368 Part III: Muscle Foods
hydrophobic or hydrophilic sites of the protein mol-
ecules interact with each other to form hydrophobic-
hydrophobic and hydrophilic-hydrophilic bonds,
either within the protein molecules, resulting in
deconformation of the native three-dimensional
structure of protein, or between adjacent protein mo-
lecules, causing protein-protein interactions and re-
sulting in aggregation (Shenouda 1980). Xiong
(1997) proposed that protein aggregates to maintain
its lowest free energy as water forms ice, thus result-
ing in protein denaturation. Matsumoto (1979) sug-
gested that redistribution of water during freezing
allows protein molecules to move closer together
and aggregate through intermolecular interactions.
Lim and Haard (1984) found that the loss of protein
solubility as a result of protein denaturation in
Greenland halibut during frozen storage was mostly
due to the noncovalent, hydrophobic interactions in
protein molecules. Buttkus (1970) proposed that the
formation of intermolecular S-S bonds is the major
cause of protein denaturation.
Solute Concentration Effect
As ice forms, the concentration of mineral salts and
soluble organic substances in the unfrozen matrix
increases. As a result, salts and other compounds
that are only slightly soluble (such as phosphate) may
precipitate out, which will change the pH (Einen et
al. 2002) and ionic strength of the unfrozen matrix
and cause conformational changes in proteins. Ions
in the concentrated matrix will compete with the
existing electrostatic bonds and cause the break-
down of some of the electrostatic bonds (Dyer and
Dingle 1961, Shenouda 1980). Takahashi et al.
(1993) found, in their freeze denaturation study of
carp myofibrils with KCl or NaCl, that freeze denat-
uration above 13°C is caused by the concentrated
salt solution.
Reaction of Protein with Intact Lipids
There are different views in the literature on the
effect of intact lipids (i.e., lipids that have not been
subjected to partial or total hydrolysis or oxidation)
on fish proteins. On one hand, they seem to pro-
tect proteins; on the other, they form lipoprotein
complexes, which affect protein properties (She-
nouda 1980, Mackie 1993). Dyer and Dingle (1961)
found that lean fish (fat content less than 1%)
showed a rapid decrease in protein (actomyosin)
extractability when compared with fatty fish species
(3–10 % lipids). They therefore hypothesized that
moderate levels of lipids may reduce protein denatu-
ration during frozen storage. In contrast, Shenouda
and Piggot (1974) observed a detrimental effect of
intact lipids on protein denaturation in their study
of a model system, which involved incubating lipid
and protein extracted from the same fish at 4°C
overnight. They showed that when fish actin (G-
form) was incubated with fish polar or neutral lipids,
high molecular weight protein aggregates formed.
They suggested that during freezing, lipid and pro-
tein components form lipoprotein complexes, which
change the textural quality of muscle tissue.Reaction of Proteins with Oxidized LipidsDuring frozen storage, lipid oxidation products
cause proteins to become insoluble and harder (Ta-
kama 1974). When proteins are exposed to peroxi-
dized lipids, peroxidized lipid–protein complexes
will form through hydrophobic interactions or hy-
drogen bonds (Narayan et al. 1964), thus causing
conformational changes in the protein. The unstable
free radical intermediates of lipid peroxidation re-
move hydrogen from protein, forming a protein rad-
ical, which could initiate various reactions such as
cross-linking with other proteins or lipids and for-
mation of protein-protein and protein-lipid aggre-
gates (Karel et al. 1975, Schaich and Karel 1975,
Gardner 1979). Roubal and Tappel (1966) found
that peroxidized protein cross-links into a range of
oligomers, which are associated with protein insolu-
bility. Careche and Tejada (1994) found that oleic
and myristic acid had a detrimental effect on the
ATPase activity, protein solubility, and viscosity of
Hake muscle during frozen storage.
Secondary products from lipid oxidation such as
aldehydes react chemically with the amino groups
of proteins through the formation of Shiff base
adducts, which fluoresce (Leake and Karel 1985,
Kikugawa et al. 1989). Ang and Hultin (1989) sug-
gested that formaldehyde might interact with pro-
tein side chains and form aggregates without caus-
ing cross-linking.LIPIDOXIDATION ANDHYDROLYSISLipids degrade by two mechanisms: hydrolysis (li-
polysis) and oxidation (Shenouda 1980, Shewfelt