Food Biochemistry and Food Processing (2 edition)

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19 Biochemistry of Seafood Processing 357

hand, when the tissue is frozen rapidly, the cellular fluids do not

have enough time to migrate out to the extracellular spaces and

will freeze as small crystals uniformly distributed throughout the

tissue (Mazur 1984). Thus, rapid freezing has a less detrimental

effect on the cell or tissue (Coggins and Chamul 2004). However,

if the storage temperature fluctuates, the intracellular ice recrys-

tallizes, forming large ice crystals and causing cell disruption,

which leads to drip loss as well. Mechanical damage from ice

crystals on the texture of food during freezing and frozen stor-

age has been studied in kiwi fruit (Fuster et al. 1994), lamb meat

(Payne and Young 1995), squid mantle muscle (Ueng and Chow

1998), pork (Ngapo et al. 1999), and beef (Farouk et al. 2003).

During storage, if the product is not wrapped or is improperly

packaged, ice may sublime into the headspace and produce a

product defect called freezer burn (Blond and Le Meste 2004,

Coggins and Chamul 2004). The sublimation of ice crystals

leaves behind small cavities and causes the surface of fish to

appear grayish. It is more pronounced when the storage temper-

ature is high (Blond and Le Meste 2004). Freezer burn increases

the rate of rancidity and discoloration because of the greater

exposed surface, resulting in a “woody” (Blond and Le Meste

2004), tough, and dry texture in fish, making and leaving the fish

less acceptable (Coggins and Chamul 2004).

Dehydration Effect

Native protein is stabilized by the folding of hydrophobic chains

into the protein molecules and is held together by many other

forces, including hydrogen bonding, dipole–dipole interactions,

electrostatic interactions, and disulfite linkages (Benjakul and

Bauer 2000). The formation of ice during freezing removes water

from the protein molecules, thus disrupting the hydrogen bond-

ing network between the native proteins and water molecules

and exposing the hydrophobic or hydrophilic sites of the protein

molecules (Shenouda 1980). The exposed hydrophobic or hy-

drophilic sites of the protein molecules interact with each other

to form hydrophobic–hydrophobic and hydrophilic–hydrophilic

bonds, either within the protein molecules, resulting in decon-

formation of the native three-dimensional structure of protein, or

between adjacent protein molecules, causing protein–protein in-

teractions and resulting in aggregation (Shenouda 1980). Xiong

(1997) proposed that protein aggregates to maintain its lowest

free energy as water forms ice, thus resulting in protein de-

naturation. Matsumoto (1979) suggested 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 inter-

actions 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 breakdown 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 denaturation above− 13 ◦C is caused by the

concentrated salt solution. Effect of freeze-concentration in a

cellular structure was demonstrated in a phospholipid liposomal

model system (Siow et al. 2007).

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 to-

tal hydrolysis or oxidation) on fish proteins. On one hand, they

seem to protect proteins; on the other, they form lipoprotein com-

plexes, which affect protein properties (Shenouda 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). Therefore, they hypothesized that moderate levels of

lipids may reduce protein denaturation 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 protein components

form lipoprotein complexes, which change the textural quality

of muscle tissue.

Reaction of Proteins with Oxidized Lipids

During frozen storage, lipid oxidation products cause proteins to

become insoluble and harder (Takama 1974). When proteins are

exposed to peroxidized lipids, peroxidized lipid–protein com-

plexes will form through hydrophobic interactions or hydro-

gen bonds (Narayan et al. 1964), thus causing conformational

changes in the protein. The unstable free radical intermediates

of lipid peroxidation remove hydrogen from protein, forming

a protein radical, which could initiate various reactions such

as cross-linking with other proteins or lipids and formation of

protein–protein and protein–lipid aggregates (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 insolubility.

Careche and Tejada (1994) found that oleic and myristic acid

had a detrimental effect on the ATPase activity, protein solubil-

ity, 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)