BLBS102-c10 BLBS102-Simpson March 21, 2012 13:7 Trim: 276mm X 219mm Printer Name: Yet to Come
208 Part 2: Biotechnology and EnzymologyPROTEIN CROSS-LINKS IN FOOD
Protein cross-linking refers to the formation of covalent bonds
between polypeptide chains within a protein (intramolecular
cross-links) or between proteins (intermolecular cross-links)
(Feeney and Whitaker 1988). In biology, cross-links are vital
for maintaining the correct conformation of certain proteins and
may control the degree of flexibility of the polypeptide chains.
As biological tissues age, further protein cross-links may form,
which often have deleterious consequences throughout the body
and play an important role in the many conditions of ageing
(Zarina et al. 2000, Ahmed 2005, Nass et al. 2007, GuI et al.
2009). Chemistry similar to that which occurs during ageing
may take place if biological tissues are removed from their nat-
ural environment – for example, when harvested as food for
processing.
Food processing often involves high temperatures, extremes
in pH, particularly alkaline, and exposure to oxidising conditions
and uncontrolled enzyme chemistry. Such conditions can result
in the introduction of protein cross-links, producing substantial
changes in the structure of proteins, and therefore the functional
(Singh 1991) and nutritional (Friedman 1999a, 1999b, 1999c)
properties of the final product. A summary of protein cross-
linking in foods is given in Figure 10.1, in which the information
is organised according to the amino acids that react to form
the cross-link. Not all amino acids participate in protein cross-
linking, no matter how extreme the processing regime. Those
that react do so with differing degrees of reactivity under various
conditions.Disulfide Cross-linksDisulfide bonds are the most common and well-characterised
types of covalent cross-link in proteins in biology. They are
formed by the oxidative coupling of two cysteine residues that
are close in space within a protein. A suitable oxidant accepts the
hydrogen atoms from the thiol groups of the cysteine residues,
producing disulfide cross-links. The ability of proteins to form
intermolecular disulfide bonds during heat treatment is consid-
ered to be vital for the gelling of some food proteins, including
milk proteins, surimi, soybeans, eggs, meat and some vegetable
proteins (Zayas 1997). Gels are formed through the cross-linking
of protein molecules, generating a three-dimensional solid-like
network, which provides food with desirable texture (Dickinson
1997).
Disulfide bonds are thought to confer an element of thermal
stability to proteins and are invoked, for example, to explain
the stability of hen egg white lysozyme, which has four
intramolecular disulfide cross-links in its native conformation
(Masaki et al. 2001). This heat stability influences many of the
properties of egg white observed during cooking. Similarly, the
heat treatment of milk promotes the controlled interaction of
denaturedβ-lactoglobulin withκ-casein through the formation
of a disulfide bond. This increases the heat stability of milk
and milk products, preventing precipitation ofβ-lactoglobulin
(Singh 1991). Disulfide bonds are also important in the forma-
tion of dough. Disulfide interchange reactions during the mixingof flour and water result in the production of a protein network
with the viscoelastic properties required for bread making
(Lindsay and Skerritt 1999). The textural changes that occur in
meat during cooking have also been attributed to the formation
of intermolecular disulfide bonds (Singh 1991).Cross-links Derived from DehydroproteinAlkali treatment is used in food processing for a number of rea-
sons, such as the removal of toxic constituents and the solubilisa-
tion of proteins for the preparation of texturised products. How-
ever, alkali treatment can also cause reactions that are undesir-
able in foods, and its safety has come into question (Savoie et al.
1991, Shih 1992, Friedman 1999a, 1999c). Exposure to alkaline
conditions, particularly when coupled to thermal processing, in-
duces racemisation of amino acid residues and the formation
of covalent cross-links, such as dehydroalanine, lysinoalanine
and lanthionine (Friedman 1999a, 1999b, 1999c). Dehydroala-
nine is formed from the base-catalysed elimination of persulfide
from an existing disulfide cross-link. The formation of lysinoala-
nine and lanthionone cross-links occurs throughβ-elimination
of cysteine and phosphoserine protein residues, thereby yielding
dehydroprotein residues. Dehydroprotein is very reactive with
various nucleophilic groups, including theε-amino group of ly-
sine residues and the sulfhydryl group of cysteine. In severely
heat- or alkali-treated proteins, imidazole, indole and guanidino
groups of other amino acid residues may also react (Singh 1991).
The resulting intra- and intermolecular cross-links are stable, and
food proteins that have been extensively treated with alkali are
not readily digested, reducing their nutritional value. Mutagenic
products may also be formed (Friedman 1999a, 1999c).Cross-links Derived from TyrosineVarious cross-links formed between two or three tyrosine
residues have been found in native proteins and glycoproteins,
for example in plant cell walls (Singh 1991). Dityrosine cross-
links have recently been identified in wheat and are proposed to
play a role in the formation of the cross-linked protein network
in gluten (Tilley et al. 2001). They have also been formed indi-
rectly by treating proteins with hydrogen peroxide or peroxidase
(Singh 1991) and are implicated in the formation of caseinate
films by gamma irradiation (Mezgheni et al. 1998). Polyphenol
oxidase can also lead indirectly to protein cross-linking, due
to reaction of cysteine, tyrosine, or lysine with reactive benzo-
quinone intermediates generated from the oxidation of phenolic
substrates (Matheis and Whitaker 1987, Feeney and Whitaker
1988). Such plant phenolics have been used to prepare cross-
linked gelatin gels to develop novel food ingredients (Strauss
and Gibson 2004).Cross-links Derived from
the Maillard ReactionThe Maillard reaction is a complex cascade of chemical
reactions, initiated by the deceptively simple condensation of an
amine with a carbonyl group, often within a reducing sugar or fat