Denaturation cryoprotectants

Sugar alcohols have also found appHcation in foods containing sugars. Sorbitol is an effective cryoprotectant in surimi, preventing denaturation of the muscle protein during fro2en storage. 

Surimi is fish paste from deboned fish used to make simulated crab legs and other seafood. For preservation the paste is blended with cryoprotectants, such as sucrose, sorbitol and phosphates, and frozen. To make the final product, the frozen paste is thawed, blended with starch and extruded as a film onto a belt. The belt takes the film into an oven that heat-denatures the fish protein and cooks the starch. The film is then rolled to form striations, shaped, colored and cut. Depending on the required distribution, the product is frozen or refrigerated. Potato and tapioca starch were used in surimi products 400 years ago, since they provided a cohesive, elastic matrix consistent with seafood. Frozen distribution has made the use of highly-stabilized, moderately crosslinked tapioca starch popular, alone or with native tapioca starch. Modified waxy maize products are used, as is unmodified com starch, for increased cuttability. Kim188 reported that the gel strengthening ability of starch correlates with starch paste viscosity. 

Molecular-dynamical processes at sub-zero temperatures appear interesting for a number of reasons 1) some biological reactions including electron transfer were found to occur at low temperatures 2) cryoprotection of proteins and enzymes against denaturation and... 

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. 

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 during storage at sub-zero temperatures may be minimized by use of suitable cryoprotective agents which include several families of compounds. 

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

Figure 8 Schematic representation of model for conformational changes during freezing, drying, and rehydration. N, native U, unfolded K, conformation equilibrium upon freezing, which shifts toward the native state in the presence of a cryoprotectant k, rate constant for refolding lc2, rate constant for formation of irreversibly denatured (aggregated) forms. (From Ref. 9.)...

This argument does indeed support the contention that on a per-mole basis PEG is much more effective than sucrose at increasing protein chemical potential. And for cases where relatively high concentrations of PEG (e.g., >1% wt/vol) are needed to confer cryoprotection, the Timasheff mechanism may be applicable. However, it seems unlikely that a PEG concentration of 0.01% (wt/vol) would have a significant effect on the thermodynamics of the system. This is because the actual parameter of interest is the transfer free energy of the native versus denatured protein from water into cryoprotectant solution. The difference between the values for the two states determines the magnitude of the effect on the free energy... 

T. Tanaka, T. Takeda, and R. Miyajama, Cryoprotective effect of saccharides on denaturation of catalase during freeze-drying. Chem. Pharm. Bull. 59.-1091-1094 (1991). 

Membrane inactivation depends on how closely anions can approach cationic binding sites. Poorly solvated ions show the strongest binding. They are also known to be the most effective protein denaturants (65). The Stokes law hydrated radius of the toxic bromide anion is about 1.2 A, that of the relatively nontoxic fluoride about 1.6 A and that of the cryoprotective acetate anion 2.2 A. Biological membranes usually appear in thin sections as three-layered structures 60 to 100 A thick. In view of this relatively large cross section, accessibility of binding sites becomes of obvious importance. 

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). 

Controlling freeze denaturation, which appeared very difficult only a short time ago, is now within reach as far as minced muscle is concerned. MSG has proved to be an especially effective cryoprotective agent. Still to be accomplished is the control of protein denaturation in intact fish muscle. 

Badii, F. and Howell, N.K. 2002a. Effect of antioxidants, citrate and cryoprotectants on protein denaturation and texture of frozen cod (Gadus morhua). Journal of Agricultural and Food Chemistry 50 2053-2061. 

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