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 215that engenders a propensity to form stable covalent homodimers
(Morgan et al. 1999b). From this point, it is thought that
polymerisation occursviahydrophobic interactions between
unfolded homodimers and modified monomers (Morgan et al.
1999b). Experiments undertaken by Bouhallab et al. (1999)
showed that this final polymerisation process was not due
to intermolecular cross-linking via the Maillard reaction, but
did confirm that increased solubility of the protein at high
temperatures (65–90◦C) was associated with polymerisation,
presumably invoking disulfide linkages. Some recent data, how-
ever, suggests that Maillard cross-links may also be important in
the polymerisation process (Chevalier et al. 2001). Interestingly,
Pellegrino et al. have observed that an increase in pentosidine
formation coincided with heat-induced covalent aggregation
ofβ-casein, which lacks cysteine, but could not account for
the extent of aggregation observed, suggesting the presence of
other cross-links that remain undefined (Pellegrino et al. 1999).
The glycation approach has also been also employed as a tool
to improve gelling properties of dried egg white (Handa and
Kuroda 1999, Matsudomi et al. 2002), a protein which is used in
the preparation of surimi and meat products (Weerasinghe et al.
1996, Chen 2000, Hunt et al. 2009). Some have suggested that
the polymerisation may occur via protein cross-links formed as
a result of the Maillard reaction, but disulfide bonds may still
play some role (Handa and Kuroda 1999); the exact mechanism
remains undefined (Matsudomi et al. 2002).
The effect of the Maillard reaction and particularly pro-
tein cross-linking on food texture has received some attention
(Gerrard et al. 2002a). Introduction of protein cross-links into
baked products has been shown to improve a number of proper-
ties that are valued by the consumer (Gerrard et al. 1998b, 2000).
In situ studies revealed that following addition of glutaraldehyde
to dough, albumin and globulin fraction of the extracted wheat
proteins were cross-linked (Gerrard et al. 2003a). Inclusion of
glutaraldehyde during bread preparation resulted in the forma-
tion of a dough with an increased dough relaxation time, relative
to the commonly used flour improver ascorbic acid (Gerrard
et al. 2003b). These results confirm that chemical cross-links
are important in the process of dough development and suggest
that they can be introduced via Maillard-type chemistry.
The Maillard reaction has also been used to modify properties
in tofu. Kaye et al. (2001) reported that following incubation with
glucose, a Maillard network formed within the internal structure
of tofu, resulting in a loss in tofu solubility and a reduction in
tofu weight loss. Furthermore, Kwan and Easa (2003) employed
low levels of glucose for the preparation of retort tofu, which re-
sulted in the production of firmer tofu product. Changes in tofu
structure have also been observed in our laboratory when in-
cluding glutaraldehyde, glyceraldehyde or formaldehyde in tofu
preparation (Yasir et al. 2007a, 2007b). However, these results
suggested that protein cross-linking agents may change the func-
tional properties of tofu via non-cross-linking modifications of
the side chains of the amino acid residues, perhaps by changing
their isoelectric point and gelation properties.
Caillard et al. (2008) have also observed interesting results
with soy protein that forms hydrogels in the presence of the
cross-linking agents glutaraldehyde and glyceraldehyde. Theirstudies found that glutaraldehyde was the more efficient cross-
linker, because it was able to form stronger hydrogels with mod-
ifiable properties than glyceraldehyde. More recently, Caillard
et al. (2009) investigated the photophysical and microstructural
properties of soy protein hydrogels cross-linked with glutaralde-
hyde in the absence or presence of salts.
The covalent polymerisation of milk during food processing
has been reported (Singh and Latham 1993). This phenomenon
was shown to be sugar dependent, as determined in model studies
withβ-casein. Further, pentosidine formation paralleled protein
aggregation over time at 70◦C (Pellegrino et al. 1999).
The Maillard reaction has been studied under dry conditions
to gain an understanding of the details of the chemistry (Kato
et al. 1988, French et al. 2002, Oliver et al. 2006). Kato et al.
(1986b) reported the formation of protein polymers following
incubation of galactose or talose with ovalbumin under desic-
cating conditions: an increase in polymerisation, relative to the
protein only control, was observed. This study was extended us-
ing the milk sugar lactose, as milk can often be freeze dried for
shipping and storage purposes. In this study, it was shown, in a
dry reaction mixture, that ovalbumin was polymerised following
incubation with lactose and glucose (Kato et al. 1988). In model
studies with the milk proteinβ-lactoglobulin, the formation of
protein dimers has been observed on incubation with lactose
(French et al. 2002).
Proof of principle has thus been obtained in several systems,
demonstrating that reactive cross-linking molecules are able to
cross-link food proteins within the food matrix and lead to a no-
ticeable change in the functional properties of the food. However,
much work remains to be done in order to generate sufficient
quantities of cross-linking intermediates during food processing
to achieve a controlled change in functionality.Enzymatic MethodsThe use of enzymes to modify the functional properties of foods
is an area that has attracted considerable interest, since con-
sumers perceive enzymes to be more “natural” than chemicals.
Enzymes are also favoured as they require milder conditions,
have high specificity, are only required in catalytic quantities,
and are less likely to produce toxic products (Singh 1991). Thus,
enzymes are becoming commonplace in many industries for im-
proving the functional properties of food proteins (Chobert et al.
1996, Poutanen 1997, Oliver et al. 2006, Hiller and Lorenzen
2009).
Because of the predominance of disulfide cross-linkages in
food systems, enzymes that regulate disulfide interchange reac-
tions are of interest to food researchers. One such enzyme is
protein disulfide isomerase (PDI; Hatahet and Ruddock 2009).
PDI catalyses thiol/disulfide exchange, rearranging ‘incorrect’
disulfide cross-links in a number of proteins of biological in-
terest (Hillson et al. 1984, Singh 1991, Hatahet and Ruddock
2009). The reaction involves the rearrangement of low molecu-
lar sulfhydryl compounds (e.g. glutathione, cysteine and dithio-
threitol) and protein sulfhydryls. It is thought to proceed by the
transient breakage of the protein disulfide bonds by the enzyme
and the reaction of the exposed active cysteine sulfhydryl groups