Info

Note: See Table 2 for abbreviations.

"Measurements for S0 determination were carried out for protein solutions in 0.01 M sodium phosphate buffer, pH 7.0-7.4; 0.6 M NaCl and 0.3 M NaCl were included in the buffer for salt extracts of chicken breast muscle and for isolated chicken myosin, respectively.

Note: See Table 2 for abbreviations.

"Measurements for S0 determination were carried out for protein solutions in 0.01 M sodium phosphate buffer, pH 7.0-7.4; 0.6 M NaCl and 0.3 M NaCl were included in the buffer for salt extracts of chicken breast muscle and for isolated chicken myosin, respectively.

tative importance of the physicochemical properties in functionality is well recognized, quantitative prediction based on QSAR analysis of food proteins is still not a facile task, being limited by the need to measure relevant parameters, including hydrophobic interactions, which can take into account the effects of processing for incorporation into structure-functionality models. The following highlight the role of hydrophobic interactions in explaining some of the important functional properties of food proteins.

Solubility

Solubility has long been considered a critical property because of its effect on many other functional properties, such as emulsifying, foaming, and gelling properties. Charge frequency and hydrophobicity have been proposed to be the two major factors affecting protein solubility (17). Generally speaking, proteins are soluble in aqueous media when electrostatic repulsive forces between protein molecules are greater than the driving forces for hydrophobic interactions. Insolubility is more likely when the repulsive forces are at a minimum, near the isoelectric pH of the protein. However, proteins with relatively few exposed hydrophobic regions on the molecular surface may remain soluble even at pH near their isoelectric point. For example, the isoelectric precipitation of casein proteins during acidification of milk is attributed to their high surface hydrophobicity. On the other hand, the globular proteins in the whey fraction remain soluble. Denaturation of whey proteins, such as may occur by heat treatment, leads to exposure of previously buried hydrophobic groups and consequently to insolubilization upon acidification.

Aromatic amino acids have been suggested to play a greater role than aliphatic amino acids in contributing to the hydrophobic interactions affecting insolubilization

(28). At pH values where charge effects are minimized (zero zeta potential), the insolubility of some food proteins has been correlated to hydrophobicity parameters measured using the aromatic fluorescence probe ANS or by reverse phase chromatographic behavior on phenyl Sephar-ose (PSC), but not to hydrophobicity measured using the aliphatic probe CPA. At other pH values, charge frequency measured as zeta potential and hydrophobicity measured by ANS or PSC are both significant parameters to explain the insolubility of proteins. Although calculations of hydrophobicity scale values based on free energy of transfer from organic solvent to water indicate that aromatic amino acids are more hydrophobic than aliphatic amino acids, statistical scales based on frequency of location of amino acid residues at the surface versus the interior of protein molecules indicate that aromatic amino acids are often considered less hydrophobic than aliphatic ones. While inherently more hydrophobic, the bulky nature of the aromatic residues may discourage their effective burial in the protein interior (29). These surface-exposed residues determine the strength of hydrophobic interactions that affect properties such as solubility.

The effects of various compounds on protein solubility can be explained on the basis of their opposite effects on electrostatic and hydrophobic interactions of the proteins. Salts may be ranked in terms of a lyotropic series describing their water structure-making or -breaking effects based on molal surface tension values (25). At high ionic strength, ammonium sulfate and sodium chloride increase the surface tension of water, whereas tetraethylammon-ium chloride and guanidinium chloride reduce it. The salting-in or salting-out of proteins by different salts can thus be explained theoretically by the lyotropic series. The salting-out constant of a protein is a function of its surface hydrophobicity, calculated as the hydrophobic contribution of each amino acid (7) and the fraction of exposed hydrophobic residues (10). Similarly, the stabilizing effects of sugars and polyols such as glycerol may be related to their water structure-enhancing effects, which intensifies the intramolecular hydrophobic interactions that stabilize protein structure. The effect of urea in solubilizing proteins has been explained by solvation of the hydrophobic moieties in urea, as well as by water structure-breaking effects. Precipitation by trichloroacetic or sulfosalicylic acid has been postulated to arise from an increase of accessible hydrophobicity of peptide moieties (64).

Emulsifying and Foaming Properties

Emulsion and foam-related properties depend on interactions of protein molecules at the oil-water interface and air-water surface, respectively. Although protein solubility is a key determinant in these functionalities, additional important considerations are molecular flexibility and hydrophobic interactions of the protein at the surface or interface, which result in formation and stabilization of the emulsion or foam. It has been reported that the decrease in interfacial tension and improvement in emulsifying activity are not related to hydrophobicity values calculated from the total content of hydrophobic amino acids, but rather to the effective or surface hydrophobicity of proteins measured by hydrophobic partition, hydrophobic interaction chromatography, or fluorescence probes (24,55). The extent of unfolding to expose areas for hydrophobic interactions is expected to be greater for foaming than emulsifying properties, which may be related to the higher tension at an air-water surface than an oil-water interface. The values of exposed hydrophobicity, measured by fluorescence probe assay after denaturation of protein molecules by heating in the presence of sodium dodecyl sulfate, were found to be correlated with foaming capacity (65). An example of the industrial relevance of hydrophobic interactions in surface properties is demonstrated in the positive correlation between the content of hydrophobic protein fractions separated from beer by hydrophobic interaction chromatography and beer foam stability (66).

In analogy to the hydrophile-lipophile balance (HLB) concept originally developed for nonprotein, synthetic emulsifiers, the amphiphilic nature of proteins is vital to their function as surface active agents. A balance in accessible hydrophobic and hydrophilic areas is required for optimal functionality. Solubility of the protein molecules facilitates diffusion to the surface or interface. However, once there, the ability of the protein to interact with oil or other protein molecules to form an interfacial layer depends on the flexibility and accessibility of surface groups, especially through hydrophobic interactions. The importance of hydrophobic interactions is illustrated in the observation that functionality can often be improved by mild denaturing treatments that increase surface hydrophobicity without impairing solubility (55,67,68). When solubility is a constant parameter, emulsifying properties are improved by increasing surface hydrophobicity, up to some critical point. However, as predicted by the HLB concept, excessive hydrophobicity is detrimental to functionality, and emulsifying properties are poor when proteins have excessively high values of hydrophobicity (17,62).

Thermal Functional Properties

The importance of intramolecular hydrophobic interactions in the stability of proteins to thermally induced denaturation has been assumed, due to the increasing strength of these interactions with increasing temperature up to 60 to 70°C. However, extensive comparison of hydrophobic indices in thermophilic versus mesophilic proteins has not demonstrated any definitive relationship between hydrophobicity and thermal stability. Stabilization is promoted by greater internal or intramolecular hydrophobic interactions and lower external or surface hydrophobicity. Because aliphatic residues have a greater tendency to be located in the protein interior than bulky aromatic side chains, it has been suggested that an aliphatic index may be more relevant to protein stability than a general hydrophobicity index (29).

Thermally induced gelation or coagulation are important in affecting textural characteristics of foods. Coagulation-type (concentration-dependent) and gelation-type (concentration-independent) proteins have been differentiated depending on molecular weight and relative content of hydrophobic amino acid residues. One hypothesis proposes that proteins having over 31.5 mol % of hydrophobic

(Val, Pro, Leu, lie, Phe, and Trp) residues would be of the coagulation type, whereas those with less mol % of those residues would form translucent-type gels (69). However, such generalizations assume that denaturing treatments prior to gelation result in total exposure of the hydrophobic residues. The balance of electrostatic repulsive forces and hydrophobic attractive forces on the molecular surface determine the formation of particulate, fine-stranded, or mixed structure gels, with distinctive textural characteristics and appearance (70,71). A striking example of the involvement of hydrophobic interactions is the setting phenomenon observed in particular fish species, wherein an elastic gel forms at relatively mild heating temperatures. Introduction of aromatic or aliphatic hydrophobic groups through chemical modification can induce nonsetting species to behave like easily setting fish species, exhibiting characteristics of increased viscosity and gelling on low temperature heating (72,73).

Hydrophobic interactions may be involved in the initial intramolecular stage of unfolding as well as the later stage of network or aggregate formation through intermolecular interactions. Both surface and exposed hydrophobicity may be important in the nature of the resulting product. It has been suggested that a low value of surface hydrophobicity of the native protein, coupled with a high value of exposed hydrophobicity after thermal denaturation, may lead to the formation of strong gels (74). The ability to unfold, or molecular flexibility, may be hindered by intramolecular stabilization through noncovalent interactions as well as covalent bonds, particularly of the disulfide type. The involvement of intermolecular sulfhydryl-disulfide reactions usually appears in later stages of these thermally induced phenomena and may be concentration dependent. At temperatures below 70°C, sulfhydryl reactivity is not typically involved and hydrophobic interactions may be the major driving force in network formation, with hydrogen bond formation during the cooling phase contributing to strengthening of the final gel structure (17).

Flavor

Although most proteins do not have a strong intrinsic flavor, they do influence flavor due to their ability to bind flavor compounds. This property can play an important role in the transmission of undesirable off-flavors, for example, in some foods containing soy proteins; conversely, binding of some flavor components may lead to a reduction in the perceived desirable flavor (75). The nonpolar nature of many flavor components such as aldehydes and ketones promotes hydrophobic interactions that may be critical in their binding to proteins. The binding affinity of aliphatic ketones with /i-lactoglobulin increases proportionally with chain length of the ketone, suggesting that the association is primarily hydrophobic in nature (75). Influences of processing such as heat denaturation may be expected by consideration of their impact on the hydrophobicity of the protein and consequently, the flavor binding sites. Oddly, very little interest appears to have been devoted to the study of flavor retention and release, compared with the bulk of work on molecular interactions (76). Systematic study in this area should allow optimal design of flavors for new formulated foods, elimination of transmitted off-flavors and development of efficient flavor carrier systems (75).

Hydrolysis of proteins to form peptides also has a great influence on flavor. Formation of bitter peptides from casein and soy proteins, for example, is detrimental to the flavor acceptability of hydrolysates. Bitterness appears to be related to the average hydrophobicity (HQ) of the peptides, with those peptides having HQ values above 1400 cal/mol residue being bitter. However, the extent of exposure of hydrophobic residues and their incorporation into peptides in contrast to their existence as free amino acids also affects bitterness. Thus bitterness depends not only on the content of hydrophobic amino acids in the original protein, but also on the degree of hydrolysis and the location of the hydrophobic residue on the peptide sequence (77).

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