Denaturation during frozen storage

When 0.1 M sodium glutamate was added to carp actomyosin, denaturation during frozen storage was almost eliminated, as measured by changes in solubility, viscosity, ultracentrifugal behavior, ATPase activity and electron microscopic profiles (66,72) (Figure 3). This protective effect of sodium glutamate will be discussed below. 

The concentrated salt solution may denature the proteins (9-17, 169-177). Whereas experiments with isolated muscle protein preparations cannot exclude the effects of salts such as NaCl or KC1 (since they are required to solubilize the proteins), denaturation during frozen storage has been decreased or prevented completely when an efficient cryoprotectant such as sodium glutamate or glucose was added (66,67,82,93,145-150). Hence, the effect of salts may not be of primary importance, though they may contribute. 

The mechanisms of denaturation during frozen storage and of the cryoprotective effects have been discussed and a hypothetical model has been presented. 

Nevertheless after reviewing the literature, Connell concluded that protein-lipid interactions do not appear to be a major cause of protein denaturation during frozen storage of fish (2). 

Shenouda, S.Y. Theories of protein denaturation during frozen storage of fish flesh. Adv. Food Res. 1980, 26, 275-311. 

Sugar alcohols have also found application in foods containing sugars. Sorbitol is an effective cryoprotectant in surimi, preventing denaturation of the muscle protein during frozen storage. 

Khan, M. A. A., Hossain, M. A., Hara, K., Osatomi, K., Ishihara, T., and Nozaki, Y. 2003. Effect of enzymatic fish-scrap protein hydrolysate on gel-forming ability and denaturation of lizard fish Saurida wanieso surimi during frozen storage. Fish. Sci., 69,1271-1280. 

Figure 2. Hypothetical mechanisms of aggregation of fish actomyosin during frozen storage. (A) King, 69 (B) Connell, 61 (C) Matsumoto ( proposal in the present paper). AM, actomyosin M, myosin MD1 and MDt, denatured myosin A, actin.

Jarenback and Liljemark (75,76) found similar changes in cod actomyosin solution and cod muscle during frozen storage. The denatured myosin was not extracted with salt solution. 

Connell has proposed that insolubilization of actomyosin during frozen storage of cod muscle is attributable to the denaturation of myosin rather than actin (89). During 40 weeks storage at -14°C, extractability of actomyosin and myosin decreased in parallel, while that of actin appeared to remain constant. The decrease in extractability of myosin was biphasic, while that of actomyosin followed an exponential curve. 

Freezing and storage after addition of sodium glutamate decreased the rate of denaturation. The solubility did not decrease and the F-actin filaments kept their fine structures during frozen storage (Figure 6). 

Tropomyosin and troponin. Tropomyosin is apparently the most stable of the fish fibrillar proteins during frozen storage. It can be extracted long after actin and myosin become inextract-able however, it does denature gradually (90). 

These results led to the conclusion that denaturation and/or insolubilization of actomyosin and myosin during frozen storage is a result of aggregation caused by the progressive increase in intermolecular crosslinkages due to formation of hydrogen bonds, ionic bonds, hydrophobic bonds and disulfide bonds. 

Among the above hypotheses, effects of lipids (4-17,59-62, 69-71,155-159), formaldehyde (160-166), and gas-solid interface TMJ appear to be very important in Gadoid fishes. Denaturation of myofibrillar proteins caused by free fatty acids and/or lipid peroxides must occur during frozen storage. To prove this, Jarenback and Liljemark have shown by electron microscopy that, in muscle stored frozen with added linoleic and linolenic hydroperoxides, myosin became resistant to extraction with salt solution (168). 

The water-activity relations, effects of displacements of water or effects of changes in the state of water must be the most important factors to trigger and to promote the denaturation of muscle proteins during frozen storage. 

As described by Fennema (9J, several refined hypotheses such as "physical barrier and structured water hypothesis" (134,178, 179), "ice-moderator hypothesis" (180-183), and "minimum cell volume hypothesis" (184) have been proposed. However, the author will take a more naive approach in interpreting the results on denaturation of muscle proteins during frozen storage at the same time taking advantage of the basic ideas of the above hypotheses. 

Figure 9. A schematic model of denaturation of a-helical proteins during frozen storage and its prevention by cryoprotectants. The case with dianionic cryopro-...

Denaturation of Fish Muscle Proteins During Frozen Storage... 

Dyers outstanding work stimulated many studies of a similar nature leading to the firm conclusion that denaturation of actomyosin occurs at a significant rate during frozen storage of fish muscle and that the rate of denaturation can be used to estimate the rate of quality change of the fish. 

Other Proteins. Since Reay and Dyer discovered that denaturation of myofibrillar proteins is of such profound importance, little attention has been given to the water-soluble proteins including enzymes and other proteins in the sarcoplasm, subcellular organelles, and cell membranes. Recently reports have appeared on the freeze denaturation of enzymes. These studies involved enzymes such as catalase, ADH, GDH, LDH, and MDH from sources other than fish (88,89,90) and attention was given to the effectiveness of various cryoprotective substances (89, 90). Comparable studies with enzymes from fish muscle are few in number (91). Studies on fish muscle proteins must be extended to this area if a complete picture of the freeze denaturation of fish muscle is to be obtained. It should be noted that freeze stable enzymes might have important effects during frozen storage of fish (92,93). 

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