al. 1998b, 2000)
have also been analysed at a molecular level. In particular, the en-
zyme produced a dramatic increase in the volume of croissants
and puff pastries, with desirable flakiness and crumb texture
(Gerrard et al. 2001). These effects were later correlated with
cross-linking of the albumins and globulins and high-molecular
weight glutenin fractions by transglutaminase (Gerrard et al.
2001). Subsequent research suggested that the dominant effect
was attributable to cross-linking of the high-molecular weight
glutenins (Gerrard et al. 2002). However, as noted above, the
use of transglutaminase for wheat-based produce is not recom-
mended (Gerrard and Sutton 2005).FUTURE APPLICATIONS OF PROTEIN
CROSS-LINKINGAlthough protein cross-linking is often considered to be
detrimental to the quality of food, it is increasingly clear that
it can also be used as a tool to improve food properties. The
more we understand of the chemistry and biochemistry that
take place during processing, the better placed we are to exploit
it – minimising deleterious reactions and maximising beneficial
ones.
Food chemists are faced with the task of understanding the
vast array of reactions that occur during the preparation of food.
Food often has complex structures and textures, and through
careful manipulation of specific processes that occur during
food preparation, these properties can be enhanced to generate
a highly marketable product (Dickinson 1997). The formation
of a structural network within food is critical for properties such
as food texture. From a biopolymer point of view, this cross-
linking process has been successfully applied in the formation
of protein films, ultimately for use as packaging. Enhancing tex-
tural properties, emulsifying and foaming properties, by protein
cross-linking has been a subject of interest of many working in
this area, as exemplified by the number of studies detailed above.