Physical Dnaturants

Heat. Application of thermal energy is the most common means by which food is processed. Among the major components in food, proteins may be the most sensitive to temperature extremes. Thermal denaturation of proteins may be detrimental to product quality, as reflected in their reduced functional properties (Table 1), or desirable, as in the heat processing of whey for use in confections. The denaturing effects of increased temperature are dependent on many factors, including protein type and concentration, water activity, pH, ionic strength, and the nature of ions present. For most chemical reactions, rate increases approximately twofold for each 10°C rise in temperature. However, the rate of protein denaturation may increase 600-fold. This large difference in reaction rate is attributable to the low-energy bonds/interactions stabilizing protein conformation (Table 2). The dependence of the rate of protein denaturation on temperature can be determined from (first-order) thermal denaturation curves derived using any of a number of different methods to monitor changes in protein conformation (Table 3). These curves provide information needed to calculate such kinetic/ processing parameters as activation energy and z values. These values are related by

where Ea is the activation energy (kJ/mol), R is the universal gas constant, T is temperature (Kelvin), T1 is the temperature 10°C above T (Kelvin), 1/z is the slope of the denaturation curve, and z is the temperature change (°C) required to change the thermal denaturation rate by a factor of 10 (42,43). The energy required to denature the pro tein is represented by activation energy (Fig. 3, Table 4), which can be calculated from the Arrhenius law

where kD is the equilibrium or denaturation rate constant and T is absolute temperature (Kelvin). The values for transition-state enthalpy (AH) of various proteins listed in Table 4 are related to Ea by equation 8:

Values of Ea depend on the extent of denaturation, ie, the nature of the D state. For example, the Ea values associated with the thermal denaturation of proteins, from the N state to a completely unfolded state (eg, random coil), are large relative to other chemical reactions. Although co-valent bonds other than disulfide cross-links are not broken during denaturation, a large number of low-energy noncovalent bonds and interactions are broken. The Ea values associated with thermal denaturation (ie, inactivation) of enzymes are relatively low, however, because the active sites of most enzymes are dependent on only a few low-energy bonds and/or interactions. Enzyme inactivation may be a direct consequence of thermally induced modification of protein conformation; however, the effects of temperature on substrate(s), activators, and inhibitors must also be considered.

Thermal denaturation of food and food-related proteins generally takes place between 55 and 80°C; enzymes tend to be more sensitive to the effects of heat and may begin to denature at a temperature as low as 45°C. Decreased solubility may accompany protein denaturation as heat-induced exposure of hydrophobic groups to solvent (ie, water) may result in the aggregation of unfolded protein molecules (7). A reduction or loss of biological activity and increased water absorption, susceptibility to protease digestion, and intrinsic viscosity may also accompany (thermal) denaturation. In addition, heat-mediated chemical alteration of proteins and their constituent amino acids can result (5,10). For example, dehydrogenation of serine, deamidation of glutamine and asparagine, and formation of new intra- and/or intermolecular covalent cross-links (eg, y-glutamyl-e-TV-lysine) can significantly decrease the nutritional quality of proteins.

Cold. At some stage between postharvest/postslaugh-ter and consumption, many foods are subjected to refrigeration and/or freezing so that product quality be maintained. However, just as the application of thermal energy can result in denaturation of proteins in foods, so too can the removal of thermal energy. Low-temperature denaturation of proteins is largely mediated by a reduction in hydrophobic interactions in conjunction with enhanced hydrogen bonding (Table 2). Thus, low temperatures can lead to aggregation and precipitation of proteins and/or to alteration of quaternary structure. For example, cold inactivation (ie, 10°C) of the glycolytic enzyme phosphofructo-kinase occurs by dissociation of the tetramer to two dimers as a result of weakened hydrophobic interactions between subunits.

The principal injurious effect of freezing is not low temperature per se, but the concomitant concentration of all soluble species as pure ice separates from the mixture. Concentration of acids and/or salts, resulting in large changes in pH and ionic strength, can have profound effects on proteins. At high subzero temperatures, the extent of protein denaturation is greatest, whereas at or below the eutectic temperature of the food system, minimal damage occurs (4). Fish proteins are particularly susceptible to destabilization by freezing temperatures. As a result, fish may become tough and exhibit excessive drip loss on thawing. Similarly, caseinate micelles of milk, which are relatively stable to heat, may be destabilized and coagulate during freezing. Not all proteins, however, are sensitive to freezing temperatures. In fact, several lipases and oxidases are resistant to freezing and remain active at subzero temperatures. In order to inactivate (ie, denature) these enzymes, foods are heat-treated (eg, blanching of vegetables) before frozen storage.

Pressure. Generally, proteins are not sensitive to pressure, and only when large pressures are applied do they exhibit changes. It appears that in most cases the denaturing effect is dependent on applied pressure, exposure time, pH, protein concentration, and temperature (44). Pressure-induced denaturation is thought to result from a decrease in protein volume accommodated by holes in the native protein structure and exposure of hydrophobic groups to solvent (45). The effects of shear on proteins are not unrelated to those for pressure. Shearing action during extrusion processing contributes to the texture of the final product; however, the denaturing effect of the shear plates is most likely secondary to the high temperatures and pressures used during texturization of the proteins. This subject has been reviewed (46). Shear denaturation can also occur during protein purification (eg, at pump heads and in chromatographic columns) and in immobilized enzyme reactors (4).

Interfaces. Gas-liquid, liquid-liquid, and liquid-solid interfaces commonly occur in food systems, eg, emulsions, foams, and aerosols. These interfaces are thermodynami-cally unstable; however, compounds may be present or added, with an affinity for each phase, that reduce energy at the interface and stabilize the system. Proteins, owing to their amphoteric nature and relatively high molecular weights, tend to migrate to interfaces and, in so doing, reduce interfacial tension between phases. The protein thereby adopts a higher energy state, ie, becomes denatured. This phenomenon is technologically exploited in the processing of such food products as milk, ice cream, butter, finely comminuted meats, cakes, and salad dressings. Yet denaturation of proteins at interfaces may also be detrimental, a phenomenon to be avoided or at least controlled during food processing.

The mechanism of denaturation at interfaces is a two-step process (Fig. 5), the first step involving rapid and diffusion-controlled sorption at the interface until a monolayer of concentration 2 to 3 mg/m2 is attained. The pro-

pensity of different proteins to become sorbed at interfaces is dependent on their structures. Proteins that do not contain sizable hydrophobic or hydrophilic regions, or that possess disulfide cross-linked stabilized structures, tend not to adsorb at interfaces. When proteins are sorbed at interfaces, their sorbed structure depends on the stability of the native conformation. Once sorption has occurred, protein molecules reorganize (eg, unfold) and become denatured. This second step is rate-limiting, the rate being dependent on the conformational potential of the protein and its concentration. Detailed information on the dena-turation behavior of //-casein, bovine serum albumin, and lysozyme at interfaces is reported in References 47 and 48.

Irradiation. Ionizing radiation (eg, y, high-energy electrons) has been proposed for application in several areas of food processing, eg, inhibition of sprouting in onions, potatoes, and carrots; sterilization; and pasteurization. The effectiveness of irradiation as an antimicrobial agent has been well documented. However, at the levels of ionizing radiation required to destroy microorganisms (eg, 10 kGy), deterioration of sensory and nutritive quality may still result from autolysis (49). In general, the dose of ionizing radiation required for complete enzyme inactivation in situ is about 10-fold greater than that necessary to destroy microorganisms (50,51). Denaturation of proteins by irradiation is analogous to denaturation by other means; however, the specific effects are dependent on the wavelength and energy of the applied field. Other factors include the nature of the protein (enzyme), water activity, protein concentration and purity, oxygen tension, pH, and temperature (50). The structural alterations that irradiation can induce in proteins (eg, amino acid oxidation, ionization, free-radical formulation, polymerization) are often mediated by radiolysis of water (43). If the applied energy is sufficiently high, covalent bonds may be ruptured.

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