9 Protein Cross-linking in Food 229Melanoidins can have molecular weights of up to
100 kDa (Hofmann 1998a). Due to their sheer chem-
ical complexity, it has been difficult to isolate and
characterize these molecules (Fayle and Gerrard
2002). However, some recent work in this area has
led to some new hypotheses. Hofmann proposed
that proteins may play an important role in the for-
mation of these complex, high molecular weight
melanoidins (Hofmann 1998b). Thus, it was pro-
posed that a low molecular weight carbohydrate-
derived colorant reacts with protein-bound lysine
and/or arginine, forming a protein cross-link. Form-
ation of color following polymerization of protein
has indeed been observed following incubation with
carbohydrates (Cho et al. 1984, 1986a,b; Okitani et
al. 1984). Hofmann tested this hypothesis by reac-
tion of casein with the pentose-derived intermediate
furan-2-carboxaldehyde and subsequent isolation of
a melanoidin type colorant compound from this
reaction mixture (Fig. 9.3). Although non-cross-
linking in nature, the results encouraged further
studies to isolate protein cross-links that are mela-
noidins, and possibly deconvolute the chemistry of
melanoidin from these data. Further studies by
Hofmann in the same year proposed a cross-link
structure BISARG, which was formed on reaction of
N-protected arginine with glyoxal and furan-2-
carboxaldehyde (Hofmann 1998a) (Fig. 9.3). This
cross-link was proposed as plausible in food sys-
tems due to the large amount of protein furan-2-
carboxaldehyde and glyoxal that may be present in
food (Hofmann 1998a).
A lysine-lysine radical cation cross-link, CROS-
SPY (Fig. 9.3C), has been isolated from a model
protein cross-link system involving bovine serum
albumin and glycoladehyde, followed by thermal
treatment (Hofmann et al. 1999). CROSSPY was
also shown to form from glyoxal, but only in the
presence of ascorbic acid, supporting the suggestion
that reductones are able to initiate radical cation
mechanisms resulting in cross-links (Hofmann et al.
1999). Electron paramagnetic resonance (EPR) spec-
troscopy of dark-colored bread crust revealed results
that suggested CROSSPY was most likely as-
sociated with the browning bread crust (Hofmann et
al. 1999). Encouragingly, when browning was inhib-
ited, radical formation was completely blocked.
A model system containing propylamine and glu-
cose was found to yield a yellow cross-link product
under food processing conditions (Fig. 9.3D) (Knerr
et al. 2001); however, this product is still to be iso-
lated from foodstuffs. In another model study under-
taken by Lerche et al., reacting butylaminammoni-
um acetate (a protein-bound lysine mimic) with
glucose resulted in the formation of a yellow prod-
uct (Fig. 9.3E); this molecule is also still to be iso-
lated from foodstuffs (Lerche et al. 2003).
The hypothesis that formation of melanoidins
involves reaction of protein with carbohydrate is
supported by Brands et al., who noted that following
dialysis of a casein-sugar (glucose or fructose) sys-
tem with 12 kDa cutoff tubing, around 70% of the
brown colored products were present in the retentate
(Brands et al. 2002).Maillard-Related Cross-linksAlthough not strictly classified under the heading
of Maillard chemistry, animal tissues such as colla-
gen and elastin contain complex heterocyclic cross-
links, formed from the apparently spontaneous re-
action of lysine and derivatives with allysine, an
aldehyde formed from the oxidative deamination of
lysine catalyzed by the enzyme lysine oxidase (Feen-
ey and Whitaker 1988). A selection of cross-links
that form from this reaction is outlined in Figure 9.4.
The extent to which these cross-links occur in food
has not been well studied, although their presence in
gelatin has been discussed, along with the presence
of pentosidine in these systems (Cole and Roberts
1996, Cole and Roberts 1997).CROSS-LINKSFORMED VIA
TRANSGLUTAMINASECATALYSISAn enzyme that has received extensive recent atten-
tion for its ability to cross-link proteins is transglut-
aminase. Transglutaminase catalyzes the acyl-trans-
fer reaction between the -carboxyamide group of
peptide-bound glutamine residues and various pri-
mary amines. As represented in Figure 9.1, the -
amino groups of lysine residues in proteins can act
as the primary amine, yielding inter- and intramo-
lecular -N-(-glutamyl)lysine cross-links (Motoki
and Seguro 1998). 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