BLBS102-c10 BLBS102-Simpson March 21, 2012 13:7 Trim: 276mm X 219mm Printer Name: Yet to Come
214 Part 2: Biotechnology and Enzymologyγ-carboxyamide group of peptide-bound glutamine residues
and various primary amines. As represented in Figure 10.1,
theε-amino groups of lysine residues in proteins can act as
the primary amine, yielding inter- and intramolecularε-N-(γ-
glutamyl)lysine cross-links (Zhu et al. 1995, Motoki and Seguro
1998, Yokoyama et al. 2004, Jaros et al. 2006,Ozrenk 2006). ̈
The formation of this cross-link does not reduce the nutritional
quality of the food as the lysine residue remains available for
digestion (Seguro et al. 1996).
Transglutaminase is widely distributed in most animal tissues
and body fluids and is involved in biological processes such
as blood clotting and wound healing.ε-N-(γ-glutamyl)lysine
cross-links can also be produced by severe heating (Motoki and
Seguro 1998), but are most widely found where a food is pro-
cessed from material that contains naturally high levels of the
enzyme. The classic example here is the gelation of fish muscle
in the formation of surumi products, a natural part of traditional
food processing of fish by the Japanese suwari process, although
the precise role of endogenous transglutaminase in this process is
still under debate (An et al. 1996, Motoki and Seguro 1998) and
an area of active research (Benjakul et al. 2004a, 2004b).ε-N-(γ-
Glutamyl)lysine bonds have been found in various raw foods in-
cluding meat, fish and shellfish. Transglutaminase-cross-linked
proteins have thus long been ingested by man (Seguro et al.
1996). The increasing applications of artificially adding this en-
zyme to a wide range of processed foods, along with safety
aspects, are discussed in detail below.Other Isopeptide BondsIn foods of low carbohydrate content, where Maillard chemistry
is inaccessible, severe heat treatment can result in the formation
of isopeptide cross-links during food processing, via condensa-
tion of theε-amino group of lysine, with the amide group of an
asparagine or glutamine residue (Singh 1991). This chemistry
has not been widely studied in the context of food.MANIPULATING PROTEIN
CROSS-LINKING DURING FOOD
PROCESSINGA major task of modern food technology is to generate new
food structures with characteristics that please the consumer,
using only a limited range of ingredients. Proteins are one of the
main classes of molecule available to confer textural attributes,
and the cross-linking and aggregation of protein molecules is
an important mechanism for engineering food structures with
desirable mechanical properties (Dickinson 1997, Oliver et al.
2006). The cross-linking of food proteins can influence many
properties of food, including texture, viscosity, solubility, emul-
sification and gelling properties (Kuraishi et al. 2000, Motoki
and Kumazawa 2000, Oliver et al. 2006). Many traditional food
textures are derived from a protein gel, including those of yo-
ghurt, cheese, sausage, tofu and surimi. Cross-linking provides
an opportunity to create gel structures from protein solutions,
dispersions, colloidal systems, protein-coated emulsion dropletsor protein-coated gas bubbles and create new types of food or
improve the properties of traditional ones (Dickinson 1997). In
addition, judicious choice of starting proteins for cross-linking
can produce food proteins of higher nutritional quality through
cross-linking of different proteins containing complementary
amino acids (Kuraishi et al. 2000).Chemical MethodsAn increasing understanding of the chemistry of protein cross-
linking opens up opportunities to control these processes dur-
ing food processing. Many commercial cross-linking agents are
available, for example from Pierce (2001). These are usually
double-headed reagents developed from molecules that deriva-
tise 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 been cast as to the accuracy with which reactivity of these
reagents can be predicted (Green et al. 2001) but they remain
widely used for biochemical and biotechnological applications.
Unfortunately, these reagents are expensive and not often ap-
proved for food use, so their use has not been widely explored
(Singh 1991). They do, however, prove useful for ‘proof of
principle’ studies to measure the possible effects of introduc-
ing specific new cross-links into food. If an improvement in
functional properties is seen after treatment with a commercial
cross-linking agent, then further research effort is merited to
find a food approved, cost-effective means by which to intro-
duce 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. 2002, 2003a,
2003b). The cross-linking of hen egg white lysozyme with a
double-headed reagent has also been used to show that the pro-
tein is rendered 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, particularly in the milk processing industry, some
have harnessed the Maillard reaction to produce more stable
proteins following incubation with monosaccharide (Aoki et al.
1999, Bouhallab et al. 1999, 2001, Handa and Kuroda 1999,
Morgan et al. 1999a, Shepherd et al. 2000, Chevalier et al.
2001, Matsudomi et al. 2002). Increases in protein stability at
high temperatures and improved emulsifying activity have been
observed (Aoki et al. 1999, Bouhallab et al. 1999, Shepherd et al.
2000), and dimerisation and oligomerisation of these proteins
has been noted (Aoki et al. 1999, Bouhallab et al. 1999, Morgan
et al. 1999a, Pellegrino et al. 1999, Chevalier et al. 2001, French
et al. 2002). Inβ-lactoglobulin, it has been suggested that
oligomerisation is initiated by glycation of aβ-lactoglobulin
monomer, resulting in a conformational change in this protein