Thermodynamic aspects of dough formation and functionality

Vol. 11 no. 2 pp. 181-193, 1997

Food Hydrocolloids

-

Thermodynamic aspects of dough formation and functionality Vladimir Toistoguzov

Nestle Research Centre, PO Box 44, Vers-Chez-Les-Blanc, CH-1000 Lausanne 26, Switzerland

Abstract Thermodynamic and microrheological approaches providing information on mechanisms involved in dough formation and functionality are proposed The phase behaviour of biopolymer mixtures and excluded volume effects of macromolecules are critical factors influencing the functionality of dough. Milk proteins and flour proteins have similar structures and fun ctionalities. therefore skimmed milk protein-polysaccharide systems can be used to model wheat dough. During mixing offlour with water, albumins, globulins, water-soluble starch (from damaged starch granules) and pentosans form a liquid aqueous phase. This is immiscible with glutelins and gliadins which form a separated gluten phase. Aggregation of the gel particles of the gluten phase minimizes contact with the non-wetted liquid phase and results in the formation of dough structure. At first, bread dough contains two continuous protein phases: a gluten thixotropic gel phase and a liquid phase. Phase equilibrium of the co-existing phases is controlled by mechanical treatment of the dough and by dough additives and ingredients, such as salt, sugars, lipids, surfactants and alcohol produced by fermentation. Mechanical treatment greatly affects the structure-property relationship of doughs by: establishing an equilibrium between the co-existing phases; transformation of the continuous liquid protein-polysaccharide phase into the dispersed phase; orientation of polypeptide chains of the gluten phase, intensification of their hydrophobic interactions and formation of hydrophobic structural domains adsorbing lipids. Basic mixing of doughs includes: ( i) deformation and breaking down of the liquid and gas dispersedparticles; (ii) spinneretless spinning of gluten strips formed between oriented capillary-like particles of the liquid and gas phases; (iii) decreasing the size of liquid, gas dispersed particles and the thickness of gluten strips; (iv) revolving starch granules in shear flow providing high fluidity of doughs due to a 'ball-bearing' effect; and (v) migration of starch granules towardsa higher shearing gradient providing a decrease in water content of the central layers and the formation of 'starch-empty' surface layers of lower stickiness. Starch gelatinization upon heating results in dewatering of the protein phases and fixation of structure, shape and volume of the loaf A large body of experimental evidence supports the mechanisms of dough formation andfunctionality proposed here.

Introduction Food structures are normally formed by proteins, polysaccharides, their mixtures and complexes interacting with water and other food components. However, in real foods, the functional properties of proteins and polysaccharides are not only governed by basic physicochemical properties of food macromolecules and their environment. The complex hierarchy of food structures complicates the study of mechanisms of formation of structure-property relationships (I -5). This structural hierarchy reflects the difference in size and interactive forces between structural elements. Four levels of

© Oxford University Press

food structural hierarchy analogous to the structure of globular protein molecules can be distinguished, namely submolecular, molecular, supermolecular and macroscopic. The size of the corresponding structural elements changes from that typical of a monomer unit, through that of macromolecules to colloidal and coarser particles. The primary submolecular level covers chemical modifications of macromolecules, their branching and cross-linking, such as intra- and intermolecular disulphide cross-linking of proteins. The molecular and supermolecular levels cover non-covalent interactions of biopolymers between each

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other and with the solvent, conformational changes, aggregation and dissociation of macromolecules and molecular aggregates, inter-biopolymer complexing, formation of three-dimensional gel networks and twodimensional interfacial layers stabilizing emulsions and foams. The superior macroscopic level corresponds to foods as composites. Solid foods are usually multicomponent, heterogeneous, heterophase composites of isotropic or anisotropic structures, containing gas, liquid and/or solid dispersed particles, and one or more continuous phases. Supermolecular and macroscopic levels contribute greatly to food system functionality. There is still little insight, however, into how these superior structures are formed and how their contribution to a food system is co-ordinated. This is in contrast to globular proteins where the primary structure directly determines other levels of structural arrangement of the protein. It has been shown that thermodynamic and microrheological approaches are highly promising for analysing the formation of food structures (1-5). The thermodynamic approach provides information about the possible state and potential behaviour of a multicomponent food system, including the formation of its structural hierarchy (1). The thermodynamic incompatibility of macromolecular components greatly determines food structure and properties. Biopolymer incompatibility is a thermodynamic phenomenon typical of foods. It determines the heterophase nature of many food systems (3-7). Moreover, it has been shown that phase separation in biopolymer mixtures is a key parameter determining food structural hierarchy. Accordingly, phase-separated systems are helpful for modelling, understanding and improving conventional foods, as well as for the development of novel foods (1-10). The rheology of dispersions can be considered at two levels: macro- and microrheological. The first deals with the dispersion as a whole, its viscosity, compliance and other physical characteristics, but not with its structure. The second, microrheological approach aims at understanding the movement, deformation and interaction of individual dispersed particles. This approach is applicable to structure formation in flowing food dispersions (11-14). The thermodynamic and microrheological approaches have been used for understanding food hydrocolloid functionality and structure-property relationship in meat extenders, thermoplastic extrudates and low-fat spreads (13-20). The aim of this paper is to apply the same approaches to the most traditional and complex of food systems: wheat flour doughs and bread. In other words, our objective is to consider the formation and contributions of different structural levels to dough functionality. The paper is arranged in the following way: the initial section is devoted to basic information on the thermodynamic incompatibility of food biopolymers used for the study of the formation of structural hierarchy of doughs. The second section considers

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excluded volume effects of food macromolecules. The third section continues considerations of dough structure formation during mixing and shaping. This means that we will start with phase-separated systems as a whole, continue with their separated phases and return to microrheological properties of dough as an example of a phase-separated system in flow. In other words, we will start with the macroscopic structural level, continue to the supermolecular and molecular levels and then return to the macroscopic level.

Incompatibility of biopolymers One hundred years ago, in 1896, Professor WBeijerinck discovered that two aqueous solutions of biopolymers cannot be mixed together (Fig. 1). Even extensively mixing a gelatin solution and an agar or starch solution could not provide a homogeneous mixture. These two aqueous solutions formed a water-in-water emulsion. It is of importance that both biopolymers are mainly in different phases. This means that biopolymers are only partially co-soluble in the common solvent, water. It was the first observation of incompatibility between biopolymers (21,22). Fifty years later, it began to be clear that this phenomenon is typical of polymer mixtures (23-27). During the last 20 years, it has been found that, normally, chemically or structurally unlike biopolymers have limited thermodynamic compatibility or co-solubility in aqueous media (1-5,28-40). These findings are only just beginning to attract the attention of food scientists. Food systems are usually non-transparent, therefore the heterophase nature of aqueous food systems is often not recognized and taken into account when their structure-property relationships are discussed. Co-solubility of a protein with a polysaccharide or another protein can be characterized by their phase diagram. Figure 2 shows a typical phase diagram of mixed solutions of a protein and a polysaccharide. The bold curve is a

Thermodynamics of doughformation andfun ctionality

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binodal curve. It separates composition regions of a single-phase and a two-phase state of mixed solutions. Different biopolymers are completely miscible or co-soluble in the concentration region under the binodal. Above the curve, any biopolymer mixture demixes and forms a two-phase liquid system, i.e, a water-in-water emulsion. Figure 2 shows that a mixture of composition C is obtained by mixing protein solution A with polysaccharide solution B in the proportion BC/AC. This mixed solution breaks down into two liquid phases, D and E. One of the phases, D, is rich in protein, while the other phase, E, is rich in polysaccharide. The tie-line (ED) connects the binodal points representing the compositions of these co-existing phases. Any point on the same tie-line corresponds to the composition of the systems breaking down into phases of the same composition (D and E) . The dashed line passing through the mid-tie-lines is the rectilinear diameter. The rectilinear diameter presents the composition of systems breaking down into phases of the same volume. Near the rectilinear diameter, inversion of the phases can take place. Phase separation of a mixed biopolymer solution can give rise to a strong, up to several-fold increase in the concentration of one of the system phases and a corresponding dilution in the other phase (Fig. 2) . Water redistribution between the phases results in a higher protein concentration in phase D than in the initial protein solution A and the mixture C. A difference in concentration between the equilibrium phases corresponds to the difference between biopolymers in their affinity for the solvent, water, and in the occupancy of solvent volume by their macromolecules. These two fa ctors determine the difference between biopolymers in competitiveness for water and in the water-holding capacity of the food system's phases. The process of water redistribution between co-existing phases of a food is calle d membraneless osmosis (1-4,41,42). Membraneless osmosis Figure 3 illustrates differences between diffusion, osmosis and membraneless osmosis (42). Diffusion is a process by which different substances mix

Figure 3 Diffusion, osmosis and membraneless osmosis processes.

due to random thermal motion. Diffusion takes place when two solutions of different concentrations enter into contact. Conventional osmosis involves the passage of a solvent through a semi-permeable membrane separating two solutions of different concentration. A semi-permeable membrane is one through which the solvent molecules can pass, but macromolecules cannot. As a result, the solvent, water, flows unilaterally. Membraneless osmosis uses solutions of two immiscible biopolymers. Since the biopolymer solutions are not miscible, a semi-permeable membrane is no longer necessary. Because of the absence of the semi-permeable membrane, exchange between the co-existing phases involves all components. Owing to the highly developed interfacial area, typical of aqueous biopolymer systems, equilibrium between the phases is rapidly established and determines water distribution between food system phases (1-4,20,42). We now turn to the phase behaviour of wheat flour doughs. The complexity of dough composition necessitates the use of model systems. Model systems Thermodynamic aspects of bread dough functionality can be modelled by mixtures of skimmed milk with polysaccharides. There are several reasons for this. First, both gluten proteins and milk casein have similar structural features because of their similar functions. Gluten proteins are seed storage proteins. This means that these proteins are deposited in the wheat seed to meet future requirements of the germinating seed for amino acids and nitrogen. The low water solubility of most seed storage proteins of cereals favours protein deposition and drying of the seeds. They do not have enzymatic functions typical of seed albumins and globulins. Storage proteins constitute the bulk of total seed proteins and are a raw material reserve which can be efficiently mobilized to provide the initial growth of the plant. Milk casein also serves as a source of amino acids,

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nitrogen and short peptides. Caseins are the group of milk proteins which are precipitated from milk by acidification to pH 4.6 at 20°e. Seed storage proteins and casein both have mainly unfolded structures, and tend to form aggregates consisting of many polypeptide chains (subunits). The unfolded structure of both of these proteins is necessary for efficient hydrolysis. The use of many associated subunits favours changes in the amino acid composition of seed storage proteins during evolution. Milk protein mixtures with polysaccharides are used to model the effect of polysaccharide components of doughs. Second, milk casein and wheat flour glutenin are soluble in basic solutions, i.e. they are glutelins. It should be stressed that gluten proteins, and especially glutenin subunits, are closely linked to dough functionality (43-47). The Osborne classification proposed for vegetable proteins is based on protein solubility and thermodynamic by nature (1-4). Third, doughs and skimmed milk both contain the main Osborne protein classes, i.e, albumins, globulins, glutelins and prolamines. The ratio of these protein fractions is nearly the same in both products. Casein is -80% of milk protein. Wheat flour also contains -85% seed storage glutenins and gliadins, and 15% albumins and globulins. This similarity of milk and wheat flour in their protein fraction ratio is important thermodynamically. The second model system is a mixture of starch-like polysaccharides with gelatin, i.e. a protein of unfolded structure. This system will be used for modelling the rheological properties of doughs. Figure 4 illustrates the incompatibility of skimmed milk proteins with solutions of high-ester pectins, gum arabic and arabinogalactan (41,42). Point (a) corresponds to 3% protein content of skimmed milk and point (b) to 1.5% pectin content. Their mixture (c) containing -2% milk protein and 0.7% pectin spontaneously breaks down into two liquid phases: phase (d) containing -20% casein, and phase (e) containing -0.5% pectin and milk whey proteins. Figure 4B and C shows gelation of the dispersed casein phase obtained by adding gum arabic or arabinogalactan to skimmed milk (41,42). Tie-lines converge to a single point corresponding to a gel-like dispersed phase. This point represents the critical concentration for gelation of the protein phase. After formation, the concentration of the protein gel does not change with an increase in concentration of the polysaccharide. Membraneless osmosis stops when gel-like dispersed particles are formed. Gel particles in an aqueous medium behave as an osmometric cell with elastic walls. The deswollen gel could create an elastic back-pressure compensating the osmotic pressure of the surrounding liquid phase. Parts A, Band C of Figure 4 differ in the partition coefficient of Ca ions between the phases resulting from phase separation. The casein phase is gelled (Fig. 4B and C) due to a strong increase in concentration of both casein and Ca ions. Figure 4D, E and F confirms the role of Ca ion partition. Sodium caseinate solutions can be concentrated without gelation (2,32,33,42).

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Dough formation: contributions of different factors An understanding of the phase behaviour of skimmed milk-polysaccharide mixtures can be used for the interpretation of dough composition-property relationships. The following assumptions can be made.

Incompatibility of biopolymers Gliadin and glutelin fractions of wheat flour are not miscible with albumins, globulins, soluble starch and pentosans. Phase separation of gliadin and glutelin fractions of wheat flour from water-soluble proteins, starch and pento sans seems to contribute to the reproducibility of the physicochemical behaviour of wheat flour components, e.g. minimizes changes resulting from a modification of the composition of the dough proteins and aqueous medium. This immiscibility of seed biopolymers could also be of importance thermodynamically for the physiological function of seed storage proteins during germination. The concentration of the casein reaches those typical of the swollen gluten phase of doughs. Moreover, the concentrated casein phase is formed when the concentration of added pectin corresponds to the minimal content of soluble pentosans in wheat flour doughs. In the model milk protein-pectin system, the concentration of the casein-rich phase reaches those typical of the swollen gluten phase of

Thermodynamics of doughformation andfunctionality

doughs. Moreover, the concentrated casein phase is formed when the concentration of added pectin corresponds to the minimal content of soluble pentosans in wheat flour doughs. Adding 1-2% of pectin to the skimmed milk results in -25-30% of casein in the protein-rich phase. Good-quality powdered vital wheat gluten containing -80% protein (and 15-18% starch on a dry basis) adsorbs about twice its weight of water. In the swollen state, its water content increases up to 70%, while its protein content decreases to 25-30%. In wheat flour dough, swelling of the gluten phase, hydration and dissolving of other components are competitive processes. The concentrated phase of gluten proteins can be in equilibrium with a relatively lower concentrated polysaccharide-rich phase containing water-soluble wheat flour biopolymers. Wheat (bread) flour (with -11% protein) usually contains -0.8-1.2% soluble pentosans, 3% damaged starch and -2% (15% of the total protein of wheat flour) water-soluble proteins (albumins and globulins). Damaged starch content can vary widely, e.g. from 2 to 10%. Both thermodynamic and kinetic factors, however, contribute to the competition for water between dough components. For instance, for this reason mechanical treatment can affect the water-binding capacity of doughs and water partition between dough phases. At least two aqueous phases containing proteins are formed in doughs. The first is the concentrated protein viscoelastic phase containing gliadins and glutelins, called gluten . The second co-existing phase is a viscous mixed solution of albumins, globulins, neutral and charged polysaccharides. This leads to the hypothesis that the composition of the dough co -existing phases containing proteins is changeable and that gluten is not a protein complex, as is widely believed , but rather a highly concentrated protein phase. The gluten phase is a thixotropic gel. The second protein-polysaccharide phase may be treated as the liquid phase. These assumptions are in good agreement with experimental evidence about the composition of the liquid and solid protein-containing phases of doughs presented in Figure 4G. This fundamental information about the heterophase nature of wheat flour doughs was first obtained by Baker et at. (48). The authors separated dough into several phases by high-speed centrifugation. They obtained two protein-containing phases: a solid phase containing gluten and starch, and a liquid phase. These findings were confirmed by Mauritzen and Stewart (49,50), MacRitchie (51 ), and Larsson and Eliasson (52,53). It has been shown that the dough phases (liquid, pentosan gel, gluten and starch) cannot be separated by ultracentrifugation at water Contents below 34.5% (51) and can only partially be separated at water contents corresponding to the farinograph water absorption. The efficiency of phase separation of doughs by ultracentrifugation increases with an increase in Water content (52). Therefore, low-viscosity doughs have Usually been used for investigation of the composition of the separated dough phases. MacRitchie (51) showed that dough

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containing 7.9% proteins, 44% polysaccharides (mainly starch) and 46% water is separated by centrifugation into a liquid phase and a gel-like phase. The liquid phase contained 3.4% proteins, 7.0% soluble polysaccharides and 86% water, while the gel-like phase contained 8.9% gluten proteins, 55% starch and 34.5% water. Figure 4G shows that the composition of the gel and the liquid phases obtained by dough ultracentrifugation by MacRitchie can be presented in the form of a 'phase diagram'. The starch can be regarded as an inert filler; therefore, on a starch-free basis, the dough containing 14.1% protein is separated into two phases: a gluten gel with 19.8% protein and a liquid phase containing 4.0% protein and 8.1% soluble polysaccharides. Since there is usually an increase in co-solubility of biopolymers when one of them is gelled, Figure 4G shows an arbitrary phase diagram in soluble polysaccharides-total protein or soluble biopolymer-swollen gluten co-ordinates. Figure 4A-G shows that milk protein-polysaccharide mixtures and wheat flour-water mixtures have quite similar phase behaviour. This similarity between casein and gluten mixtures with variou s polysaccharides, in spite of their large differences in chemical composition, illustrates the more general character of the thermodynamic properties of unfolded proteins. Low compatibility and low co-solubility are typical of such food systems. These mixtures exibit a lower critical point and a decrease in biopolymer compatibility when the temperature rises (19). Similarity in the phase behaviour of proteins with unfolded unordered structures from milk and wheat in mixtures with polysaccharide (Fig. 4) seems to be the basis of an historically wide application of milk and milk products in bread making and a great variety of culinary products, such as pizza, cakes and pastries. Similarities in technological approaches between milk and grain processing also stem from this similar phase behaviour. For instance, pasta and cheeses are traditionally made by compression of gel particles, i.e. of hydrated semolina and casein gel particles, respectively, to produce monolith gels (solids). The ease of washing starch out of wheat flour dough reflects a low affinity between starch granules and the gluten phase and, therefore, incompatibility of these biopolymers. This process is a separation of the phases, but is not a phase separation . Starch granules initially form a separate phase in both wheat flour and wheat flour dough. Another possible reason for the ease of starch separation may be the higher affinity of the granules for the water-soluble polysaccharides and proteins of the dough that are also washed out with the starch granules. The separate phase of insoluble pentosans seems to be responsible for binding an excess of water. Presumably, the gel-like pentosan phase acts as a water reserve, like that of the swollen gels of synthetic polymers used for providing soils with water. This can contribute to dissolving and partitioning of enzymes between the dough phases.

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Flour protein, %

Swelling and dissolving of macromolecular components

At an early stage of mixing flour and water, swelling of gluten phase proteins and dissolving of water-soluble proteins, starch and pentosans occur simultaneously, but at different rates. Since the diffusion rate of the water molecules is considerably greater than that of the macromolecules, swelling and formation of a gel is the first stage in the formation of biopolymer solutions. Swelling and formation of the gluten gel results in the exposure of hydrophilic side groups at the gel/water interface. Water surrounding the gluten gel particles is replaced by an unwettable biopolymer mixed solution. This leads to the agglomeration of dispersed particles in the gluten phase. Agglomeration also results from swelling gluten phase particles. They grow, collide, compress with and adhere to each other in the bulk of the unwetted medium during dough mixing. In other words, dissolving of water-soluble proteins and polysaccharides provides a medium of reduced wetting for the gluten phase particles. This means a transition from hydrophilic gluten dispersed particles to hydrophobic gluten phase particles aggregating and forming a new continuous dough phase. This results in a decrease in dough stickiness. The formation of a gluten gel network and rearrangement of the gluten network microstructure and surface are important contributions of dough mixing. The effect of an added polysaccharide is more strongly pronounced in colloids than in molecular protein dispersions. Aggregation of biopolymers normally contributes to their incompatibility, while dissociation of aggregates and macromolecules favour co-solubility of biopolymers. For instance, addition of phosphate or citrate ions to a skimmed milk-pectin solution mixture gives rise to dissociation of casein micelles and an increase in the phase separation threshold and co-solubility of these biopolymers. In contrast, incompatibility increases on cooling the gelatin-dextran-water system from 40 to 18°C where gelatin macromolecules are aggregated (4,5). Owing to an excess of surface free energy, colloidal dispersions are thermodynamically unstable and flocculate (aggregate) in unwettable media. Unlike mixed protein-polysaccharide solutions, where the phase separation threshold usually exceeds 4%, polysaccharides are normally used as flocculants of protein suspensions at concentrations below 0.3%. Accordingly, albumins and globulins may be co-soluble with soluble pentosans in the liquid phase of doughs. The formation of a gluten phase network with a minimal interfacial surface is thermodynamically preferential. A significant energy input is necessary to increase interfacial surface area in doughs. Thixotropy of gluten gel phase

The separation of the starch granules, the pentosan gel and the liquid phase from the gluten phase by dough ultracentrifugation (48-53) results from the thixotropy of

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gluten gel, i.e. its reversible change in liquid state on mechanical treatment. Since the gluten phase forms a thixotropic gel, an equilibrium between the solid and liquid dough proteineous phases can be established during mechanical treatment of the dough when both these phases are liquid. Unlike Figure 4B and C, an equilibrium between co-existing liquid phases means that there are relationships between dough composition and phase volume ratio and composition of the liquid and the gluten phases. For this reason, dough mixing is of great importance for controlling the compositionproperty relationship of dough phases. This is in agreement with results obtained by Baker et at. (48). Figure 5 schematically represents the data [determined by Baker et al. (48)] on the yield and composition of the liquid dough ultracentrifugates obtained from five individual representative flours, and the average, maximum and minimum values obtained from a series of 25 flours of 9.8-14.9% protein content. Figure 5 shows that according to ref. 48, protein content in the flour and the yield (i.e. volume fraction) and composition of the liquid phase of doughs are correlated. The formation of a three-dimensional network inhibits the establishment of a thermodynamic equilibrium between the co-existing dough phases (phase diagrams 4B and C). Determination of the equilibrium swelling of gluten gel in a standard biopolymer solution is important for the prediction of both dough quality and effects of additives. Dough mixing

Dough mixing leads to the formation of a concentrated gluten phase and the development of non-covalent interactions, mainly hydrophobic interactions, between polypeptide chains oriented in shear flow. The formation of hydrophobic structural domains of oriented densely packed polypeptide chains during dough mixing can result in a

Thermodynamics of dough formation andfunctionality

decrease in dielectric permeability and the degree of dissociation of ionizable side chains within an interior of the aggregates. A decrease in the mobility of macromolecular chains resulting from hydrophobic and hydrogen bonding within the aggregates can be compensated by liberation of water molecules from hydrated non-polar groups of the unfolded polypeptide chains. The formation of protein aggregates is also accompanied by sulphydryl-disulphide exchange. Scission of disulphide bonds between chains can lead to a reduction of the length of the chains and change the size of their aggregates. The increase in a number of disulphide bonds within the aggregates decreases the contribution of disulphide cross-links to the formation of both the gluten phase network and rheological properties of the dough. Low-molecular-weight components can be non-uniformly distributed between protein phases of doughs, and change osmotic pressure and equilibrium between dough phases. These dough components can affect the hydrophobic, electrostatic and hydrogen bonding of gluten proteins. A pronounced dynamic hydrophobic-hydrophilic competition takes place during the formation of dough structures. A hydrophobic-hydrophilic balance similar to that responsible for the tertiary structure of globular proteins occurs. Presumably, there is a similarity in driving forces of structure formation between collapsed (e.g. overmixed) doughs of densely packed aggregated polypeptide chains and the densely folded polypeptide chain of a globular protein. The competition between hydrophobic and hydrophilic contributions to the formation of structure-property relationships in doughs can make dough highly sensitive to its composition and processing conditions. Hydrophobichydrophilic balance in the continuous gluten phase of doughs can be controlled by temperature, water content, pH, salt concentration, sugars, alcohol (produced by fermentation), lipids and surfactants. A shift in hydrophobic-hydrophilic balance towards the van der Waals interactions between non-polar side groups (intensified by mechanical treatment, heating and some hydrophobic additives) may lead to a more or less pronounced collapse or shrinkage of the matrix gluten phase that can occur on dough handling and heating. For instance, this occurs on heating of biscuit doughs (54). Dough additives and ingredients

The functionality of dough ingredients can be studied based on their effects on structure-forming proteins and polysaccharides in a given food system. Many substances, such as salts and sugars, are components of the complex solvent for biopolymers (5,6). They affect the solubility, co-solubility and conformational stability of biopolymers (1-6). Alcohol could control: swelling degree and solubility of gluten proteins; partition coefficient of lipids, and other components between dough phases; conformational stability of globular proteins; and contribute to an increase in gas bubble pressure and loaf volume. Sucrose solutions are a

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better solvent than water for many biopolymers (18). Sugar contributes to an increase in gluten solubility and the compliance of shaped biscuit doughs. Addition of both sugar and salt decreases the free water content, and increases the denaturation temperature of globular proteins. This also means that sugars, e.g. maltose, produced by enzymatic and mechanochemical starch degradation, could contribute to an increase in stability of enzymes in the dough. Since hydrophobic interactions in aqueous media are intensified by an increase in salt concentration, addition of salt results in an increase in dough rigidity. Lipids can plasticize highly hydrophobic gluten proteins in low-moisture doughs. Another effect may be a reduction in the conformational stability of globular proteins. Lipids can form complexes with amylose and proteins, decrease the solubility and co-solubility of biopolymers and provoke phase separation of biopolymer mixtures. Addition of oxidants and reducing agents causes changes in the sulphydryl-disulphide bond ratio and provokes sulphydryl-disulphide exchange. These effects can be interpreted as changes (an increase or a decrease) in the molecular weight of gluten phase proteins (polymerization or depolymerization) accompanied by changes in viscosity, relaxation time and thixotropic properties. Heating

Baking is accompanied by water evaporation, denaturation of proteins and starch gelatinization. This radically changes water partition between the dough phases. Starch is the main storage polysaccharide of higher plants and the main (-70% wt) dough component. It is more hydrophilic than proteins. Because starch granules are a filler of both the liquid and gel-like protein phases of dough, starch gelatinization may provoke dewatering of both dough phases. Glass transition of the gluten phase within the exterior layer of the loaf fixes its structure, shape and volume, and retards staling. The Maillard reaction: flavour formation and release

Breads are notable for both intensive and reproducible flavours inspite of low lipid content. The Maillard reaction is of great importance for food flavour. The amount and distribution of Maillard reaction reagents and products between the system phases, interfacial layers and hydrophilic interior of macromolecules are of great importance. In bread, the gluten phase could be both a source of flavour formation and a carrier of flavour (hydrophobic volatiles). Similarity in behaviour of proteins with unfolded structure in different foods implies: (i) an increase in the rate of protein disintegration (enzymatic and mechanochemical) contributing to formation of flavour by the Maillard reaction and (ii) a flavour reinforcement due to the reversible absorption of flavours by these proteins (solvents for flavour compounds, some lipids and surfactants) and to a strong development of the interfacial area in phase-separated systems.

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Interactions of flavour components with macromolecules and other food components can be competitive, reversible or irreversible. The hydrophobic interior of gluten protein aggregates can be regarded as a droplet of solvent for flavours with an intermediate dielectric permeability close to that of alcohol. In other words, the three main types of solvent for food flavours are: lipids, aqueous phases and hydrophobic structural domains formed by proteins of unfolded structure. Unlike heat-set globular proteins, casein, gluten and gelatin seem to be able to form gels with a low extent of irreversible flavour adsorption. Presumably, this determines the wide variety, robustness and reproducibility of cheese and bread flavours, as well as the extensive use of gelatin in various jelly-like products containing meat, fish, milk ingredients, etc. The Maillard reaction: protein-polysaccharide conjugates

The unfolded structure of gluten phase proteins could result in an increase in the rate of chemical interactions between these proteins and polysaccharides (by the Maillard reaction) with the formation of protein-polysaccharide conjugates. Protein-polysaccharide conjugates (also called hybrids or duplex compounds) are very efficient stabilizers for oil-in-water emulsions (55-58). Their molecules contain the protein and polysaccharide parts covalently bound together. The conjugates have an affinity to both protein and polysaccharide phases. This increases adhesion between both aqueous and non-aqueous phases of foods and therefore the stability of food systems (17,18). High viscosity, emulsifying and other functional properties of protein-polysaccharide conjugates reflect the transition from demixing of biopolymers in the bulk of mixed solutions to segregation of protein and polysaccharide parts of the same hybrid macromolecule (1,18). Protein-polysaccharide conjugates could be formed during wheat flour ageing and storage, dough making and baking, bread staling and drying. From phase-separated systems, we now turn to their phases and to single-phase systems. To aid understanding, excluded volume. effects of macromolecules will be briefly discussed.

Excluded volume effects Excluded volume effects are based on spatial limitations. Macromolecules and their segments cannot occupy the same volume (5,6). These requirements result in small repulsive intermacromolecular interactions. In dilute biopolymer solutions, excluded volume interactions prevent interpenetration (or overlapping) of the effective macromolecular volumes of biopolymers. The first molecule already present in a solution excludes the second one from a certain volume, U, called the excluded volume. The volume of dilute solution available for the second protein molecule, \)2 = A(V - V), is less than the entire volume, V, which was initially free for occupation. A is the proportionality factor. For the third molecule, the excluded volume will be \)3 = A(V - 2V), etc.

radiusR

A~OO

Figure 6 Schematic representation of the excluded volume for compact spherical molecules of a protein.

For very concentrated solutions, when the concentration increases the excluded volume approaches the real volume of macromolecules. This is due to an overlapping of the volumes excluded by individual molecules (17). Figure 6 illustrates excluded volume effects for the simplest case of a globular protein (17). Each spherical macromolecule occupies space in the solution which cannot be occupied by another macromolecule. This means that around each spherical protein molecule there is a spherical volume from which the centres of all other protein molecules are excluded. Accordingly, a minimal distance between two neighbouring protein molecules, A and B, equals the sum of their radii or the diameter of one of them. In other words, the excluded volume (V) around each protein molecule is eight times greater than that of the protein molecule itself. Excluded volumes are significantly greater for non-spherical macromolecules, especially linear rigid polysaccharides. In food systems, excluded volume effects have the following manifestations (3-6,20): (i) enhancement of association of biopolymers, including denatured globular proteins; (ii) reduction of the solubility and co-solubility of macromolecular solutes; (iii) destabilization of biopolymer suspensions (by depletion flocculation), e.g. formation of gluten gel phase; (iv) increase in gelation rate and size of fibrils of gel networks; (v) intensification of the adsorption of proteins (or protein complexes) at oil/water and gas/water interfaces; (vi) changes in partitioning of functional additives and ingredients, including wheat flour enzymes during dough formation; (vii) enhancement of enzymesubstrate complex formation and increase in the thermodynamic activity of enzymes due to highly concentrated substrates. Excluded volume effects of macromolecules determine the degree of volume occupancy in mixed biopolymer solutions and, hence, the phase separation threshold of biopolymer mixtures. A difference in excluded volume effects of biopolymers (reflecting the ratio of molecular masses and the shape of their molecules) also determines the relative hydrophilicity of biopolymers and therefore water partition between the phases. Figure 7 shows that critical biopolymer concentration, corresponding to total occupancy of the solution volume, is usually below 1%for polysaccharides and varies from 10 to 30% for globular proteins. This critical value occurs at an abrupt increase in the slope of the curve of

Thermodynamics of dough formation andfunctionality

Apparent specific Viscosity Polysaccharide

Elastic modulus Optical Rotation

Globular protein

fA\ V

~+~-"

100

Gelatin

10

1 234

189

Elastic modulus sWgletwop a~e phase

"

1

20 kD 65kD 3000 kD

0.5 Days

2 Dextran, % wt

1

Figure 8 The effect of additives of dextran on the rate of gelation 0.1 0.5 1 1.5

30

100

and theelastic modulus of gelatin gel.

Concentration, %

Figure 7 Apparent specific viscosity versus concentration of biopolymer solution.

specific viscosity versus biopolymer concentration. It reflects the transition from a dilute to a semi-concentrated solution. In dilute solutions, macromolecules are kinetically independent. In concentrated solutions, macromolecules interact. The phase separation threshold for proteinpolysaccharide mixtures is -4% or higher, while for polysaccharide mixtures it usually exceeds 1%. The phase separation threshold is -2-4% for mixtures of gelatin with linear anionic polysaccharides and exceeds 12% for mixtures of globular proteins. Phase separation thresholds are usually below biopolymer concentrations typical of many food systems. In other words , many food systems are phase separated. Le Chatelier's principle determines the reaction of food system ph ases upon an applied change. According to Le Chatelier's principle: any system in equilibrium shifts the equilibrium, when subjected to any constraint, in the direction which tends to nullify the effect of the constraint. For instance, an increase in concentration of biopolymer ingredients or additives usually leads to a reduction in excluded volume effects due to a decrease in the amount of space-filling particles by biopolymer aggregation, gelation and/or phase separation of mixed biopolymer solutions. Excluded volume effects of incompatible biopolymers affect both bulk properties and surface properties of food systems (5,6,20). Gelation of biopolymer mixtures

The most significant manifestation of excluded volume of food macromolecules is the synergistic effect of gelling agents (20). Excluded volume effects result in a mutual concentration of all biopolymers in their mixed solutions. Each incompatible biopolymer behaves as if it was in a more concentrated solution. Since the shear modulus of a gel is usually proportional to the square of its concentration, small additions of a hydrocolloid can increase several-fold the elasticity modulus of the gel (20). The effect is more pronounced in the case of biopolymers with higher excluded volumes, such as gelatin (12,60--62).

The relationship between co-solubility limits and critical concentrations for gelation of biopolymers determines the structure-property relationship of multicomponent gels. The critical concentration for gelation does not usually exceed 0.1-0 .3% for anionic polysaccharides, while for proteins this value varies from 1 to 13%. Comparison of these values with the mentioned phase separation thresholds shows that combinations of gelling agents can form mixed gels on both sides of the binodal curve. The critical concentration for gelation is lower in mixed solutions of incompatible biopolymers than for each of them individually. Because of the absence of data on the co-solubility of flour protein, and co-solubility of soluble proteins and polysaccharides, the formation of mixed gels can only be broadly discussed using model systems. Some examples of excluded volume effects are illustrated in Figure 8. Twenty-five years ago, it was shown that the addition of a small amount of dextran results in a strong increase in the gelation rate and elasticity modulus of gelatin (12). The effects are dependent on the molecular weight of the dextran and phase behaviour of dextran-gelatin mixed solutions. Above the binodal, the elasticity modulus of the gel decreases with the volume fraction of liquid dispersed particles acting as a filler of the gel. Similar effects could be of importance for dough rheology. Gelatinization of starch leads to conversion of the starch granules from solid to liquid droplets and then again to a solid filler of the gluten matrix. The rate of starch recrystallization and the glass transition temperature of starches strongly depend on the moisture level. For instance, different water contents cause remarkable differences in texture and staling rate between pan breads and Chinese steamed breads. Similarly to starch, the liquid protein phase of doughs could also act a filler of the gluten phase. The liquid phase particles can act as a lubricant. An increase in the volume fraction of the liquid phase particles decreases the effective cross-section of the filled gel (Fig. 8). Accordingly, extraction of the water-soluble components of the dough results in a considerable increase in the elasticity modulus of the gluten phase (63). Emulsifying and foaming properties of biopolymer mixtures

The addition of a polysaccharide decreases protein

190

V. Tolstoguzov

solubility. This encourages protein adsorption at the non-polar phase/water interfaces and reduces the protein concentration required for protein multilayer formation (1,5,6,17). These effects have been found using mixtures of the lIS broad bean globulins with dextran (5,6,64). Effects are proportional to the excluded volumes of the polysaccharide molecules. It can also be assumed that multilayer protein adsorption at the non-polar phase/water interfaces is mainly due to phase separation of protein-polysaccharide solutions (1,6,17). Concerning wheat flour doughs, the presence of soluble pentosans and starch could result in high foaming of the liquid dough phase (3,5,65). The liquid phase is a saturated mixed solution in which total biopolymer concentration is close to the phase separation threshold of the proteinpolysaccharide mixture. The liquid protein-polysaccharide phase could separate into two phases during processing. This may result from: changes in dough composition, e.g. in amount of damaged soluble starch, salts, lipids, surfactants, alcohol (produced by fermentation), etc.; mechanical treatment of the dough; formation of protein-lipid and lipid-polysaccharide complexes of lower solubility; and protein denaturation. Owing to phase separation of the liquid phase of dough, gas bubbles can be entrapped (encapsulated) by the new protein phase. Protein precipitation on the surface of gas bubbles changes the equilibrium between the gluten and the liquid phase. The fibrous gluten network formed during dough mixing can act as a beater to whip the liquid phase, and to beat dough during dough handling. Presumably, another function of the gluten phase is similar to that of the leather covering the rubber-like bladder inside a football: two membranes of different permeability and mechanical properties are formed by adsorption of proteins from the liquid phase and by the gluten phase of doughs. We will now discuss dough at the macroscopic structural level.

Some features of aqueous heterophase systems Low interfacial tension and interfacial depletion layers

Specific features of phase-separated aqueous systems are low interfacial tension, interfacial depletion layers between phases and easy deformability of liquid dispersed particles. These favour mass exchange between phases and provide formation of fibres and layers by system deformation (1,14,17). A common solvent, water, and marked co-solubility of biopolymers are the main contributory factors to low interfacial tension and easy deformability of dispersed particles of two-phase aqueous systems in flow. The presence of interfacial or depletion layers between immiscible biopolymer solutions reflects the tendency for incompatible biopolymers to minimize contacts. Depletion of the macromolecules from the interfacial layer and the spherical shape of the dispersed particles lead to

== =.

.---=..., Wheat flour

-

'"-.......r-'

Gluten Fonnatlon of

fibrous and lamellar structure

Water

Baking Figure 9 Deformation, breaking down and coalescence of dispersed gas and liquid particles during mechanical treatment of doughs. Spinneretless spinning gluten fibres.

surroundings of the same type for each kind of macromolecule in the bulk of the phases. The adsorption of non-polar food components within the interfacial layers between immiscible aqueous phases is another interesting feature of phase-separated food systems. Adsorption of lipids can take place around starch granules, forming honeycomb-like structures within the gluten phase (1,17,19). This type of heterogeneity could be an important factor in the texture and fat-like mouthfeel of many dough products. Spinneretless spinning

Shearing has a pronounced effect on the structure of phase-separated systems, particularly doughs (14-17). From the microrheological view point, wheat flour doughs can be treated as a dispersion of deformable gas and liquid particles, and non-deformable rigid semi-crystalline starch granules. Figure 9 illustrates spinneretless spinning of deformable dispersed particles (1-3,14-17). When a dispersion is flowing, the stresses arising in the dispersion medium tend to deform and orient liquid and gas dispersed particles. However, fibre-like dispersed particles are unstable. They break down into smaller spherical particles. These latter can be deformed and break down to even smaller droplets. They may also coalesce to form larger droplets and longer fibres. A dynamic equilibrium involving droplet deformation, break down and coalescence may establish itself during mixing of a dough. This could be exploited to control the formation of dongh structural properties. Three main structural elements of doughs may be formed by spinneretless spinning, namely gluten fibres, spherical liquid and gas particles, and oriented liquid- and gas-filled filaments (capillaries). The continuous gluten gel phase between oriented capillary structures is made of non-cylindrical fibres typical of gluten strips. Normally, an increase in mixing intensity leads to mechanical degradation of liquid filaments and the formation of smaller spherical particles. This corresponds to the main objectives of dough mixing, punching and

Thermodynamics of dough formation andfunctionality

remixing, which are: (i) to increase the quantity and decrease the size of gas bubbles ; (ii) to establish an equilibrium between the liquid and gel phases of the dough by thixotropic destruction-regeneration of the gluten gel network; and (iii) to stretch gluten strands. Larger filamentous gluten particles may be formed more easily when the gluten phase and the liquid proteinpolysaccharide phase have approximately the same volume fraction . In this area of composition, deformation could lead to the formation of a system with two continuous phases. The liquid dispersed phase seems to be partly squeezed onto the surface of the dough during mixing and moulding . This affects the dough stickiness. The formation of capillary structures during dough sheeting and rolling, together with squeezing from capillaries and dewatering of the liquid continuous phase during baking, can lead to an open porous bread structure. In the case of 100% humidity of steamed bread , this could result in the formation of a smooth bright surface on the loaf by the liquid phase components. Another result of dough mixing and handling is the thinning of gluten fibrils by spinneretless spinning, and an increase in the orientation of polypeptide chains during deformation of the fibrils. The density of cohesion energy of protein-protein interactions increases during the formation of dough from hydrated flour. The intensity and time of dough shearing are responsible for the formation and reinforcement of the hydrophobic network of the gluten phase, and for the size and shape of the liquid protein-polysaccharide phase. The shear force gradient (mixing speed and work input) during mixing is directly related to the quality of dough and bread . An excessive elongation of viscoelastic gluten strands could result in elastic recovery, e.g. shrinkage of the overmixed doughs and dough contraction following sheeting. Granule migration Figure 10 gives a descriptive model of starch granule migration towards the central layers of a flowing suspension. This figure shows that shear flow of a dough is fastest through the central part of the tube and slowest near the walls. According to Bernouilli's principle, when the velocity of shear flow is greatest, the pressure is least. This means that pressure is lowest in the centre and highest close to the walls. Since there is a difference in flow velocity on both sides of the starch granule, a net 'upward' force is experienced by the granule moving it to the centre. In other words, starch granule migration in flowing doughs results in starch concentration increases in central layers and decreases in surface layers. An increase in concentration of solid semi-crystalline starch granules in the centre of extruded pasta products means a decrease in water content at the centre of the product and favours drying . Granule migration can also form 'starch-empty' and 'protein-full' surface layers. This is of great importance for, for example, cooking quality and reducing the stickiness of pasta products. Because of the velocity gradient in viscous shear flow, a

I---!!.!!!!!'----

191

Greater pressure

shear flow

Figure 10 Scheme of Bernouilli's principle. An upward force (lifting) moving starchgranules.

solid starch granule could rotate in the flowing dough, act as a lubricant and thereby decrease the viscosity of the dough . Rotation of granules can be regarded as a ' ball-bearing' effect, providing high fluidity of doughs. This effect could be responsible for a decrease in internal stresses in the gluten matrix resulting from rapid drying of pasta products, and prevention of the formation and propagation of cracks. This 'ball-bearing' effect (produced by small granules) can also be of importance for fat mimetics in foods and for cosmetic starch powders.

Conclusion This paper is an attempt to link the structure formation processes and contributions of structural elements at different structural levels (molecular, supermolecular and macroscopic) to dough functionality. The objective of the paper is to stimulate interest in thermodynamic and microrheological approaches, using model systems, for a better understanding of structure formation in foods. This study suggests that the phase behaviour of biopolymer mixtures and excluded volume effects of macromolecules are the key factors influencing structure formation in foods.

Acknowledgements The author is indebted to Mr Stephen G.Collyer and Dr Elizabeth Prior for valuable discussions and editing of the manuscript.

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