Food Hydrocolloids 17 (2003) 123 www.elsevier.

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Review

Some thermodynamic considerations in food formulation

Vladimir Tolstoguzov*
Research Centre, P.O. Box 44, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland Nestle Accepted 12 October 2001

Abstract Several factors underlie the thermodynamic similarity of foods. First is the common physiological storage function of most important components of food raw materials. Second is the general character of non-specic intermolecular interactions of food macromolecules. Third is the mimicry of biopolymers that underlie quite similar physico-chemical properties of biopolymer species. Molecular mimicry and molecular symbiosis affect phase behaviour and rheology of biopolymer mixtures. Molecular mimicry implies the chemical and structural similarity of hydrophilic surfaces of globular proteins with their chemical information hidden in the hydrophobic interior, and the low excluded volume of the globules. Another mimicry technique is the binding of different biopolymers into a new hybrid (e.g. conjugates) macromolecule acquiring an afnity to the macromolecular constituents as co-solutes. Molecular symbiosis means that interactions (attraction or repulsion) between biopolymer molecules greatly differing in conformation (globular and rod-like), favour the biological efciency of one of them, at least. Thermodynamic incompatibility is typical of food macromolecules, whose denaturation, association, complexing and chemical modication reduce their mimicry and co-solubility. Biopolymer incompatibility, self-association and interbiopolymer complexing contribute to synergistic and antagonistic effects of food formulation. The thermodynamic approach is highly promising for modelling of food formulation. Food formulation aims to control interactions between proteins, polysaccharides and their interactions with other food components. Thermodynamic aspects of food digestion mechanisms and the multifunctionality of exopolysaccharides, prebiotics, storage proteins and other chyme components are considered in terms of food formulation. Normally, food and chyme are phase-separated systems. Thermodynamic similarity, which is a fundamental feature of processed food systems determines the high efciency of empirically developed food technologies and the low sensitivity of structural and mechanical properties of the chyme to the composition of diets. q 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Biopolymers; Proteins; Polysaccharides; Molecular mimicry; Molecular symbiosis; Food avours; Exopolysaccharides; Octopus effect; Thermodynamic similarity of foods

1. Introduction: thermodynamic similarity of foods Agricultural raw materials are, generally, perishable, non-standard, seasonal goods subjected to long-distance transportation, long-term storage and a wide variety of treatments. Yet this is not so, they are usually very consistent and sufciently reproducible. The main questions considered in this paper are: why can raw materials of non-standard composition and properties be transformed into standard foods? Why can Macdonald's foods be recognised throughout the whole world? Why do artisanally prepared foods have a high and reproducible quality? Why is culinary education, e.g. by TV broadcasting, possible and effective? What is the role of food formulation relative to food quality? The obvious answer on these questions, which has become
* Tel.: 141-21-785-8638; fax: 141-21-785-8554. E-mail address: vladimir.tolstoguzov@rdls.nestle.com (V. Tolstoguzov).

clear only quite recently, is the thermodynamic similarity of foods (Tolstoguzov, 1998a, 2000a, 2001). It is the subject of this paper. The most important factors contributing to the thermodynamic similarity of processed foods are the common behaviour features of structure-forming food macromolecules that are the result of the common physiological storage function of the main biological systems currently used as food raw materials. These are seeds (cereals, leguminous and oil-seeds), milk and eggs, whose storage components are responsible for nutrition during germination, or birth and the initial stages of growth. Muscle tissue weight loss (e.g. under starvation or stress conditions) reects its storage functions. Since the storage ingredients of seeds and milk are widely used in food formulation, doughs and ice-cream mixes have, therefore, been selected to illustrate the thermodynamic similarity of formulated foods. To illustrate the main idea behind this consideration, let us consider a football game, where the functionality of all

0268-005X/03/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0268-005 X(01)00 111-4

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Fig. 1. Schematic representation of the complexing between oppositely charged proteins and polysaccharides.

digestion. Formation of structureproperty relationships in two systems, formulated foods and chyme (i.e. the mass of digesting food transported along the intestine) will be discussed. These systems were chosen because food formulation contributes to both functional and nutritional qualities and, together with food processing (mainly shearing and heat treatment), provides the texture, avour, palatability of a food and affects its digestion. The paper is organised into three interrelated topics. The rst is general thermodynamic properties of biopolymer mixtures. The second is the effect of macromolecular components on structure property relationship in food. The third is devoted to the effect of biopolymer incompatibility on food digestion.

components is of importance. The ball can be made of leather or synthetic materials, can be kicked by a boot or by the head. However, for an effective football team, the material of the ball, boots, and heads are not of so great importance as the interactions between them. By whatever means the ball is propelled, it will move according to Newton's laws. This example is to stress that for functionality of a multicomponent physical system the interactions between components are more signicant than the composition of the components. The same situation is typical of food systems, where structures are mainly arranged by noncovalent, non-specic interactions of proteins and polysaccharides in aqueous medium. For instance, the most studied structural food macromolecules are soybean proteins, gluten, casein, milk proteins and starch. In spite of the detailed information available about individual components, the control of dough and milk system functionality remains empirical. The reason is that the properties of a food system reect more the interactions between its components than the properties of these individual components. Indeed, the high heterogeneity of food raw materials containing denatured, hydrolysed and aggregated biopolymers (their molecular, colloidal or coarser particles) favours phenomena such as thermodynamic incompatibility, interbiopolymer complexing, protective colloids, bridging and depletion occulation, which are typical of foods. This paper reviews the general features of mixtures of dissimilar food macromolecules in food formulation and

2. Interactions between food macromolecules The repulsive and attractive forces between different macromolecules underlie two opposite phenomena: biopolymer incompatibility and complex formation (Burova, Grinberg, Grinberg, Leontiev, & Tolstoguzov, 1992a; Ledward, 1994; Tolstoguzov, 1986, 1990, 1996, 1997a, 1998b; Tolstoguzow & Wajnerman, 1975; Tolstoguzov, Grinberg, & Gurov, 1985). Fig. 1 shows interbiopolymer complexing of oppositely charged proteins and anionic polysaccharides. The complexes can be both soluble and insoluble. Mutual neutralisation of chains bearing opposite charges decreases the net charge and hydrophilicity of forming junction zones. Fig. 2 shows the consequence of the mutual neutralisation of macromolecular segments and a decrease in hydrophilicity of junction zones: a compact conformation of the complex with the junction zones hidden within its hydrophobic interior (i.e. a globule protein-like construction). The neutralisation of charges of anionic polysaccharide can also reduce the rigidity of backbone chains due to a decrease in repulsive interactions of like-charged groups. The stoichiometry of an insoluble electrostatic interbiopolymer complex (e.g. protein/anionic polysaccharide weight fraction ratio) tends to satisfy the condition of its electroneutrality, i.e. complete mutual neutralisation of macromolecular reagents. Like the net charge of biopolymers, the stoichiometry of their electrostatic complexes is affected by pH. When the pH decreases, the insoluble complexes are enriched with the anionic polysaccharide. After precipitation of the insoluble complex of a constant composition corresponding to a certain pH, an excess of one of the biopolymer remains in solution. The stoichiometry of an electrostatic complex also depends upon the conformation and net charges of the reactants. Owing to topological limitations, protein globules and rigid anionic polysaccharide chains cannot achieve contact between all their charged groups. On the contrary, unfolded structure proteins (such as gelatin, casein and seed storage proteins denatured in acid media) tend to form a maximum number of contacts with an oppositely charged polysaccharide. Therefore, proteins of unfolded structure usually trend to form electrically neutral

Fig. 2. Compaction of proteinpolysaccharide complexes.

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Fig. 3. The behaviour of mixed biopolymer solutions.

insoluble complexes. Aggregation of insoluble complex particles is mainly due to electrostatic and hydrophobic interactions. Normally, soluble complexes are formed as an excess of one of the biopolymers and their relatively low bulk concentrations. The stability of junction zones mainly depends on the nature of interbiopolymer interactions (electrostatic, hydrophobic and H bonding). Freshly prepared electrostatic complexes can be dissolved in salt solutions and at pH values above the isoelectric point (IEP) of the protein, where both macromolecular reagents have like charges. The stability of electrostatic complexes usually increases due to co-ordination binding of polyvalent cations (Ca,

Fig. 4. Schematic illustration of the excluded volume for globular protein molecules.

Fe, Cu, etc.) and irreversible thermal denaturation of the bound globular protein (Gurov & Tolstoguzov, 1988; Gurov, Larichev, Krylov, & Tolstoguzov, 1978; Gurov, Larichev, & Tolstoguzov, 1983; Gurov, Gurova, & Tolstoguzov, 1986; Gurov, Gurova, Leontiev, & Tolstoguzov, 1988; Sherys, Gurov, & Tolstoguzov, 1989; Tolstoguzov & Wajnerman, 1975; Tolstoguzov et al., 1985). Fig. 3 shows typical results of mixing biopolymer solutions (Tolstoguzov, 1986, 1991). Unlike interbiopolymer complexing, phase separation caused by biopolymer incompatibility leads to an increased concentration of the biopolymers in the different phases (Fig. 3). Generally, when interbiopolymer attraction is inhibited, biopolymers are cosoluble only in a dilute solution. When biopolymer concentration increases and exceeds a certain critical value, the biopolymers become limitedly co-soluble (Antonov, Pletenko, & Tolstoguzov, 1987; Grinberg & Tolstoguzov, 1972; Grinberg & Tolstoguzov, 1997; Polyakov, Grinberg, & Tolstoguzov, 1997; Tolstoguzov, 1986, 1988b, 1991, 1997a, 1998b, 1999a, 2000b; Tolstoguzov et al., 1985). The reason is the large size and rigidity of biopolymer molecules. Therefore, the entropy of mixing of biopolymers is several orders of magnitude smaller than that of the monomers. Biopolymer incompatibility occurs even when the corresponding monomers are miscible in all proportions. For instance, sugars of low molecular weight are normally co-soluble in aqueous media, but polysaccharides differing by structure and/or composition usually have a low phase separation threshold. In spite of a co-solubility of monomer sugars and hydrophilic amino acids, proteins and polysaccharides are normally limitedly compatible, i.e. limitedly

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Fig. 5. The mechanical models for phase behaviour of macromolecular mixtures in common solvent.

miscible on a molecular level. This means that macromolecules in mixed solution show a preference to be surrounded by their own type. Consequently, their mixtures separate into liquid phases. Normally, the excluded volume of macromolecules determines their phase separation conditions.

3. Space occupancy concept Fig. 4 shows the idea of excluded volume for globular protein molecules (Johansson, Brooks, & Haynes, 2000; Launay, Doublier, & Cuvelier, 1986; Minton, 1997; Rha & Pradipasena, 1986; Tanford, 1961; Tolstoguzov, 1991, 1992, 2000b; Yu & de Swaan Arons, 1996). It shows two
Table 1 Phase separation thresholds of some protein- and proteinpolysaccharide mixtures Protein-I 1 protein-II (or 1polysaccharide) polymer-I 1 polymer-II. Phase separation conditions Ovalbumin 1 soybean globulins pH 6.6; 40 8C Ovalbumins native 1 thermodenatures pH 6.7; 20 8C Casein 1 soybean globulins pH 6.9; 20 8C Gelatin 1 legumin pH 7.0; 40 8C Casein 1 Na alginate pH 7.2 Gelatin MW 170 kDa) 1 Na alginate MG 50% pH 6.0; 40 8C Gelatin MW 170 kDa) 1 Na alginate MG 50% pH 6.0; 40 8C Phase separation threshold (%)

19.7 13.3 12 8.4 3.0 1.67 1.36

adjacent spherical molecules of the same radius R. They are not penetrable by each other. Consequently, a minimal distance between two protein molecules equals the sum of their radii or the diameter (D) of one of them. This means that the radius of the excluded volume around each protein molecule equals the diameter of the macromolecule. In other words, the excluded volume, from which the centres of other protein molecules are expelled, is eight-fold greater than that of the molecule itself. Excluded volume is signicantly greater for non-spherical macromolecules, e.g. linear rigid polysaccharides. In dilute solution, stiff rod-like macromolecules are relatively independent when the distance between them equals or is larger than their length. Excluded-volume effects reect mutual competition between macromolecules for solution space. A decrease in the excluded-volume with increasing concentration of macromolecules results in small repulsive interactions between them. Fig. 5 shows the simplest mechanical models for competition for solution space between differently sized macromolecules in thermal movement (Tolstoguzov, 2000b,d). This may be represented by the trafc ow of vehicles differing in size, shape and interactions with the road. On an overloaded motorway, two opposite measures can make trafc more uid. The rst (Fig. 5(1)) is the collective movement of vehicles by car-transporters. This model corresponds to associated biopolymers and soluble interbiopolymer complexes. The second and third models (Fig. 5(2),(3)) are `phase separation' of vehicles between motorway lanes, which results in a more homogeneous composition of these motorway lanes. Fig. 5(2),(3) display an important feature of phase separated systems. This is the

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Fig. 6. Phase diagram typical of proteinpolysaccharide mixed solutions.

presence of interfacial (or depletion) low-density layers between motorway lanes, i.e. biopolymer phases. The second model (Fig. 5(2)) corresponds to phase separation in proteinprotein and proteinpolysaccharide mixtures, while the third corresponds to incompatibility in polysaccharidepolysaccharide mixtures and in mixtures of interbiopolymer complexes. Phase separation in wheat our dough and ice-cream mixes may be illustrated by the second model. Phase separation in tea, coffee and sour rye dough could be presented by the third model (Tolstoguzov, 1997b, 1998c, 2000a,d, 2001). Excluded volume determines space occupancy in biopolymer solutions and the phase separation threshold. Phase separation occurs (Table 1) at about 24% for mixtures of gelatin or casein with linear polysaccharides, about 4% or higher for globular proteinpolysaccharide mixtures and more than 12% for mixtures of globular proteins. The specic feature of foods in terms of biopolymer incompatibility is that foods are usually highly volumeoccupied, phase-separated systems. The excluded volume of biopolymer molecules implies that most water is present as non-solvent water, i.e. inaccessible to macromolecular solutes.

The points of the binodal connected by the tie line represent the compositions of the co-existing equilibrium phases. For instance, on mixing a protein solution A with a polysaccharide solution B, the mixed solution C obtained breaks down into two phases. These liquid phases are: phase D enriched in protein and phase Eenriched in polysaccharide. The line DE is the tie line. The phase D to phase E, volume ratio is estimated by the inverse lever rule, i.e. by the ratio of the tie line segments: EC/CD. When the composition of the system C is shifted along tie-line DE, the volume ratio of the co-existing phases D and E is changed, but their compositions remain constant. The line passing through the mid-tie-lines and the critical point G is the rectilinear diameter. It gives the composition of systems splitting into phases of the same volume. Point F represents the phase separation threshold, i.e. the minimal critical concentration of biopolymers required for phase separation to occur (Table 1). Normally, in terms of biopolymer incompatibility, foods are phase separated and highly volume occupied systems. This means that the concentration of the biopolymers in a food usually exceeds their phase separation threshold.

5. Aqueous phases of formulated foods: W/W emulsions 4. Thermodynamic incompatibility of food macromolecules Fig. 6 shows a phase diagram typical of biopolymer mixed solutions (Antonov et al., 1987; Grinberg & Tolstoguzov, 1972; Grinberg & Tolstoguzov, 1997; Polyakov et al., 1997; Tolstoguzov, 2001; Tolstoguzov et al., 1985). The solid line is the binodal. The binodal branches, which do not coincide with the phase diagram axes, correspond to a limited co-solubility of the biopolymers. Compositions of biopolymer mixtures lying under the binodal curve correspond to single-phase solutions. In the concentration region lying above the binodal, the biopolymers form two-phase systems. They are water-in-water (W/ W) emulsions. A W/W emulsion is a disperse system, where droplets of one of the immiscible aqueous solutions are dispersed throughout another aqueous biopolymer solution. We now turn to the features of W/W emulsions in terms of food formulation. First feature is that co-existing phases can be in equilibrium. The thermodynamic equilibrium principally distinguishes W/W emulsions from both classical oil-in-water (O/W) and water-in-oil (W/O) emulsions. Fig. 6 can also be used to illustrate several typical mistakes of food formulation. It is widely believed that mixing of biopolymer solutions results in their mutual dilution. However, a mixed solution of composition C is not stable. It breaks down into the phases corresponding to point D and point E. Protein concentration in phase D is higher than in the initial protein solution A and the mixed solution C. Accordingly, the initial polysaccharide solution B is diluted to point E. Competition between macromolecules for space determines the water partition between the phases. The phase diagram asymmetry can be characterised (Fig. 6) by

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Fig. 7. Formation of a honeycomb-like lipid structure.

the angle made by the tie line with one of the concentration axes. Because of marked phase diagram, asymmetry food formulation highly inuences rheological and other physical properties of the system. Fig. 6 also shows that a polysaccharide B added to a food system, may perform its functional properties either within the bulk of a system in the concentration range below the binodal (e.g. B1) or within the volume of system phases in the range above the binodal (e.g. in B2 and B3). Phase inversion usually occurs near the mid-tie-lines and greatly changes the system properties (e.g. between B2 and B3). Both possible effects on food texture, synergistic or antagonistic, result from a strong change in the composition of the continuous phase of the system, which is primarily responsible for its properties. The phase diagram shows that minor changes in food formulation, especially near the critical point and near the rectilinear diameter, can change the composition of the continuous phase, and consequently the texture, avour and other qualities of the food. This illustrates the fact that the functionality of an added biopolymer will greatly depend upon food formulation. Unfortunately, however, Fig. 6 does not show the partitioning of all other components between the phases, which may be of key importance for qualities of a food. The phase separation threshold of a food system is determined by its least compatible macromolecular components. After phase separation, the two phases obtained act as two complex solvents for other components of the system. The difference in composition and in solvent properties between the coexisting phases increases with the distance from the critical point (Fig. 6). Thus, food formulation can induce phase

separation and partitioning of the components between the phases and the interfacial layer of the system. The fractionation can cover the biopolymers, enzymes, and reagents of the Maillard reaction, its low molecular weight products and other components. This greatly affects avours, texture and other qualities of the food, since the composition of continuous phase of an emulsion is normally most signicant for its organoleptic perception. The second feature of W/W emulsions is the relatively low density and viscosity of the interfacial or depletion layer between immiscible aqueous phases (Fig. 5). Its thickness exceeds the macromolecular size, since it is formed to diminish unfavourable interactions between dissimilar macromolecules. The depletion layer is signicant rheologically (Suchkov, Grinberg, & Tolstoguzov, 1981; Tolstoguzov et al., 1974a; Tolstoguzov, Mzel'sky, & Gulov, 1974b). The third feature, shown in Fig. 7 is that the interfacial layer can adsorb hydrophobic particles, e.g. cells, cellular organelles and lipids (Tolstoguzov, 1993a, 1994a, 1998a). Albertsson has developed the partitioning technique for separation and fractionation of macromolecules, cells and cell organelles between two aqueous polymer phases. He found that this is a highly efcient method for fractionation of biological particles depending on their surface properties. Zaslavsky (1995) systematically investigated analytical applications of the method for low molecular weight soluble compounds. Albertsson and Walter showed that unlike the partitioning of soluble materials (macromolecules) which distribute according to their solubility between the two bulk phases, colloidal-size and coarser particles is

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Fig. 8. Schematic representation of ball-bearing effect.

partitioning among the two bulk phases and the interface and, as their size increases, between one bulk phase and the interface. The partitioning behaviour of particles can be used to study, e.g. differences between surface properties of cells and afnity between cells, and for the industrial isolation of cellular particles. The biopolymer pair most widely used for partitioning is a mixture of dextran and polyethylenglycol (Albertsson, 1958a,b, 1972; Brooks, Sharp, & Fisher, 1985; Johansson & Walter, 2000; Walter, 1994a,b; Walter & Larsson, 1994; Walter & Brooks, 1995; Walter, Johansson, & Brooks, 1991; Zaslavsky, 1995). The interfacial adsorption of hydrophobic particles, most importantly lipids, could greatly contribute to formation of food structures (Tolstoguzov, 2000a, 2001). Fig. 7 shows that coalescence of adsorbed lipid droplets can result in a lipid capsule around the aqueous dispersed particle. A surfactant stabilising W/O emulsions favours the encapsulation of aqueous droplets. Coalescence of the lipid layers covering aqueous particles, can lead to a so-called honeycomb-like lipid construction. This is a three-dimensional lipid network lled with aqueous droplets, which can be solidied by cooling due to gelation of the aqueous phases and crystallisation of the lipids. This gives butter replacers containing 2030% lipids and encapsulating up to 7080% aqueous phases. Surface properties of the gel granules determine the formation of honeycomb-like construction. Since surface properties of gels depend on the medium in which gelation occurred, lipids are added to a biphasic liquid food system (Tolstoguzov, 1999a). For instance, butter replacers

Fig. 9. General scheme of the deformation of dispersed particles in a owing W/W emulsion.

may be produced by mixing lipids with two biopolymer solutions, e.g. gelatine and maltodextrin. In this type of low-fat spread, small aqueous gel granules separated by thin lipid layers have a low adhesion to each other. Fig. 8 shows that under shearing conditions the low adhesion of rigid granules immersed in a viscous liquid to each other and to the medium results in their rotation. The rotation provides the uidity of a mass of small granules, which mimic the surface and rheological properties of fat globules. This lubricant effect, called a `ball-bearing' effect, can be responsible for fat mimetics of many foods and cosmetics, such as cosmetic rice starch powder. Honeycomb-like structures are, probably, involved in the fat-like mouthfeel of, e.g. micro-particulated gels and fat substitutes based on polysaccharide (Tolstoguzov, 1997a, 1998a, 2001). The next feature of W/W emulsions is low interfacial tension. The reason is the similar composition of the two co-existing phases. Also, the main component, and common solvent, is water. The biopolymers are also co-soluble in both phases (Albertsson, 1972; Tolstoguzov, 1993a; Tolstoguzov et al., 1974b). The two other features of W/W emulsions, which are of importance for mixing of formulated food components, are shown in Fig. 9. They are the high deformability of dispersed particles and their coalescence in a owing W/ W emulsion (Tolstoguzov, 1988a,b, 1993c; Tolstoguzov et al., 1974b). These features are due to the low interfacial tension, the similar viscosity of the co-existing phases and the low viscosity of the interfacial layer. Dispersed particles can be easily deformed in ow when their viscosity is lower than that of the continuous phase. Spherical dispersed droplets can greatly deform, orient, coalesce and form long liquid laments. The latter are not stable and break up into smaller drops. Deformation of a W/W emulsion followed by gelation of its phases results in anisotropic materials of brous or lamellae structure. Generally, as it is shown in Fig. 9, the structure of the material depends upon the volume fraction of the dispersed phase, the phase viscosity ratio and shearing conditions. A relatively low volume fraction of liquid dispersed droplets in the owing emulsion results in individual bres of the same volume as the initial droplets. With an increase in concentration of dispersed particles, coalescence of limited-size droplets leads to long bres. A further increase in concentration of dispersed particles induces their coalescence in both the

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ow direction and the direction normal to the ow. This results in a layered structure of the product. An important aspect of food formulation is that since proteins and polysaccharides are mainly in the different phases, an increase in concentration of a polysaccharide increases the concentration of the co-existing protein phase and decreases its ability to be deformed under given shearing conditions. We now turn to behaviour features of proteins and polysaccharides as food ingredients. 6. Native and denatured biopolymers: molecular mimicry and symbiosis Food processing implies a conversion of biological systems based on specic interactions of the components, into food systems with properties based on the non-specic interactions of denatured components. Modern food processing uses an increasing amount of isolated food macromolecular components. These food additives can contain denatured, aggregated and hydrolysed biopolymers. Another specic trend is an increasing application (in food processing, biotechnology and medicine) of different enzymes. For these reasons compatibility and phase behaviour of biopolymer mixtures becomes of practical importance. 6.1. Molecular mimicry Table 1 (Grinberg & Tolstoguzov, 1997; Polyakov et al., 1997) shows that co-solubility of native globular proteins is surprisingly high, at least more than ten-fold higher than that of synthetic polymers. In a common solvent, synthetic polymers and biopolymers, such as gelatin, casein and polysaccharides, usually have very low phase separation thresholds and tend to be completely separated between co-existing phases. It was assumed that high co-solubility of proteins with other macromolecules is necessary for enzyme function. It arises from molecular mimicry, which implies a chemical resemblance of hydrophilic surfaces of globular proteins with most chemical information hidden in the hydrophobic interior (Tolstoguzov, 1999a,b, 2000b,d). Hydrophilic side groups, mainly concentrated on the molecular surface, determine net charge and solubility of proteins. The globular conformation gives rise to a low excluded volume favouring compatibility of proteins, which is enhanced by their polyelectrolyte nature. Counterions increase the entropy of mixing biopolymers and their cosolubility (Khokhlov & Nyrkova, 1992). Formation of a dense globule diminishes the accessibility of its interior to various reagents. Chemical stability and efciency could give globular structures some preferences in molecular evolution by natural selection. Since aqueous media are typical of biological systems, mimicry seems to start with sub-molecular helical structures. With increasing amounts of monomers with hydrophobic side groups, the mimicry started to make use of

globular conformations. The thermodynamic constraints could be a tool for evolutionary improvement of the mimicry of biopolymer constructions, e.g. viruses and genes. Phase separation could be a mechanism for rejection of molecules with insufcient mimicry, e.g. to precipitate unnecessary proteins to be degraded and re-used. The separated phase of a biopolymer with insufcient mimicry should be a better solvent for hydrolyses. This mechanism could provide raw material for the synthesis of more stable and effective biopolymers and organisms. Thus, phase separation, precipitation and hydrolysis of macromolecules could be used for different (intra- and extracellular) types of nutrition. The principle of phase separation and precipitation of macromolecules with insufcient mimicry could also be used in immune defence. Another evolutionary way to increase the co-solubility of biopolymers and to simplify recognition of mimic proteins could be based upon covalent binding of chemically different chains, e.g. polypeptides with polysaccharides. This second mimicry type could account for formation of new hybrid macromolecules, conjugates, such as proteoglycans, glycoproteins, and polypeptideRNA conjugates. Two or several chemically different biopolymers bound covalently can have an afnity to macromolecular constituents as cosolutes, and to both phases of W/W emulsions. Conjugates can, therefore, contribute to the stability of biopolymer mixtures (Tolstoguzov, 1993a,b, 1994a, 2000b). A transition from attraction to repulsion between unlike polymer parts of the same hybrid molecule (i.e. from intramacromolecular complexing to incompatibility conditions, and consequently from collapse to swelling) could greatly change the excluded volume of the combined macromolecule, its net charge, hydrophilichydrophobic balance and the viscosity of the solution. These conformational changes of proteinpolysaccharide conjugates could also be regarded as a model of the foldingunfolding behaviour of globular proteins. This is presumably, a reason for good and versatile functional properties of proteinpolysaccharide conjugates and the numerous number of biological functions of proteoglycans and glycoproteins. Synthesis and biochemical and physico-chemical properties of protein polysaccharide conjugates were systematically studied (e.g. Dickinson, 1993; Kato, Sasaki, Furuta, & Kobayashi, 1990; Kato, Shimokavwa, & Kobayashi, 1991; Kato, Minaki, & Kobayashi, 1993; Matsudomi, Tsujimoto, Kato, & Kobayashi, 1994; Matsudomi, Inoue, Nakashima, Kato, & Kobayashi, 1995; Nakamura & Kato, 2000; Nakamura, Kobayashi, & Kato, 1994). An unfolding of proteins adsorbed at oilwater interfaces and with subsequent aggregation of denatured proteins in the bulk of the solution and its gelation could be regarded as conformational adaptation of modern biopolymers to the surrounding, as a manifestation of molecular mimicry at molecular and supermolecular levels. Surface properties of macromolecular aggregates, e.g. denatured oligomeric seed storage proteins, and gels, e.g. a gelatin gel, depend on the

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medium in which protein denaturation or gelation occurred (Tolstoguzov, 1999a). For instance, gelatin gels formed in oil or air are not wetted with water, but are perfectly wetted with oil. The 11S broad bean globulin denatured in a dilute alcohol solution has markedly better emulsifying properties than of the native protein (Grozav, Danilenko, Bikbov, Grinberg, & Tolstoguzov, 1985). 6.2. Molecular symbiosis The likeness of macromolecules impoverishes properties of protein mixtures. Their properties become difcult to control by simply changing the composition and physical conditions. An additional thermodynamic tool acting in the direction opposite to molecular mimicry, might be called `molecular symbiosis'. The term molecular symbiosis implies that a mutual inuence (attraction or repulsion) of dissimilar species of macromolecules (symbionts) greatly differing in conformation (globular and rod-like) favours the biological efciency of one of them, at least. Molecular symbiosis is suggested to underlie the predominance of the two extreme macromolecular conformations (globular and rod-like) typical of biopolymers (Tolstoguzov, 1999b, 2000d, 2001). The main tool of macromolecular symbiosis is a long rigid chain. Their associationdissociation can greatly and rapidly change both the free volume and the effective concentration of other biopolymers in a mixed solution. For instance, the dissociation of supermolecular structures formed by rod-like macromolecules can increase the effective concentration of an enzyme without additional synthesis. Owing to excluded volume effects, the macromolecules behave as if they were in a more concentrated mixed solution. At higher concentrations, system phase separation is accompanied by an abrupt change in both the concentration and the weight ratio of dissimilar macromolecules in the phases (see Sections 4 and 5). It should be noted that though specicity of biopolymer interactions underlie the functionality of cells and organisms no specic interactions can exists without non-specic inter- and intramacromolecular interactions. For instance, association of enzymes can alter their catalytic properties, while binding of a substrate can inuence solubility and partitioning of the complex between the aqueous phases. The most typical of non-specic interactions are due to thermal motion of macromolecules, interactions with the solvent and excluded volume effects. Enzymesubstrate, antigenantibody, gene regulation and molecular recognition can be examples of specic interactions. The relationship between enzyme and substrate are comparable to the relationship between a key and a lock. An enzyme can select its substrate among a huge number of similar molecules. An enzyme inhibitor has the same selectivity to block enzymatic activity. Specic interactions are also widely used in vitro as immunology and different afnity methods for biopolymer identication and separation. Both these interactions have the same

nature as hydrogen bonding, hydrophobic and electrostatic interactions. There are some differences between specic and non-specic interactions. Specic interactions are mainly attractive and have a special strereogeometry arrangement of both interacting partner sites, which are chemically heterogeneous. Non-specic interactions can be both attractive and repulsive and use larger molecular surface areas. The frequency ratio between specic and non-specic interactions could be characterised by the surface area ratio between the active centre and the enzyme molecule.

7. Macromolecular additives The choice of raw materials is of great importance for processing conditions, and qualities of the nal foods. Native biopolymers are usually better co-soluble that denatured food ingredients. From the viewpoint of food formulation, the advantages of native proteins as food components, are a maximal cosolubility with other ingredients and a minimal contribution to the system viscosity. Due to molecular mimicry, globular proteins usually have quite similar physico-chemical properties, such as surface activity, conformational stability, viscosity and gelation. The main advantages of polysaccharides as food components, are the minimal phase separation threshold, low critical concentration needed for gelation, low co-solubility with other biopolymers before gelation, maximal co-solubility with other biopolymers after gelation and maximum contribution to the viscosity and gelation rate of the food system. Phase separation of proteinpolysaccharide mixtures can result in highly concentrated protein phases and an increase in both denaturation temperature and the glass transition temperature of the protein. Formation of highly concentrated protein phases (due to membraneless osmosis) could probably explain the effectiveness of the polysaccharides (e.g. maltodextrin) as cryostabilisers of minced meats (muscle tissues of sh) during frozen storage. Most published data are, however, devoted to vegetable polysaccharides (e.g. pectins, alginates, etc.) (Table 1), which are products of hydrolysis, mechano-chemical and thermal decomposition of cell wall materials. Exopolysaccharides from micro-organisms are high molecular weight copolymers including charged and neutral, branched and blockcopolymers and mucopolysaccharides. In spite of the fact that they are components of conventional and novel functional foods, their compatibility with each other, with proteins, other polysaccharides and phase behaviour of their mixtures remain unstudied. Because of very high molecular weight of many exopolysaccharides, their phase separation thresholds are presumably markedly lower than those of vegetable cell wall polysaccharides. Exopolysaccharides could affect the phase behaviour and textural properties of yoghurts, sour rye dough and other traditional

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Fig. 10. Oil-in-water emulsions stabilized by proteinpolysaccharide mixtures.

and novel functional foods fabricated using lactic acid bacteria (Tolstoguzov, 2001). But what are the consequences of an increase in biopolymer concentration in a food system? They obviously correspond to Le Chatelier's principle, which states that a system at equilibrium, when subjected to a perturbation responds in a way that tends to minimise the effect of this perturbation. According to Le Chatelier's principle, an increase in concentration of food macromolecules in a highly volume occupied system favours a reduction of the excluded volume (Tolstoguzov, 1997a, 1998a, 2000d). Processes, which can provide this, are self-association, interbiopolymer complexing, gelation, crystallisation of macromolecules, and their adsorption on the interfaces. The latter effect is presented in Fig. 10. It shows several factors affecting the stability of O/W emulsions (Tolstoguzov, 1998a, 2000a). Lipids are the principal components of food emulsions. The mixing of lipids with aqueous biopolymer solutions can be accompanied by the formation of protein lipid and/or polysaccharidelipid complexes of a reduced co-solubility and an increased surface activity. Precipitation of these complexes could result in multilayer coverage, i.e. the encapsulation of the O/W emulsion droplets. Fig. 10 also shows that the interaction with fatty acids reduces conformational stability of both single chain protein and oligomeric globular protein (Tolstoguzov, 1991, 1992, 1993b). This could favour unfolding of protein molecules at the oil water interface. Consequently, protein molecules adsorbed and partly unfolded at the oilwater interface and molecules of the same protein dissolved in the dispersion medium cannot recognise each other as being the same. This means that adsorbed protein molecules form a monomolecular layer inhibiting further adsorption at the oil/water

interface. Addition of a polysaccharide does not only increase the viscosity of the continuous aqueous phase, but also reduces the protein concentration required for its precipitation on the O/W interface. The comparison of the binodal with the protein adsorption isotherm shows that formation of protein multilayers is due to phase separation in the aqueous phase of the O/W emulsion. In this connection, a recently found phase separation within adsorption lm formed by a mixture of as-casein/b-caseins is of great interest (Sengupta, Razumovsky, & Damodaran, 2000). In mixed solutions, an excluded volume of polysaccharide molecules does not inuence the thermal denaturation of proteins, since thermal denaturation does not substantially increase the volume of molecules. Denaturation favours aggregation and incompatibility of proteins. Since formulated foods usually contain denatured proteins, incompatibility of biopolymers is one of the most common features of foods (Burova et al., 1992a; Tolstoguzov, 1991). According to Le Chatelier's principle, mutual concentration of biopolymers in mixed solutions increases the rate of their gelation and/or crystallisation. The reason is that these processes reduce the amount, mobility and volume of spacelling particles and their excluded volume. Their mechanical model may be a collective movement of vehicles by car-transporters (Fig. 5). For instance, the presence of gelatin (solutions or gels) favours crystallisation of both amylopectin and maltodextrin (Doi, 1965; Doi & Nikuni, 1962; Kasapis, Morris, & Nortom, 1992; Kasapis, Morris, Nortom, & Gidley, 1993). Crystallisation of amylopectin in gelatin solutions and gels could be a model for amylopectin retrogradation in the process of bread staling (Tolstoguzov, 1997a, 1998a). Accordingly, an increase in concentration of dough favours bread

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11

Fig. 11. Thermograms of bovine serum albumin (BSA) and BSA complexes with pectin (BSAP), 2-octanone BSA (BSAOc) and triple complexes BSA pectin2-octanone (BSA 1 P 1 Oc) at pH 6.4 and pH 4.3 (0.01 M NaCl, rate of heating 2 8C/min from 5 to 100 8C). Binding of 2-octanone is accompanied with an increase in the conformational stability of BSA. The change in pH from neutral to acid results in a marked decrease in the temperature and the enthalpy of denaturation of BSA and its complexes. Electrostatic complexing of BSA with pectin results in destabilisation of the protein. The denaturation peak practically disappears in the acid medium. Accordingly, 2-octanone bound by BSA/pectin complexes in neutral aqueous solutions is released by a shift of pH to the acid pH region. B.- BSA denaturation temperature versus concentration of 2-octanone is the calibration curve for determining the amount of 2-octanone.

staling and an increase in the amount of resistant starch in pasta products (Fardet et al., 1998; Yue, RayasDuarte, & Elias, 1999). Presumably, this is also a reason for an increased staling rate of Chinese steamed bread, where the dough contains less water (Tolstoguzov, 1997a, 1998c, 2001). Addition of a biopolymer (gel-forming or not) to a solution of a gel-forming biopolymer does not increase only the rate of the gelation. Since the elastic modulus of a gel is usually proportional to the square of its concentration, the elastic modulus of a mixed gel can be several times higher than those of gels of its individual components. On the contrary, for two-phase mixed solutions the higher the volume fraction of dispersed particles, the lower the elastic modulus of the gels (Braudo, Gotlieb, Plashina, & Tolstoguzov, 1986; Gotlieb et al., 1988; Moritaka, Nishinari, Horiuchi, & Watase, 1980; Morris, 1990, 1998; Tolstoguzov, 1990, 1995; Tolstoguzov & Braudo, 1983; Tolstoguzov et al., 1974; Watase & Nishinari, 1980, 1983; Zasypkin, Braudo, & Tolstoguzov, 1997). This reects a low adhesion between the ller and the matrix (similar to that in low-fat spreads. How can immiscible biopolymers form gels with interpenetrating networks? The existence of interpenetrating networks was shown for the gelatinCa-alginate mixed gel prepared by cooling a mixture of gelatin and sodium alginate solutions. The gel obtained was then immersed

into a solution of calcium acetate. In this mixed gel each of the two networks can be destroyed without destruction and changing the shape of the gel sample. The gelatin network is destroyed by heating or by hot water treatment. Immersing in 0.5 M NaCl or alkali solutions (Tolstoguzov, 1990, 1995; Tolstoguzov et al., 1974a) destroys the Ca-alginate network. The formation of interpenetrable networks is based on the fact that gelation usually includes a two-stage aggregation process (primary aggregation of macromolecules followed by the secondary aggregation of primary aggregates or dispersed phase particles), both of which decrease the amount and mobility of space-lling particles forming the gel. Thus, gelation increases the free volume, i.e. the volume fraction of `solvent water' accessible to other macromolecular solutes. The miscibility of biopolymers is increased due to the gelation of one of them. Compared to the initial mixed solution of gelling agents, the dispersion medium of a gel is always a better solvent for other biopolymers. Dry gel particles are widely used for formulation and preparation of foods and beverages. Because of the memory of glassy state dry food ingredients, their processing and storage history are of importance for re-hydration behaviour. Slow relaxation processes of highly viscous and glassy structural elements of networks underlie the memory phenomena (Tolstoguzov, 1999a, 2000c,d). For instance, a set of gelatin gels of different concentrations can be dried at

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Fig. 12. Flavouring of gelatin gel.

room or lower temperature and ground to have a set of gelatin powders of the same moisture content. However, on re-hydration, the swelling degree of these dry powders is inversely proportional to the concentration of the initial gels. The maximal swelling degree corresponds to the powder obtained from the gel with the lowest concentration. Memory of gel networks is of special importance for low-moisture foods. For instance, mechanical energy accumulated by a loaded viscoelastic food system can be memorized by its rapid vitrication (shorter than relaxation time) and be released (elastic recovery) above the glass transition temperature. The effects of memory and elastic recovery could be of importance for producing dry foods changing their form on re-hydration in hot water, e.g. selfstirring instant foods. Glassy biopolymer phases, which are poor solvents for avour provide their encapsulation and affects their release.

8. Low molecular weight additives: avouringavour binding and release We now turn to food avouring in terms of food formulation. The main contributors to food avour are, presumably, the Maillard reaction, oxidation, hydrolysis, fermentation, various enzymatic processes, the use of natural and articial avouring compositions and the mentioned partitioning of avours between the phases and the interfacial layers of the system. However, in any case, the reversibility of binding avours by food macromolecules during processing and storage is of key importance. Only an equivalent binding of avour components and complete release of all of them can provide processing without distortion of avours. Unfortunately, data about avour binding by biopolymers is still limited. These include the capacity, reversibility and competitivity of avour binding by different biopolymers on molecular and supermolecular levels and depending on the medium. We will consider some examples (Burova et al., 1999; Mikheeva, Grinberg, Grinberg, & Tolstoguzov, 1998; Tolstoguzov, 1999a, 2000a). Fig. 11 shows the release of avour (2-octanone) bound

by soluble bovine serum albumin (BSA)pectin complexes in a neutral medium. The avour release results from a shift of pH from neutral (pH 6.4) to acid (pH 4.3) medium typical of yoghurt fabrication. Fig. 11 presents thermograms (at pH 6.4 and 4.3) of BSA, its complexes with pectin, and 2-octanone and triple complexes of serum albumin/pectin/2-octanone (Burova et al., 1999). BSA comprises over 60% of proteins in blood serum and is responsible for controlling osmotic pressure and for adsorption and transportation of low-molecular weight organic compounds to be evacuated. Unlike most other globular proteins (Fig. 11), the conformational stability of BSA is increased by the adsorption of hydrophobic ligands. This is, presumably, to inhibit losses of various hydrophobic ligands transported by this versatile absorbent. The release of 2-octanone was due to BSA unfolding on the oppositely charged rigid polysaccharide matrix (a single-dimensional model of kidney membrane) at pHs below BSA's isoelectric point. Fig. 11B shows that an increase in the denaturation temperature is proportional to the amount of 2-octanone bound by the protein. This is the calibration curve, which enables the use of BSA as a molecular-sensor to detect minute concentrations of avours. To explain avour adsorption and release by BSA it was assumed that a relatively small BSA molecule with 17 disulde bridges contains a densely cross-linked intramolecular network, which has probably a low excluded volume (relative small molecules) and can act as a `drop' of `good organic solvent'. The polypeptide chain may act as a surfactant encapsulating droplets of the ne `nanoemulsion'. Accordingly, avour release may result from deformation of the droplets on a pectin matrix. This means that the deformable network of a BSA molecule acts as a solid `nano-sponge solvent'. It should also be noted that protein molecules adsorbed on the oilwater interface could release the avour due to their partial unfolding. This means that the function of the pectin matrix in proteinpolysaccharide complexes providing partial unfolding of the protein molecules could be fullled by the oilwater interface (Tolstoguzov, 1999a). Fig. 12 shows a difference in the avour binding capacity between a solution and a gel of the same biopolymer,

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13

typical of globular proteins. The same approach was then applied in the technology of meat extenders (Tolstoguzov, 1998a). Now, to illustrate thermodynamic similarity of concrete foods, the compositionproperty relationship of some of them will be considered.

Fig. 13. Experimental data (Table 2) on the phase separation in wheat our doughs.

9. Conceptual thermodynamic models of doughs and icecreams Thanks to experimental ndings the similar phase behaviour of biopolymer mixtures and the similarity of compositionproperty relationships in many foods have become more comprehensible. Conceptual thermodynamic models of doughs and ice-cream mixes could serve as examples. Actually, there are several reasons for believing that a mixture of skimmed milk with polysaccharides may serve for thermodynamic modelling of wheat our dough. First is a common physiological function of gluten proteins and casein, which are storage proteins, i.e. raw materials for early stages of growth. This results in structural and thermodynamic similarity of these proteins. Second, milk and doughs both contain the same protein classes according to the Osborne classication. Moreover, nearly the same proportion of Osborne's protein fractions is typical of both doughs and milk. Third, both casein and gluten form dense chemical gel networks with covalent (disulphide) and coordination (with Ca-ions) cross-links. These chemical networks, which are not typical of other biopolymer gels, can decrease the hydrolysis rate of these unordered structure proteins (Tolstoguzov, 1997b, 1998c). Fig. 13 shows phase equilibria in a mixture of skimmed milk with a pectin solution (Fig. 13A) and in wheat our dough (Fig. 13B). The skimmed milkpectin mixtures are broken down into two liquid phases highly different in concentration and viscosity. The lower phase can contain from 20 to 30% and more casein, while the upper phase has a markedly weak concentration and contains most of the pectin and the milk whey proteins. It is of importance that 1% pectin concentration in the upper phase corresponds to the concentration of soluble pentosans in wheat doughs, while the concentration of the co-existing casein-phase, corresponds to that of the gluten phase in doughs. These model systems are useful for interpretation of phase behaviour during formulation of many foods. For instance, common features of ice cream stabilisers, i.e. polysaccharides and gelatin, are a high excluded volume, a hydrophilicity, a low phase separation threshold and a high phase diagram asymmetry. To minimise the growth of ice crystals, the stabilisers are used in small concentrations from 0.1 to 0.5 wt%, which, however, exceed the phase separation thresholds for their mixtures with casein (under casein concentration typical of ice-cream mixes). Fig. 13 shows that such amounts of apple pectin added to skimmed milk results in phase separation. In other words, the function of

gelatin. This study was necessary to develop an analogue of caviar consisting of gelatin granules containing casein and covered by two membranes (Tolstoguzov, 1995, 2000a). The binding of amines as main shy avour components, by a liquid solution and a gel of gelatin (at pH 57 and at different salt concentrations) was investigated using electrical conductivity. The device shown in Fig. 12 is a ball viscometer supplied with two electrodes and a water jacket. It was used for the assessment of both the gel melting temperature and the free amine concentration by conductivity (Tolstoguzov, 2000a). In the rst approximation, the conductivity is proportional to the amount of amines added to the gelatin gel. On heating, the conductivity slowly increases. Above the melting temperature of the gel, melting the knots of the network and especially melting of structural network elements (macromolecular aggregates) is accompanied by a great decrease in conductivity at the temperature. The conductivity value is not restored on cooling. The macromolecular aggregates forming the network have a lower binding capacity than exible freely accessible macromolecules in the gelatin solution. This effect may possibly reect a great contribution of intermacromolecular interaction into the glassy state of network elements of the gel, which are a poorer solvent for avour than the gel melt. In other words, biopolymer-binding sites are used either for gelatin aggregation and formation of network, or for binding amine molecules. Accordingly, avouring compositions was added to the gel granules at the end stage of technology. A casein-stabilised oil-in-water emulsion was used to simplify the addition and uniform distribution of the avouring compositions. Additionally, the following three effects underlie the avouring technique. The rst is the well-known phenomenon that when sh and milk curd are kept together in a refrigerator, the milk curd will acquire a shy avour. Second is that perfume is signicantly more intensive when the bottle of perfume is broken than when it is partially opened. Third is that cheeses and fresh breads always have highly pronounced avours, presumably, caused by a unordered structure of the main proteins, casein and gluten, respectively. Consequently, O/W emulsions were used to have a highly extended interfacial surface area of the two solvents for water-soluble and oil-soluble avour components. Casein was used as an emulsion stabiliser, to minimise irreversible avour adsorption

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Fig. 14. Schematic representation of the rolling-pin and `extrusion-drying' effects.

stabilisers is, presumably, to induce phase separation in the aqueous phase of ice cream mixtures. Interfacial layers between aqueous immiscible phases may be partially lled by lipids (Fig. 7) and form honeycomb-like structures. Ice crystal growth may be stopped by highly developed thin lipid layers, air bubbles and by an adsorption of stabiliser macromolecules on surfaces of the growing ice crystals (Tolstoguzov, 1998a, 2000a). The freezing of water favours phase separation and gelation of the aqueous phases. The freeze concentration of Ca-caseinate phase determines heatshock stability of the product. A quite similar gelation process forms a continuous gluten phase of wheat our doughs and breads. Fig. 13B and Table 2 show the results of experimental study of phase separation in wheat our dough (Baker, Parker, & Mize, 1946; MacRitchie, 1976; Mauritzen & Stewart, 1965; Larsson & Eliasson, 1996a,b). On mixing wheat our with water, at least two continuous aqueous phases are formed. The rst is a concentrated phase of gluten proteins. The second is a viscous mixed solution of polysaccharides containing most of the soluble proteins. Presumably, the liquid aqueous phase containing globular proteins, soluble pentosans and starch, is responsible for high foaming properties of doughs (Tolstoguzov, 1997b, 1998c). The bulk biopolymer concentration of this phase (4% and higher) is close to the phase separation threshold typical of proteinpolysaccharide mixtures. An additional amount of polysaccharides dissolved during dough kneading and their interactions with the lipids and other components could result in phase separation and encapsulation of the gas bubbles. Thus, presumably, two membranes are formed around gas bubbles in doughs. The rst is formed by the precipitation of the liquid phase components and the second by the gluten phase due to its thixotropic gelation (i.e. a reversible liquidsolid transformation under loadingunloading conditions, respectively). It should be noted that similar textural properties of doughs and icecream mixes could reect their structural similarity. They are thixotropic emulsions, suspensions and foams at the same time.

10. Functional contributions of dough ingredients Fig. 9 shows that when dough is owing, the liquid and gas dispersed particles can form an oriented capillary structure. Presumably, therefore, the continuous gluten phase between oriented capillaries consists of non-cylindrical strips. The liquid phase squeezing from the oriented capillaries onto the surface affects dough stickiness. Starch granules comprising about 70% of the dough, are rheologically multifunctional. Fig. 14 shows that the granules rotated between adjacent gluten layers owing at different rates, could act as `rolling-pins'. Starch granules could roll out the gluten strips. The rolling-pin effect could contribute to a homogeneous aeration and homogeneous gas retention by thixotropic solidication of the dough. The next feature is migration of starch granules towards the central layers in owing dough. A starch granule behaves like an aeroplane wing between the neighbouring gluten layers owing with a different velocity. According to Bernoulli's principal: pressure is least where ow velocity is greatest. An `upward force' moves a granule towards faster owing dough layers. Migration of starch granules could result in formation of a `starch-lled' central layer and a `starch-reduced' surface layer. The revolving of starch granules can essentially decrease the friction between adjacent gluten layers owing at different rates (Fig. 8). This ball-bearing effect results in unusually high dough uidity. Under shearing conditions, the gel granules behave like ball bearings and provide a fatlike texture to many foods. During pasta drying, this effect could decrease internal stresses in the gluten matrix. The ball-bearing effect may also be responsible for uidity of many foods and cosmetic powders.

11. Food formulation and mechanism of food digestion Incompatibility could play an important role in the structural arrangement of the cell cytoplasm (Brooks, 2000; Johansson & Walter, 2000; Walter, 2000; Walter & Brooks,

Table 2 Separation of dough phases Mauritzen and Stewart (1965) 57 3 4 MacRitchie (1976) Larsson and Eliasson (1996a)

Authors

Baker, Parker, and Mize (1946)

The number of separated dough phases (fractions or layers) (i) Lipids (ii) Liquid phase (iii) Pentosan gel (iv) Gluten gel (i) Viscous liquor (ii) Gluten 1 starch

Layers (i) Viscous liquor (ii) Gluten 1 starch intermediate (iii) Starch

(i) Liquid phase (ii) Pentosan gel (iii) Gluten gel (iv) Starch chemical analysis of the phases did not made

V. Tolstoguzov / Food Hydrocolloids 17 (2003) 123

Dough composition

73% H20; 2% NaCl; 25 ours

3850% H20; 6 ours 100,000 g for 1 h

Centrifugation conditions

40,000 g, 20 min, 29.4 ^1 8C

(v) Starch (vi) and (vii) Two intermediate layers containing damage starch granules 63.4% H20; 0.5 M NaCl solution at 30 8C, 7 ours 105,000 g for 70 min. at 30 8C

46% H20, 2% NaCl, our 13.4% protein 25,000 4 407,000 g, 50 min

15

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Fig. 15. Schematic illustration of the common origin of both non-specic immune defence and nutrition. The barrier solution layer of exopolysaccharides surrounding the cell can be regarded as a bulk phase which can, presumably, bind some proteins, and is immiscible with other proteins of both colloidal and molecular degree of dispersity. This protective layer (an alimentary capsule) is penetrable by low molecular weight substances and impenetrable for most protein molecules, could provide a universal defence of the cell during its nutrition.

1995), in many biological processes, including food digestion (Tolstoguzov, 1999a, 2000d, 2001). Hydrolysis serves to eliminate any memory concerning the foreign origins of food macromolecules, and is the main activity within the alimentary canal. Since the digestive system faces a great variety and amount of foreign biopolymers, it has been assumed that at the earliest evolution stages, both nonspecic immune defence and nutrition could be the same process (Tolstoguzov, 1999b, 2000d). 11.1. Exopolysaccharides. Multifunctionality of food components Fig. 15 shows this idea for bacteria. Exocellular polysaccharides, which are secreted by many bacterial species, form an exocellular layer acting presumably like a phase enriched in polysaccharide (Tolstoguzov, 2000d). This exopolysaccharide capsule could defend the cell against foreign proteins (non-specic immunological protection) and provide both hydrolysis of the bound foreign proteins and separation of the hydrolysates (as nutrients for the cell). The `alimentary' capsule of exopolysaccharides surrounding the cell could function due to: (i) compatibility of polysaccharides with low molecular weight substances (nutrients), (ii) incompatibility with proteins, and (iii) complexing with some of them. The mechanism of protection against foreign enzymes could also be based on a shift in pH-optima of enzymatic activity in the micro-environment of an anionic exopolysaccharide (Braudo, Streltsova, & Tolstoguzov, 1975; Streltsova & Tolstoguzov, 1977). The exopolysaccharide phase around the cell could also play the role of a good solvent (better than water) for sugars (as an energy

source for the cell). For instance, this was found for dextran (Tolstoguzov, 1994b). It has also been proposed that the exopolysaccharide capsule (Fig. 15) surrounding the cell could have developed during evolution into the vegetable cell wall polysaccharides and into the mucopolysaccharide brush membrane lining the gut. (Tolstoguzov, 2000d). Consequently, as it is shown in Fig. 15, a relative compatibility between bacterial exopolysaccharides, mucopolysaccharide brush membrane and food bres could be of importance for a competitive selection of the probiotic, intestinal micro-ora. The competitive interactions between polysaccharides of the chyme, exopolysaccharides of the micro-ora, and mucopolysaccharides of the gut could underlie functioning of food bres as prebiotics important for intestinal micro-ora, for our nutrition and immunity. It should be noted that cross-linking of rigid polysaccharide chains results in a local increase in the concentration of the chain segments, which increases the probability of further cross-linking close to this rst cross-link (e.g. Fig. 2). Consequently, exopolysaccharide and exomucopolysacchride chains can be co-operatively cross-linked, e.g. by some other sugars, polypeptide and/or metal cations and form dense networks. The cross-linking of their chains bound to the cell surface could result in a dense additional membrane around the cell. This additional solid lm could evolve into the protective membrane of yeast cells and vegetable polysaccharide cell walls functioning as prebiotics. Exopolysaccharides and exomucopolysaccharides bound to the cell surface, could full several other functions using different segments of the chains. These may include controlling the immediate (short-distance) surroundings, cell-to-cell and cell-to-substrate adhesion, enzymatic and

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17

antimicrobial activities (Gonzalez, York, & Walker, 1996; Johnson, 1990; Lloyd, Kennedy, Methacanon, Paterson, & Knill, 1998; Mizuno et al., 1990; Quintero & Weiner, 1995; Read, 1990; Selvendran & Robertson, 1990; Southgate, 1990; Van-Den-Berg et al., 1993; Vimr, Steenbergen, & Cieslewicz, 1995). Fig. 15 also shows, that if the exopolysaccharide capsule can act as a phase incompatible with foreign biopolymers of the medium, this capsule has to be surrounded by an interfacial layer (Section 5, Fig. 7). This means that secreted macromolecules could form an interfacial (with the medium) or depletion layer of low concentration around the cell. This interfacial low viscosity layer could transform the three-dimensional random diffusion of nutrients into a two-dimensional faster diffusion around the cell within this layer. Thus this `corridor' surrounding the cell could accelerate the concentration and re-distribution of nutrients consumed by the cell near the cell surface and thereby intensify the nutrition of the cell. Another contributory factor to nutrition of the cell could be the reversible interaction of exopolysaccharide segments with nutrients, e.g. oppositely charged or hydrophobic nutrients. Mutual neutralisation of the reagents forms more hydrophobic, less soluble junction zones, which tend to interact with the hydrophobic surface (e.g. precipitate on the surface) of the cell. The mutual neutralisation of the charges within junction zones formed by complexing with low or high molecular weight compounds could also contribute to the chain exibility (see Section 2, Figs. 13). Thus, the reversible, competitive binding of nutrients (such as sugars, lipids, peptides, etc) by exopolysaccharide segments could result in their preferential transportation by the surface of the cell. Fig. 15 shows this idea. This mechanism of cell nutrition due to interactions between nutrients, exopolysaccharide segments and the cell surface could possibly be called an `octopus' effect. The exopolysaccharide segments could select nutrients from the depletion layer surrounding the barrier polysaccharide layer around the cell to transport them directly to the surface of the cell. For instance, as it was presented in Figs. 1 and 2, the binding of nutrients could reduce hydrophilicity and solubility of the chain segment and provide folding of the exopolysaccharide chain. Segments of the exopolysaccharides could function similarly to the known prebiotic dietary bres. Prebiotic dietary bres (mainly their hydrolysates comprising disaccharide to ten sugar-moiety oligosaccharides, and other sources such as inulin and resistant starch) increase the number of health-promoting organisms (lactic acid bacteria), stimulate their growth, increase their activity and suppress potentially harmful bacterial species in the gastrointestinal tract (Crittenden, 1999). Thus, protection and nutrition of the cell are presumably the main functions of exopolysaccharides, which work like the arms of an octopus. An important function of exopolysaccharides may be that the hydrophilicity of their chains (like tentacles of an octo-

pus) helps to maintain the suspended state of the soft body of the cell in the aqueous medium. This also increases the number of exopolysaccharide segments binding nutrients, i.e. acting as suckers, around the cell. Presumably, the branched structure of many exopolysaccharides is to control their water-binding capacity and to minimise their crystallization. The hydrophilising effect of exopolysaccharides on the cell seems to be similar to an increased solubility and an improved functionality of proteinpolysaccharide conjugates compared to their independent components. Exopolysaccharides leaving the surface of the cell could have some other functions. Their diffusion into the medium could be inuenced by the relative thermodynamic compatibility with the fractions of exopolysaccharide covering the cell and with the medium. This could result in a collective diffusion of exopolysaccharides into the medium. The collective diffusion provides a like macromolecular surrounding within the bulk of the medium and could lead to phase separation caused by incompatibility in the surrounding of the cell. Because of a high excluded volume typical of exopolysaccharides, their phase separation threshold with the medium could be very low. Therefore, the phase separation may result in the aqueous dispersed particles, which may be a low concentrated exopolysaccharide solution and could act as transport vehicles for water and nutrients. These aqueous particles can extract nutrients from the medium to transport them collectively to the cell. The transport to the cell could be caused by the relative better thermodynamic compatibility of the dispersed particle material with the fractions of exopolysaccharide covering the cell than that with the medium. The sensitivity of biopolymer incompatibility to the composition and conformation of biopolymers could be of importance for mutual recognition of the exopolysaccharide of the dispersed particles and the exopolysaccharide capsule of the cell (the target) and their coalescence. Because the exopolysaccharides leaving the cell and forming dispersed droplets could be thermodynamically compatible with the exopolysaccharides covering the cell, the transported nutrients can be accessible only to the cell. Thus, compatibility between exopolysaccharides leaving the cell and covering the cell, together with very low interfacial tension and the low viscosity interfacial layer could play an important role in improving the nutrition of the cell. Phase separation and formation of dispersed particles in the surroundings of the cell could also increase concentration, and viscosity, provide gelation of the medium and mechanical protection to the cell. It could even be assumed that the exopolysaccharides leaving the cell could be developed through evolution into the gums exuded by certain plants. Some additional functions of the exopolysaccharides could be the binding of excessive amounts of cations and especially heavy metal cations by co-ordination. The very complex stereochemical arrangement and branching of heteropolysaccharide chains (controllable ecologically) may be aimed at the successful competition for metal

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V. Tolstoguzov / Food Hydrocolloids 17 (2003) 123

Table 3 Thermodynamic characteristics of conformational stability of legume seed storage proteins Protein Number of poly-peptide chains Number of co-operative domains Melting of domains Temperature (8C) 11S globulin from: Soya Broad bean Pea Sunower Pumpkin 7S globulin from french beans 12 12 12 12 12 3 12 12 12 12 12 6 85.5 87 97.2 95.0 107.0 85.0, 91.0 Enthalpy (kJ/mol)

600 550 560 800 640 540, 880

cations and water with other macromolecules, to protect and provide homeostasis of the cell. The precipitation of toxic cations, peptides, polypeptides and cells by the exopolysaccharides leaving the cell could be a component of non-specic immunity. The two other possible consequences of specic coordinative binding metal cations could be noted. The rst is that like charges on the chain affect its shape, intermolecular interaction, viscosity and gelation. The repulsion of like charges (due to charged side groups and/or specically bound metal cations) increases the rigidity of the polysaccharide chains and controls the rheology of the surroundings of the cell. This could contribute to mechanical protection of the cell. Actually, the systematic study of exopolysaccharides as food thickening and gel-forming agents showed that metal cations and sugars greatly affect their gelation (Miyoshi & Nishinari, 2000; Miyoshi, Takaya, & Nishinari, 1996; Morris, 1991, 1998; Morris, Gothard, Hember, Manning, & Robinson, 1996; Watase & Nishinari, 1993). The second consequence is specic adsorption of an additional sugar molecule from the medium and increased stability of a symmetric metal complex formed by a sugar molecule and the monomer units of the chain. This reversible and preferential binding of sugars of adequate stereochemistry corresponds to the basic principle of ligand exchange chromatography (Davankov, Navratil, & Walton, 1989). From this viewpoint it could be assumed that stereochemistry of molecular and supermolecular structures formed by an exopolysaccharide chain aims at controlling its secondary interactions with nutrients, whose chirality and molecular weight can affect a co-operativity of their binding. Consequently, exopolysaccharide chains could contain information about the ideal nutrients for the cell. Exopolysaccharides leaving a cell could interact with exopolysaccharides of other cells. This could lead to cross-linking and precipitation of the foreign cells (due to bridging occulation), to change the surroundings of these cells (induce their depletion occulation), and to provide a mimicry of the foreign cells. On the contrary, thermodynamic compatibility and an afnity between the exopolysaccharides produced by the same bacteria could cause formation of micro-organism colonies as stable biotic communities. Thus, both exopolysaccharide fractions

bound to and leaving the cell could contribute to its protection, feeding, and its competitivity for nutrients with other bacteria species and the host organism in the case of the intestinal micro-ora. The same functions could also underlie the efciency of food bre as prebiotics. Thus, phase separation and precipitation of macromolecules with insufcient mimicry and biological efciency as an evolutionary tool, could simultaneously be used for the improvement of both nutrition and immune defence. Concerning the medium, exopolysaccharides could act as short- and long-distance analytical tools for the cell. It could be assumed that the products of an enzymatic (or acid) hydrolysis of exopolysaccharides fractions can inform the cell about danger and manage the proper reaction by the cell, i.e. act in the same way as hormones. Their evolution could result in oligosaccharins controlling plant growth, development, defence and gene expression (Cote & Hohn, 1994; Creelman & Mullet, 1997; Dumville & Fry-Stephen, 2000). Two additional possible effects of biopolymer incompatibility on the nutrition of the cell and its exopolysaccharide functions should be noted. Within a heterophase aqueous medium, the micro-organisms could be concentrated mainly within the interfacial layers (Fig. 7, Section 5) and within the bulk of a protein-rich phase. This partitioning behaviour of micro-organisms may also be typical of aqueous food phases. The low concentration of the macromolecules within the interfacial layer and its relatively low viscosity could favour the diffusion of low molecular weight nutrients into this layer and increase the concentration of nutrients around the cells adsorbed within this layer. This could favour nutrition of the cells. The competitive adsorption and concentration of cells within the interfacial layer depends on the surface properties of the cells and could contribute to the formation of the cell colonies. It should, however, be assumed that, as it is shown in Fig. 7, cells may be in competition with lipids for this preferable location within the interfacial layers. Fig. 7 is also to show a possible mechanism of controlling the metabolism of lipids by food bres. One of the aims in this paper is to show that most food components are multifunctional and, therefore, a food is not just an additive sum of its components. For instance,

V. Tolstoguzov / Food Hydrocolloids 17 (2003) 123

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mutually interacting functions of food bres presumably include: (i) binding of proteins, waterholding activity and control of the proteolysis, (ii) selecting, feeding the micro-ora and yielding short-chain fatty acids in the colon, (iii) control of peristalsis, (iv) bioavailability of many nutrients, e.g. minerals, lipid metabolism, (v) the evacuation of some non-utilised food components. Phase separation of soluble polysaccharideprotein mixtures can control the concentration of the protein-rich phases. An increase in Ca-content decreases the solubility of bres, the binding of other cations, the bioavailability of lipids due to formation of both honeycomb-like lipidpolysaccharide structures and insoluble Ca-fatty acid salts. Acid medium (in stomach and in the colon) could favour acidresistant polysaccharides, acid-resistant bido- and other bacterium and autolysis of biopolymers due to destruction of the lysosomes of food tissues. Autolytic digestion at acid pHs by enzymes of the food is probably one of the initial types of digestion. Examples of the autolytic hydrolysis could be meat `ripening' under acid conditions and activation of mothers' milk enzymes at the acid medium. 11.2. Thermodynamic similarity of food proteins The evolutionary improvement of biopolymer mimicry, presumably, also included thermodynamic stability of folded macromolecules. An interesting feature of oligomeric seed storage proteins shown in Table 3 is that their constituting polypeptide chains (e.g. 12 polypeptide chains of the 11S storage globulins from oilseeds and legume seeds), which differ greatly in molecular weight and amino acid composition, form structural domains of thermodynamically equivalent conformational stability (Burova et al., 1992b; Danilenko, Bikbov, Burova, Grinberg, & Tolstoguzov, 1986; Grinberg, Danilenko, Burova, & Tolstoguzov, 1986; Schwenke, Burova, Danilenko, Grinberg, & Tolstoguzov, 1987). After dissociation of an oligomeric protein molecule, its sub-units and constituting chains can become incompatible with each other (Tolstoguzov, 1991, 1993c). In acid media, the electrostatic repulsion of similarly charged groups at the surface of protein globules results in the denaturation of seed storage proteins at room temperature. For instance, the narrow symmetric peak of thermal denaturation of the 11S soybean globulin in neutral media becomes broader and asymmetric at pH 4.0 and broadens further and splits at pH 3.753.25. This corresponds to the dissociation of the 11S protein, i.e. dodecamer, into two hexamers at pH from 4.0 to 3.5. At lower pH values, the hexamer dissociates into the 3S form not having a cooperative conformational transition on heating (Tolstoguzov, 1988b). Another important aspect of the seed storage protein similarity is that their conformational stability decreases due to interactions with anionic polysaccharides and binding of hydrophobic ligands, such as fatty acids, different surfactants and avours (Tolstoguzov, 1988a, 1991, 1992, 1993b,c). The dissociation of oligomeric seed

storage proteins from cereals, legume and oil-seeds and the unfolding of their constituting chains could contribute to their improved digestibility and underlie their thermodynamic similarity to gluten and casein. This also explains the similar behaviour of seed storage proteins during food processing, e.g. soybean proteins and other seed storage proteins used for manufacturing vegetable milk, yoghurt, curd, cheeses, their processing by thermoplastic extrusion or co-extrusion (Burova et al., 1992a; Grinberg, Burova, Grinberg, & Mashkevich, 1989; Tolstoguzov, 1988b, 1991, 1993c). The assumed thermodynamic similarity between gluten and casein underlies a wide application of milk and milk components in dough formulation, cereal milk food compositions (e.g. for baby-foods) and the modelling of various foods. 11.3. Food composition and mechanism of digestion Incompatibility could underlie the efciency of mixed diet digestion. Fig. 13 shows that milk cannot contain a signicant amount of a soluble polysaccharide because of the low phase separation threshold for caseinpolysaccharide mixtures. Fat is, therefore, the main source of energy in milk. Accordingly, newborn mammals require rennin for concentration of casein and co-precipitation of other milk components. However, renneting is no longer necessary at later stages of life because of the transition to mixed diets, which now include polysaccharides. It is possible that the general nature of biopolymer incompatibility can ensure concentration of nutrients and their effective digestion. Accordingly, renneting of milk in cheese-making could be replaced by clotting of milk induced by an added polysaccharide (Dalan, Rivier, & Tolstoguzov, 1996). Because proteins have a relatively lower excluded volume, compared to polysaccharides, proteins, presumably, form the dispersed gel-like phase of the chyme. These dispersed protein particles can be regarded as `micro-reactors' for proteolysis. The composition of micro-reactors is controlled by their gel state. Their concentration can tend to increase due to deswelling of the gel particles in the medium containing the hydrolysis products. The phase-separated nature of foods could be of importance for partitioning the enzymes between the phases rich in corresponding substrates. Proteolytic enzymes would be expected mainly in the dispersed protein-rich phase, while amylases would be concentrated in the continuous polysaccharide-rich phase. This partitioning of the enzymes could also underlie the protective mechanism of the alimentary canal walls against self-digestion. Food formulation, presumably, affects the structure formation of both food and chyme because of the similarity of their essential treatment techniques, i.e. mixing of ingredients, mechanical and heat treatments and an acid treatment of the chyme. For instance, the unwettable surroundings of protein micro-reactors, i.e. the continuous phase rich in polysaccharides, make the chyme thermodynamically similar to wheat our dough. Like formation of gluten

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V. Tolstoguzov / Food Hydrocolloids 17 (2003) 123

networks in dough, the aggregation of gel-like microreactors to minimise contacts with an unwettable polysaccharide phase could lead to the gel-like thixotropic chyme. This thixotropy is, presumably, necessary to provide peristalsis of the chyme and to minimize the excluded volume effects. The general nature of biopolymer incompatibility providing the heterophase gel-like chyme, presumably, could underlie some other digestion features, e.g. the facts that: (i) hydrolysis of starch starts in the mouth to control phase behaviour of the chyme and protein digestion, that (ii) consumption of beverages, i.e. a dilution of enzymes during eating, does not decrease the digestion rate of the food due to gel state of the micro-reactors and the concentration of the enzymes within the phases of corresponding substrates, and that (iii) a chyme texture needed for peristalsis efciency, is preserved for a wide range of diet compositions. Thus, the thermodynamic similarity of food systems could possibly be responsible for reproducibility of structural and physical properties of the chyme. This short consideration of several specic features of biopolymers, such as competition for solution space, preferential surrounding by the same type, molecular (and supramolecular) mimicry, molecular symbiosis, honeycomb-like structures, ball-bearing, rolling-pin and octopus effects, allows to believe that a general hypothesis could be formulated by paraphrasing Feynman Lectures on Physics (All things are made of atoms and everything that living things do can be understood in the jugglings and wigglings of atoms. Feynman et al., 1965) to: everything that an animal can do, both a biopolymer molecule and a cell can do. 12. Concluding remarks In conclusion, the examples discussed here demonstrate the essential importance of non-specic interactions between components of both food and chyme. The main contributory factors to thermodynamic similarity of processed food system are: (i) the similar storage function of main components and the similar composition of biological systems used as food raw materials, (ii) the general character of biopolymer incompatibility, interbiopolymer complexing and (iii) the phase behaviour of biopolymer mixtures based on non-specic interactions of biopolymers, molecular mimicry and symbiosis. The thermodynamic similarity of food systems underlies thermodynamic and rheological reproducibility of the chyme. Thermodynamic similarity of food systems, obviously, underlies the limited number of common culinary techniques, which are highly reliable, very widely used and provide a great number of artisanally prepared high quality foods. The thermodynamic similarity of foods makes the work of a good food technologist effective. Acknowledgements The author is indebted to Dr Elizabeth Prior, Mr Stephen

Collyer and Prof. Bruce German for their help in preparing the manuscript.

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