Polysaccharide Association Structures in Food (Food Science and Technology)

Polysaccharide Association Structures Food Reginald Geneva,

Walter Yo&

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Polysaccharide association structures in food / edited Reginald H. Walter. p. cm.- (Food science and technology; 87) Includes bibliographical references and index. ISBN 0-8247-0 164-X (alk. paper) 1. Polysaccharides. 2. Macromolecules. industry and trade. I. Walter, Reginald Henry. Series: Food science and technology (Marcei kkker, Inc.); 87. TP248.65.PWP65 664-dc2 1 98-2762 CIP

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Polysaccharides are the basic skeletal component of plant tissues. Inasmuch as polysaccharides do notdepolymerizesubstantiallyduringextractionand purification, isolated polysaccharides, through their macromolecular assemblies, are exploited in the creation of structure and texture, in vitro, in aqueous media, with particular applications in fabricated foods. They frequentlyperforminterchangeably,althoughsubtledifferencesmayelicit different,sometimesunique,behavioramongmembersandisomers,under identicalcombinations stimuli.Thespontaneoustendency dispersed polysaccharidesolutes to aggregatecohesivelyandadhesivelywithother supramolecules. This treatise molecules impels them toward the creation focusesonthephysicochemicaloriginsandstructuresformedbythe aqueous,dispersedpolysaccharideswithlikeandunlike association chemical species. Reginald H. Walter

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Preface iii Contributors vii

1. Origin

PolysaccharideSupramolecularAssemblies1

Reginald H. Walter

2. PolysaccharideMolecularStructures15 David Oakenfull

3. GelFormationandUltrastructureinFoodPolysaccharides37 Tokuya Harada and Akira Harada

StructuresandPhaseTransitions of StarchPolymers 57 Costas G. Biliaderis

5.

MicrocrystallineCelluloseTechnology169 Gregory S. Buliga, George W Ayling, Gregory R. Krawczyk, and E. J. McGinley

207 6. Cyclodextrins Z. Helena Qi and Matthew L. Romberger 7. Starch-LipidInteractions

227

Ratnajothi Hoover

8. Interactions in Whey ProteinPolysaccharide Mixtures at pH 7 257

I?

B. Fernandes

9. Polysaccharide-MetalInteractions Monique A. V Axelos

273

Rheology Structured Polysaccharide Food Systems: Starch and Pectin 289 Paul E. Okechukwu and M. Anandha Rao Index 329

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Monique A. V. Axelos InstitutNational de la RechercheAgronomique, Nantes, France George W. Ayling Food Ingredients Division, FMC Corporation, Princeton, New Jersey

Costas G. Biliaderis Department of Food Science and Technology, School of Agriculture, Aristotle University, Thessaloniki, Greece Gregory S. Buliga Food Ingredients Division, FMC Corporation, Princeton, New Jersey

P. B. Fernandes*

Nestec Ltd., Nestle Research Centre, Lausanne,

Switzerland

Akira Harada Department of Macromolecular Science, Graduate School of Science, Osaka University, Osaka, Japan TokuyaHaradat

OsakaUniversity,Osaka,Japan

Ratnajothi Hoover Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland, Canada Gregory R Krawczyk FoodIngredientsDivision, Princeton, New Jersey

FMC Corporation,

E. J. McGinleyt Food Ingredients Division, FMC Corporation, Princeton, New Jersey 'Current affiliation: Friskies R&D Center Amiens, Amiens, France. 'Deceased.

David Oakenfull CSIRO Division of Food Science and Technology, North Ryde, New South Wales, Australia Paul E. Okechukwu* Department of Food Science and Technology, Corne11 University-Geneva, Geneva, New York HelenaQi Centerfor Expertise-Cyclodextrins, CerestarUSA,Inc., Hammond, Indiana M. Anandha Rao Department of Food Science and Technology, Cornell University-Geneva, Geneva, New York Matthew L. Romberger Sales and Market Development, C*CAVITRON@ Cyclodextrins, Cerestar USA, Inc., Hammond, Indiana Reginald H. Waltert Department of Food Science and Technology, Corne11 University-Geneva, Geneva, New York

*Current ufiliation: The Federal Polytechnic, Oko, Nigeria. 'Retired.

Origin of Polysaccharide Supramolecular Assemblies Reginald H.Walter* Cornel1 University4eneva Geneva, New York

INTRODUCTION Nature is adept at weaving (practically) one-, and three-dimensional structures (Fig. 1) from aqueous, dispersed polysaccharides, through whose instrumentality it imparts form and function to living plant tissues, to wit, the cellulose skeleton of fruits and vegetables. This biopolymer is deposited naturally in bundles of microfibrils that constitute the fibrous and ribbonlike suprastructures of vegetable matter.Native starch is depositedmostly as spheroidal granules. Arguably, macromolecular assemblies with aquatic and microbial polysaccharides, in vivo, have teleological significance. Polysaccharide supramolecules may also be the product of purely chemical synthesis. The natural and chemically synthesized polysaccharides and their derivatives are collectively called gums. This unconventional class of substances is increasinglyrelied on to impartcoherence,body,andtexture to fluid substrate, in vitro. The singular property common to its members is their amphiphilicity, albeit to different degrees, which enables them to associate with polar and nonpolar molecules alike in water (surfactancy). The extent of the surfactant associations is determined by the net force generated as a result of solute-solute,surfactant-solute,andsurfactant-solventinterac*Retired.

1 Natural plant structures: (left to right) partial fascicle of white pine (Pinus stobus), a portion of the leaf base of the coconut palm (Cocus nuciferu), and the loofah of a gourd (Lufla uegyptiaca).

tions and by thermal energy [l].Ionic polysaccharides experience additional coulombic attraction and repulsion. Aqueous polysaccharides display a continuum of disorder-order-disorder and order-disorder-order transitions that ultimately yield flocs, pastes, aggregates, gels, and precipitates.The final outcome is dictated more bythe solute’s aqueous environment and external stimuli than by its chemistry. It is not unusual for different gums to exhibit the same response under a given set of conditions and for the same gum to respond differently to different combinations of stimuli.

II. Some definitions are a necessary precedent to elaborating the tendency of gums to associate in aqueous media. A polymer, synonymouslycalled mucromolecule, is a molecule, 10-103 nm diameter, containing 106-1012 chain atoms [2].Thus, size is conventionally characterized by the degree of polymerization @P) as well as by the molecular weight. The DP, then, is the lengthofapolymer’sunsubstitutedprimarychain,countedinmonomer units. Oligosaccharides are differentiated from polysaccharides by a cutoff molecular weight of 10-20 monomers [3,4]. Chemically, polysaccharides mostly linear polyhydric alcohols, polyacids, or polyesters, consisting of a primary structure that is subject to various configurations (the secondary structure), depending on bond lengths

and angles, theDP, branching, heterogeneity, andsensitivity to the dispersion medium. The topology of a linear polymer is the average of multiple equilibrium geometries of the primary structure. Long, single-bondedchains are conducive to flexibility, and short chains, to rigidity. Unsubstituted primary chains facilitate parallel orientations and contacts between segments of different chains. An amphiphile, containing hydrophobic and hydrophilic groups in the same molecule, enables amphiphilicity to be empirically referenced against a quantifiable tendency to promote oil-in-water and water-in-oil emulsions; this is the hydrophile-lipophile balance [S]. Amphiphilic chain segments resultinorderedmicellaraggregation in solution or bulk[6].Dispersed polysaccharide solutes are prone to micellar aggregation, as a consequence of the high incidence of polar groups [hydroxyl (-OH),carboxyl (-COOH), and/or sulfonic (-OS0,H) groups]: -COOH is common in uronans, and -OS03H in carrageenans. micelle is defined as a spontaneously formed aggregate of tens to hundreds of surfactant molecules or ions Trifunctionality in a polysaccharide monomer accommodatesaggregation by threedimensional crosslinking. The association tendency of some polysaccharides is altered by chemical derivatization, notably esterification and etherification, and that of others by deesterification. Starch and cellulose, for example, are alkylated and hydroxyalkylated to improve performance over the parent molecules’. Pectin is a deesterified biopolymerof protopectin, the native precursor. Neitherthe original nor the derivatized species is truly water-soluble in the classical sense of a monophase; instead, they disperse to a macromolecularly heterogeneous system [8]. polymer molecule in a flow field devoid of adsorbed solvent, traveling at a different rate from the bulk solvent, is said to be free-draining. non-free-draining polysaccharide moleculehas an adsorbed envelope of water traveling at the same rate as the solid core. Non-free-draining water (water of hydration) is therefore an integral part of the discrete colloidal nucleus. Hydrodynamic interaction refers to the influence of the motion of a molecule or segment and its water of hydration on other molecules or segments. Free-draining water is the overwhelming volume of solvent in a solution or dispersion. Inasmuch as free-draining water is not in colIoida1 association with the discrete nucleus, and therefore almost always traveIs with a different velocityfrom it, any identicalness of transport of the discrete nucleus and free-draining water is purely coincidental. Free-draining water is not affected by hydrodynamic interaction. Water of hydration has astabilizing effect on dispersed through a number of different mechanisms [8].

Dispersed polysaccharides structure water at the solid-liquid interface where the water becomes non-free-draining. The influence of a polysaccharide is exercised at concentrations as low as fractions of a percent, where at the binary dispersion can assume the character of either a liquid or a solid, under the influence of solvent conditions and temperature. A dilute dispersion is considered to be one in which there is no solute-solute interaction; in such a “dilute” regime, dispersion properties are additive of the contribution of single molecules. At the highest weight or volume percentagesthe concentrated regime-each property loses its concentration dependence, and, behaving like a polymer melt, the dispersion displays thixotropy, viscoelasticity, or elasticity. At intermediate concentrations,linear dispersedsolute entangles and interpenetrates and hence exhibits unique properties. The critical micelle concentration (c*) is that concentration at which macromolecular properties of a dispersion cease to be concentration-dependent, and the properties obtain from clusters of molecules acting as single hydrocolloidal units. Molecules and segments of a dispersed polysaccharide occupy an excluded volume (vex)that is inaccessible to other segments and molecules. The v,, effect is the net of divers forces acting on the segment or molecule in vex. The free volume (v,) is that vacant volume available for occupancy by any segment or molecule. The volume of a dispersion (V) is the sum of the excluded volume and the free volume:

A large v, is indicative of much space for independent molecular motion; conversely, a small v, suggests a higher probability of polymer-polymer contacts. At the macromolecular level, Eq. (1) is temperature-dependent.The glass transition temperature (T,) is the temperature where v, is zero or constant and translational and rotationalmotions cease, relative to the expanded chain’s motion above T, where Brownian activity engenders increasing v, with increasing temperature. Below Tp v,, expands only as ‘a result of the higher amplitudes of the thermal vibrations Orderedpolysaccharide assemblies (e.g., rods andhelices)showa lower c* than do the disordered assemblies (random coils). Cellulose and cellulose derivatives are inherently stiff rods, because of the @-glycoside bonding and the steric hindrance resulting from the substituents, thereby initiating resistance to segmental rotations. Starch and noncellulose gums freely adopt flexible shapes, because of widespread amorphism. Random coils, rigid rods, stiff chains, etc., define a tertiary structure (conformation). In the vicinity of c* at a critical temperature, modulated by solution conditions, tertiary structures grow into quaternary structures, insofar as points of contact or junction zones multiply into micelles, aggre-

Supramolecular Assemblies Polysaccharide

5

gates, clusters, vesicles, gels, films, and crystals.Clusters of ordered surfactants in oil/water emulsions are typically molecules [lo]. Vesicles are self-ordering closed bilayers wherein amphiphiles are sandwiched between two liquid layers thousands of angstroms apart [ll].Gels are given many definitions, but the common feature is elasticity in the semisolid structure. Gels are the most common form of food polysaccharides. A hydrogel is a dispersion structured as a discontinuous, solid phase in a continuous water phase; a xerogel is a dehydrated hydrogel. Films are two-dimensional xerogels. The mechanics of the macromolecular associations initiating these structures include interpenetration of random coils,multiple coupling of single and double helices, orientation of rods, and electrostatic crosslinking in homogeneous and mixed junction zones. The quaternary ordering of rods and stiff chains along the molecular axis eventuates into mesomorphic phases that are intermediate between a liquid and a crystalline solid. These mesomorphs, or liquid crystals, remarkable for their appearance as a solid at rest and as a liquid when disturbed, possess unique features, e.g., dichroism and birefringence.Their generally depends on chain rigidity[l21 that in turn is a complicated function ofsideand main-chain lengths, side-chain chemistry, and temperature [13]. Mesophases are classified as lyotropic, if the self-organization results from interaction with an amphiphilic surfactant cosolute, which enables them to be disordered by solvent action; they are classified as thermotropic, if the selforganization develops in an usually narrow temperature interval. A linear, random-coil polymer has been theoretically modeled after a sphere. It should be recalled that a sphere has the lowest area/volume ratio of all known geometries. The normally hydrated polysaccharides are preferably represented by an equivalent hydrodynamic sphere that is assumed to have properties andexhibit responses identical to those of its linear counterpart. Spherical [l41 and circular [l51 polysaccharide suprastructureshave been observed experimentally.The volume of this equivalent hydrodynamic sphere is delimited by the hydrodynamic radius, Rh, the radius of gyration, R,, or the rootmean square end-to-enddistance, (r2)IR,i.e., the average distance between the two ends of a chain. The relationship between R,,, R,, and is as follows [9,16]:

The squared function averages the distances of all solute molecules in a positive and negative direction from the center of mass. Water of hydration creates long distances between non-free-draining coils, giving a longer Rh and R,. Rodlike polysaccharides are dimensionally thicker,i.e., give a shorter

Walter (?)ln for the same mass. Polyelectrolytes are postulated over a wide range of concentrations to be infinitely long cylinders on which exists a critical charge density that controls the polyanion-counterion equilibrium distance in solution [17].Because R,,, R, and are difficult and expensive to measure, dispersed polysaccharides are more frequently characterized by the easier, less expensive rheological methods. A fluid may be distinguishedby laminar shear flow-meaning a force per unit area (pressure, synonymous with stress, IT) is imposed tangentially on parallel fluid planes of unit thickness, causing each plane to be transported in a gradient ofunitdistanceperunittime,and yielding a mass.distance"time" coefficient called the shear viscosity. Rodlike polysaccharides have higher intrinsic viscosities than random-coil polysaccharides at the same molecular weight. deformation ofthe fluid by IT^ without transport, is defined as the strain (E), resulting from a series of "jumps" of the molecules from hole to hole [g] or U, subsites. Viscoelasticfluids recover E, up to a critical maximum (the yield point, IT,), whereupon viscous transport begins, in an attempt bythe fluid to assume a new equilibrium position. An elastic fluid does not have a IT,; when IT is withdrawn, it reverts to its original shape in a finite, albeit occasionally long, timeinterval (relaxation). Rods have longer relaxation times than random coils [18]. Elasticity is characterized by Hooke's equation:

IT

= GE

G is the modulus of elasticity elastic modulus, rigidity modulus, bulk modulus, stress relaxation modulus, distinguished by the experimental design). E has dimensions of linear, angular,or frequency displacement. Equation is quite sensitive to heat. Weak gels are characterized by a small G, and strong gels, by a large G . The effect of commingling unlike polymers in a single solvent may be additive, complementary, synergistic, or antagonistic. additive property is the sum of the property of individual polymers. In complementarity, one molecular species of a polymer provides a property that the other polymer lacks. Synergism is the effect of the combined population of molecules greater than the additive effect. An antugonistic ternary dispersionmanifests solute incompatibilitybyaphase-separation process called coacervation, whereby one of the two phases (coacervates) contains a major concentration in the solvent of one solute and a minor concentration of the other, and the (usuother phase, vice versa. Coacervation of a polysaccharide and a protein allygelatin) is employedindustriallyto encapsulate delicate flavorand aroma compounds. A polysaccharide polyanion is sensitive to small changes in electrolyte concentration, including acid. For this reason, ionic polysaccharides exhibit

two regions of viscosity with increasing concentration. The first region (the electroviscous region), involving polysaccharide-water interaction, declines to a minimum as the negative charges become progressively more screened by counterions; the second region involves polysaccharide-polysaccharide interaction to c*, after complete screening of the polyanionic charge. Cyclodextrins are synthetic polysaccharidesmadebycyclization of sugar monomers. The ring structure is formed in a manner that orients the hydroxyl groups on the outside and the hydrogen atoms on the inside, with the result that the hollow interior is conducive to hydrophobic interactions: for example, P-cyclodextrin is added to liquid food for the purpose of complexing and removing undesirable lyophilic compounds. Clyclodextrin molecules also aggregate to form supramolecular structures.

Entropy is an empirical index ofthe amount of energyin a system,including adispersion,that is unavailable for exchange with its environment. The higher the index, the more unavailable energy is for exchange and the more highly disorganized the dispersed solute is in the dispersion medium. The dissolution in water of a polysaccharide xerogel requires a form of supplied through heating, shearing, agitation, sonication, etc. Homogenization is amechanical process of introducing +hE by forcing a suspensionthroughmicron-sizeorifices. The process results in ahigher count of smaller particles and hence a largersurface area. Thermal and other mechanical processes defibrillate double helices. The surface tension of a solid or liquid is an unbalanced force per unit area, directed toward the solid or liquid interior, measured against air. This net force is alternatively definedas the isothermal work necessary to increase a surface by 1 cm’. Work is the product of force anddistance.Having magnitude and direction, surface tension is a vector quantity. The interfacial tension is the surface tension measured at an interface other than air, i.e., a solid-solid, liquid-solid, or liquid-liquid interface. The surface tension can be lower than 10-l’ N and still influence the properties of a hydrocolloidal system [19]. The surface tension of solids is negligible. As aresult of surface tension,many small and large molecules in solution or dispersion become adsorbed at a polysaccharide-water interface. Cosolutes, electrolytes, and solvent molecules engage in typically monopole-dipole, dipole-dipole, ion-dipole, and ion-ion interactions at the adsorbent surface. Although theseinteractions are theoretically bimolecular, the large volume of free-draining water normally present in a purely

+hE

Walter

binarypolysaccharide-water dispersion effectively makes themunimolecular. The adsorption of small molecules on a polysaccharide surface at a polysaccharide-water interface terminates at an equilibrium monolayer concentration, but the adsorption of macromolecules is multilayered and indefinite. Based on experimental observation, it is widely believed that incompletely saturated surfaces are attractive and prone to bridging flocculation, whereas completely saturated surfaces are repulsive and prone to steric stabilization [20]. The temporary storage site of +AE is the expanded surface area (A) of the dispersed, disaggregated, or defibrillated solute:

AE = uA

(5)

is the surface tension. Homogenization stabilizes a dispersion not only by reducing the size of the discrete solute particles, but also by increasing A . The process transforms a polysaccharide dispersion from a comparatively lower to a higher energy state. Notwithstanding the higher +m,the subsequent thermodynamically unstable state can remain kinetically stable for a duration by adding a protective colloid to the binary dispersion. There is a loss of entropy uponintroducing a protective colloid into the system. +AE spontaneouslydeclines,however, in conformity with the law of entropy stating that accumulated energy in any physical process is equalized with time between a system and its surroundings. condition of -hE begins to prevail by a decrease inthe areahohme ratio, as cohesion of solute particles exceeds adhesion betweenwater and polysaccharide. Says Israelachvili [19], the associated state of like molecules is energetically preferred to the dispersed state. Alternatively stated, there is always an effective attraction between like molecules or particles in a nonantagonistic, multicomponent mixture.Macromolecular assemblies are reversible with energyintervention. Micronized solute may remain suspended indefinitely, depending on the density difference between the dispersed phase and water. This is the case of an aqueous suspension of microdefibrillated cellulose [21]. Ionic polysaccharides conceptually order themselves in water into a primary layer of polyanions surrounded by a secondarylayer of counterions. This organization generates a dispersion-stabilizing electrochemical potential-the Zeta potential. Electrolytes electrokinetically disrupt this doublelayer organization (and, as a result, the Zeta potential) by shortening the interaction distance between the two layers of opposite charges. Nonsolvents, e.g., alcohol and acetone, also disrupt the organization, but by lowering the dielectric constant of the aqueous medium. Collapse of the double layer accelerates neutralization of the primary charge and an ensuing high incidence of solute-solute interaction, penultimately to aggregation, gelation, and precipitation.

Supramolecular Assemblies Polysaccharide

9

Small quantities of ionic polysaccharides may destabilize a dispersion through bridging flocculation, whereby a “bridge” is constructed between two or more primary solute molecules, causing a larger structure to precipitate. Divalent ions are efficient “bridges” for cooperatively,bonding linear polyanions (A-?. In cooperative association, the first bond autocatalyzes a series of identical interactions until A[(x/2)MJis complete. Bridging flocculation is particularly useful in water clarification.

It is universally accepted that a polymer in dilute solution behaves as an ideal gas, in compliance with the Charles-Boyle combined law stating that

is the osmotic pressure, diis the density of particles each with volume vi, and T is the absolute temperature. If ni is the number of dilutely dispersed polysaccharide random coils, then

Nci n.= ‘ M v,, = nivi

N is Avogadro’s number, and ci is in gramsiliter. Since water has a low compressibility (0.000048 per atm at 20°C), M, nivi, and T are constant, but and vi separately and in Eqs. are.variable. Accordingly, at a constant temperature, for a given quantity of solute, either ni or uichanges with changes in if n, increases, vi decreases and A increases (homogenization). +AI3 is stored in the larger surface area, after being partly compensated for by the stabilizing influence of the interfacial tension of the adsorbed water. Brownianmotion brings the equivalent hydrodynamic spheres into close proximity, where, at an equilibrium distance, the collision diameter is twice the radius (R;) of each sphere. At R;, the particles’ energy status is a primary minimum corresponding to the maximum possible state of thermodynamic stability, barring fusion. Distances shorter than R; are impermissible, except by an endothermic chemical reaction. In contradistinction

v.,

Walter

+hE

to thermodynamicstability, kinetic stability is a condition of of a dispersed solute, maintained by a variety of physical means, e.g., micronization, solvent viscosity enhancement, and protective colloid action. At constant pressure and temperature, the law of entropy is accommodated by aging-a time-dependent condition. Just how the total surface area (Ai) is affected by particle growth is illustrated in Eqs. 10-12. For a constant volume hydrodynamic spheres (2 vi = with radius R;,

(t)

3 Ai = R;

Equations 10-12 comply with the law of entropy by lowering the ni count and enlarging vi, attendant with a heightened probability of segmentsegment contacts (particlegrowth). According to Eq. (12), the changing relationship between the volume and the surface area of dispersed polysaccharide solute is governed by the factor 3/R;. The same logic applied to a double helix = ~ITR;~/[(ITRA~~] gives the factor 2/Ri. X is only approximately constant, because the shift at the particle interface of a small increment of free-draining water to and from thelarge volume of nonfree-draining water, as R; changes. The concentration-dependence of IT in dilute dispersions complies with a power-law equation:

- = KT[(ci/Mi)+ PC:

+ P‘c: + - -3

Ci

K is the gas constant, Mi is the molecular weight of component i at concentration ci, and and are interaction coefficients (also called second andthird virial coefficients). At infinitely dilute concentrations inwhich properties are assumed to be free of effects, the dispersion is characterized by and = 0. At intermediate concentrations(the semidiluteregime), entanglements of flexible,random-coilpolysaccharidemolecules impose constraints on each other’s lateral and rotational motion, thus enabling them to flow only translationally (reptation), which consequently lowers the configurational entropy and raises the free energy of the confined molecules. *Note that wRL3 contains the dimensions of work (i.e., work = length’

X

distance).

Polysaccharide Supramolecular Assemblies

11

The Huggins equation [22] is another power-law equation commonly used to characterize dilute solutions of linear polymers: = [q] + k[q12ci qJci is the viscosity number,[q] is the intrinsic viscosity, andk, the Huggins interaction coefficient, was inserted account for the sizes, shapes and cohesional properties of long-chain molecules. In the semidilute regime, the viscosity of a polysaccharidedispersion is many orders of magnitude higher than the linear relationship expected from Eq. The properties of an elastic mass are independent of ci.

The structuring of polysaccharides may be accomplished by manipulating the temperature.From 0 K upward, water and solute in mostpolysaccharide-water binary suspensions remain in a separate phase to an upper critical solution or upper consolute temperature (Tc),where they merge. In the same direction, some cellulose derivatives, for example, in a macroscopically homogeneous dispersion, remaina monophase to a lower T, where they and water exist separately. The lower T, of the cellulose derivatives is in the vicinity of 45°C.

Polysaccharide flocs, aggregates, gels, precipitates, etc., are fractal objects, not amenable to Euclidean geometric analysis, given their complex outlines. They are instead characterized mathematically by a fractal dimension [23]. The fractal dimension of a random-coil polymer is between and 2, and that of a gel (casein and gelatin) is 2-2.3 [24].

Any manipulated stimulus, e.g., high molecular weight, high concentration and high pressure, electrolytes, cosolute and nonsolvent additions, and low temperatures is capable of enhancing the probability of solute-solute interation and,ultimately, of structuring apolysaccharidedispersion on the solute’s way toward thermodynamic equilibrium. This energy state is in-

imical to functionality, conditions are made favorable for the solute to remain indefinitely in a state of kinetic equilibrium with its dispersion medium: such conditions may include homogenization, adding thickeners, and maintaining high storage temperatures. The structures formed are not easily characterized, except by rheology and fractal dimensional analysis.

1. Mukejee, P. Differing patterns of self-association and micelle formation of hydrophobic solutes. In:vanOlphen, H., andMysels, K J.,eds., Physical Chemistry: Enriching Topics from Colloid and Surface Science. International Union of Pure and Applied Chemistry Commission I. 6, Colloid and Surface Chemistry, Theorex, La Jolla, Calif. (1975). 2. Biben, T.,Hansen, J.-P., and Liiwen, H. Some aspects of the statistical mechanics of concentrated colloidal suspensions. In: Chen, S.-H., Huang, J. S., and Tartaglia,P., eds., Structure and Dynamics Strongly InteractingColloids and Supramolecular Aggregates in Solution, Kluwer Academic, London, p. 23 (1992). 3. Munk, P. Introduction to Macromolecular Science. Wiley Interscience, New York, p. 7 (1989). 4. Ikan, R.Natural Products: A Laboratory Guide. Academic Press, New York, p. 76. (1991). 5. Vold, R.D., and Vold, M. J. Colloid and Interface Chemistry.Addison-Wesley, London, p. 397 (1983). 6. Finkelmann, H., and Jahns, E. Liquid crystalline phases of polymers. In: ElNokaly, M. A., ed., Polymer Association Structures: Microemulsions andLiquid Crystals. ACS Symposium Series 384, Amer. Chem. Soc., Wash., D.C., p. 19 (1989). 7. Schramm, L.L. The Language of Colloid and Interface Science. American Chemical Society, Washington, D.C. (1993). 8. Walter, R.H. Polysaccharide Dispersions: Chemistry and Technologyin Food. Academic Press, New York (1997). 9. Cowie, J. M.G. Polymers:ChemistryandPhysics of ModernMaterials. Blackie Academic & Professional, New York (1991). 10. Domb, C., and Lebowitz, J. Preface to Vol. 16. In: Gompper, G., and Schick, M., eds., Self-Assembling Amphiphilic Systems. Academic Press, New York, p. ix (1994). 11. Gompper, G., and Schick, M. The phenomena and approaches to them. In: Gompper, G., and Schick, M., eds., Self-Assembling Amphiphilic Systems.Academic Press, New York, pp. 4ff (1994). 12. Guo, J.-X., and Gray, D. G. Lyotropic cellulose liquid crystals. In: Gilbert, R. D., ed., Cellulosic Polymers Blends and Composites.Hanser Publ., New York, p. 29 (1994).

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13. Fukuda, T., Takada, A., and Miyamoto, T. Thermotropic cellulose derivatives. In: Gilbert, R. D., ed., Cellulosic Polymers Blends and Composites. Hanser Publ., New York, pp. 47-55 (1994). 14. Stokke, B. T.,Elgsaeter, A., and Kitamura, S. Pregel cluster formation in gelling polysaccharides visualized by electron microscopy. Polym. Gels Networks 173-190 (1994). 15. Zhao, J., and Whistler, R. L. Spherical aggregates of starch granules as flavor carriers. Food Technol., July, pp. 104ff (1994). 16. Tanford, C. Physical Chemistry of Macromolecules. Wiley, New York, p. 344 (1961). 17. Eisenberg, H. Biological Macromoleculesand,Polyelectrolytes inSolution. Oxford University Press, Oxford, England, pp. 233 ff (197x). 18. Doi, M., and Edwards, S. F. TheTheory of PolymerDynamics. Clarendon Press, Oxford, England, pp. 140, 289 (1986). 19. Israelachvili, J. N. Intermolecular and&$ace Forces, 2nd ed. Academic Press, New York, p. 172 (1992). 20. La Mer, V.K, and Healy, T. W. Adsorption-flocculation reactions of macromolecules at the solid-liquid interface. Rev. Pure Applied Chem. 13: 112-133 (1963). 21. Weibel, M. K Microdisassembled cellulose as a new food ingredient. Abstract, International Hydrocolloids Conference, Sept. 6-10, Ohio State Univ., Columbus, Ohio (1994). 22. Huggins, M. L.The viscosity of dilute solutions of long-chain molecules. W. Dependence on concentration. J. Am. Chem. Soc. 64: 2716-2718 (1942). 23. Borman, S. Fractals offer mathematical tool for study of complex chemical systems. Chem. Eng. News (April 22nd): 28-35 (1991). 24. Birdi, K.S. FractalsinChemistry,Geochemistry,andBiophysics. Plenum, New York (1993).

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Polysaccharide Molecular David OakenfhH CSIRO Division

Food Science and Technology North Ryde, New South Wales, Australia

Chambers’ Dictionary of ScienceandTechnology [l1 defines molecular structure as “The way in which atoms are linked together in a moIemle.” Structure is fundamental to our understanding of biopolymers. The major biopolymers-nucleic acids, proteins, and polysaccharides-have well-defined structures. In the nucleic acids, the sequence of the nucleotide bases defines the genetic information, andthe polymer as a whole adopts the wellknown double helix structure. Proteins can have an almost infinite variety of structures, depending on the functional role the protein in the living organism. They can be as diverse as keratin (hair and fingernails) and lysozyme (the water-soluble enzyme present as an antiseptic intears) [2]. Polysaccharide structures are less varied than those uf proteins, hence the morelimited role of polysaccharides in living systems. Polysaccharides nonetheless can have well-defined structures that vary with the functional role of the polysaccharide in the living organism. These functions are nearly as varied as thase of proteins. Polysaccharides canbe structural components of the cell walls ofplants, or theycanbeinvolved in antigen-antibody recognition and hormone receptor interactions, or they may simply act as energy reserves. Because of their biologicalimportance, proteins have been studied more intensively than polysaccharides. It is therefore instructive to compare the structural chemistry of polysaccharides with the more familiar structural

chemistry of proteins. Linderstrom-Lang tural organization in proteins:

Primary structure

Secondary structure

identified four levels of struc-

The amino acid sequence. There are 20 common amino acids. The number and sequence of these linked together by peptide bonds constitutes the primary structure of the protein. The regular arrangements of the polypeptide backbone, without reference to the amino acid side chains. The most regular structures are the a-helix and the P-pleated sheet -stabilized by hydrogen bonds between the carbonyl and imide groups of the backbone:

>c=o... HN< Tertiary structure

Quaternary structure

The three-dimensional structure of the protein molecule as a whole. Typically there might be separate regions of ahelix structure or P-pleated sheet. Interactions of the individual amino acid side chains (hydrogen bonds, hydrophobic interactions, electrostatic forces) now become important in maintaining the stability and structural integrity of the folded protein. The structures of aggregates of globular proteins.Hemoglobin (a tetramer) is a well-known example of an aggregated protein.

The same levels of structural organization also occur in polysaccharides. Polysaccharides may contain a number of different monosaccharide units. (The most commonly occurring of these are shown in Fig. 1.) The numberanddistribution of these along the polymer chain constitute the primary structure. These monosaccharides can be linked in different ways that restrict their freedom of motion with respect to each other and impose secondary structure on the polymer chain. Where there are regularities in primarystructure,polysaccharidescan adopt sterically regularsecondary conformations, e.g., helices or ribbons. These ordered structures can sometimes further aggregate to form tertiary, crystal-like structures, as in cellulose. Association of ordered tertiary structures (e.g., double helices) may lead to quaternary structures, as occur when polysaccharides form gels. These four levels of structure are described in this chapter. But firstit is necessary to understand the nature of the monosaccharides units from which these structures are built.

Polysaccharide Molecular Structures

Hc)H

H

CH3

OH

A

H0

A

OH

H

H 0 OH

H

OH

a-Dglucose

H

H

a-Dmannose

H a-Dgalactose

COOH

CHpOH

NH2 a-Dgalactosarnine

Figure

BD-fructose

a-D-xylose

aD-rharnnose

H

OH

OH

a-Dgluatronic acid

a-larabinose

Structures of the more common naturally occurring monosaccharides.

II. Polysaccharides are composed linked monosaccharide units,of which the commonest is D-glucose. The structure of glucose is shown in Fig. 2. Glucose is an aldohexose, a six-carbon aldehyde, with five hydroxyl groups. The six carbon atoms are conventionally numbered from the aldehyde end, as shown in Fig. 2. This structure has four asymmetric carbon atoms:

CHO-C*HOH-C*HOH-C*HOH-C*HOH-CHZOH

A

I

2 Representations of D- and L-glucose in open chains and a-D-glucose and a-L-glucose in ring

which means that there are sixteen (24) possible stereoisomers;but only three of these occur in nature: mglucose, D-galactose, and D-mannose.Glucose is dextrorotatory; hence the designation “D.” But in other sugars the designation “D” does not necessarily mean that the compound is dextrorotatory. The convention is to use “D” to indicate sugars in the same stereochemical series, as shown in Fig. monosaccharide is of the D-family if the asymmetric carbon atom most remote from the carbonyl (reference) group has the same conformation as mglyceraldehyda (also shown in Fig. ‘The compound conventionally called D-fructose is in fact levorotatory (sometimes indicated by the name (D-(-)-fructose). Fructose is a ketohexose. It also has six carbons and five hydroxyl groups, but with a ketone group in the 2-position. When in solution, the monosaccharide chain takes upa ring form through a reversible intramolecular reaction betweenthe aldehyde group and one of the hydroxyl groups, forming a hemiacetal (also shown in Fig. 2). glycoside having a six-membered pyran ring is a pyranose, and one corre-

Polysaccharide Molecular Structures CH0

CH0

I

I

H?-OH CHpOH

HO-C-H I CHpOH

D-glyceraldehyde

L-glyceraldehyde

?H0 H-C-OH I H - C - O HH - C - O H

I

HO-C-H I

I I H-C-OH H-C-OH I I H - C - O H H - C - O HH - C - O H I I C HpOH CHzOH D-allose D-glucose

H-C-OH I HO-C-H I H-C-OH I I CHpOH

D-galactose

CH0 I

HO-C-H I HO-C-H H-C-OH I

H4j-OH CHpOH Dmannose

D-altrose

CH0CH0 CH0 I I HO-C-H H T - O HH 0 - p - IH T - O H I HO-C-H HO-C-H I I HO-C-H HO-C-H I I H-C-OH H-C-OH I I CbOH CHpOH D-talose

I

CH0 I H-C-OH I HO-C-H I H-C-OH I CH2OH Didose

Figure 3 Structures of D- andL-glyceraldehydeand from D-glyceraldehyde.

H-C-OH I HO-C-H I H-C-OH

I

CHzOH D-gulose

the D-hexoses “derived”

sponding to the five-membered furan ring is a furanose (Fig. For each of these ring forms, two isomers (anomers) are possible, because a new asymmetric center is created by the ring closure at the reducing carbon atom. h o m e r s are distinguished from each other by the Greek letters and p. For a cyclic D-aldohexose oriented in the standard way, the a anomer has

k pyranoside

furen

4 Pyranandfuranandthepyranoseandfuranose

furanoside

forms

a hexose.

the hydroxyl groups on C l projecting below the plane of the ring, while the anomer has the hydroxyl group directed above the plane of the ring. Thus for glucose, the a and forms of the ring have mirror-image stereochemistry at Cl, as shown in Fig. A freshly prepared glucose solution shows a specific rotation ([a],,) of +113O, which gradually decreases on standing to +52”. This establishment ofan equilibrium between each of the ring forms and the open chain is known as mutarotation (Fig. 5). In the case of solution of glucose at room temperature, the approximate relative proportions of each form is a-D-glucopyranose 35%, g-D-glucopyranose 61%, and a-D-glucofuranose and @-Dglucofuranose together It is clear from this distribution that the form is more thermodynamically stable than the a form, and the six-membered pyranose ring form is more stable than the five-membered furanose form. The component sugars of most food polysaccharides normally occur in the polymer chain in the six-membered pyranose ring form. The open chain form represents only 0.0026%, but it is nonetheless very important because it is this structure thatpossesses the active aldehyderesponsible for the reducing reactions of the monosaccharide. The ordinary, or a,form of glucose is obtained by crystallization from alcohol-water;the form is obtained by crystallization from a highly concentrated aqueous solution at elevated temperature (110OC). Mutarotation also occurs in oligosaccharides and polysaccharides, as long as the reducing end (aldehyde) is maintained. The ring structure is always shown in the chair conformation. Boat conformations are energetically unstable because of eclipsing of bonds and steric compression between the “peaks” (e.g., between substituents on Cl and C4), as shown in Fig. 6 The relative probability of adoption of the two chair conformations is determined by minimization of steric repulsion between axial substituents. This means that D-hexoses have almost exclusively the ,C, conformation and L-hexoses the ‘C, conformation, as shown in Fig. 7.

Polysaccharide Molecular Structures

zb I

gy

Figure Mutarotation of D-glucose in aqueous solution. At equilibrium, the approximate relative proportions of each form are a-D-glucopyranose 35%, P-D-~~ucopyranose 61%, and a-D-and P-D-glUCOfuranOSe together (see text).

Figure Pyranose ring forms. In addition to the two boatlike ring shapes shown, two further pairs of boat conformations are possible with C-2 and C-S or C-3 and C-5 above or below the plane of the other ring atoms.

a

a

a

D

Thepyranoseringgeometry of D and L sugarsintheirnormal(energetically preferred) conformations. These structures minimize steric repulsion between axial substituents (indicated by “a”). D-Hexoses have almost exclusively the 4C1conformation and L-hexoses the ‘C., conformation (see text).

Morecomplex structures are formed when hydroxyl groups from other monosaccharide molecules react with the hemiacetal, forming a glycosidic link, creating firstly a disaccharide and then ultimately a polysaccharide. In the case of D-glucose, a glycosidic link can be formed between Cl of one glucose moleculeand Cl, C2, C3,C4, or C6 or another, thatmany different structures are possible, at least in principle. The linkage is defined by the configuration at Cl (i.e., whether it is a or and the carbon atom of the ring to which it is linked via an oxygen atom. The familiar disaccharide sucrose is P-D-fructofuranosyl-a-D-glucopyranoside (Fig. It is formed from glucose and fructose linked a(l-.2), with the fructose in the furanose form (because of the positionof the keto-group)and in the p configuration. The covalent linkages between adjacent residues do not act as “universal joints” but are normally limited to a narrow range of conformational possibilities, thereby imposing preferred shapes, or secondary structure, on the geometry of the polysaccharide chain. The nature of the linkage has a dominant effecton the structure of the molecule. It is instructive to compare

CHzOH

OH

Structure of sucrose (~-D-fruCtOfuranOSyl-or-D-glucopyranoside).

O\

k

CH20H

/ wlloblose

H h

H

OH

OH

manose

cellobiose

Structures of the disaccharides maltose and cellobiose, showing how the linkage geometry influences the shape of the molecule.

the two disaccharides cellobiose and maltose. Both are dimers of glucose. In cellobiose the glucose units are linked p(1+4); in maltose theyare linked a(1-4). These molecules have different shapes, as shown in Fig. 9, and they have different physical properties (Table 1). The molecular structure of a disaccharide is therefore defined by the relative orientation of the component monosaccharide units.This, in turn, is

Maltose

Properties of Glucose Dimers: Cellobiose Linkage] Cellobiose

Melting point Taste Susceptibility to enzymic degradation

-

Maltose

~~

Decomposes at 225°C Not sweet Not fermented by yeast, maltase, or invertase

'Values before and after mutarotation.

Linkage] and

~~~~

-

Sweet Fermented by yeast and maltase

+

+,

defined by the rotational angles and as shown in Fig. 10; in addition, 1-6 linkages introduce the further possibility of rotation about the (C5)(C6) bond. For any two adjacent residues, the conformation energy associated with a particular pair of and values can be estimated using potential energy functions for van der Waals interactions, polar, torsional, and hydrogen bonding interactions. This is illustrated in Fig. 11, which shows the results of calculations for maltose and cellobiose [5]. Most conformations are energetically very unfavorable; the molecule is effectively trapped within a potential energy well that restricts its conformational freedom. The positions of the potential energy minima depend on the state of the moleculewhether it is in the crystalline state, in solution in a nonaqueous solvent, or in solution in water. (Hydrogen bonds strongly influence the conformation in nonaqueous solvents and in the crystalline state, but in water they are weaker because of competition from water molecules for hydrogen bonding sites.) The calculated positions of the potential energy minima (+ and values) agree well with experimentally determined structures. Polysaccharide structures are similarly determined bythe nature of the linkages between the monosaccharide units. The monosaccharides present are also important, but physical properties (such as solubility, viscosity, and gelation characteristics) are determined more bythe linkages than the nature of the monosaccharide units [6]. Figure 12 shows a chain of P(1+4)-1inked D-glucopyranose units. In solution the molecule is in continuous motion. If we allow free rotation about the C-0 bonds of the glycosidic linkages (as shown), the molecule is highly flexible. It can writhe and twist, continuously changing shape. A polymer molecule that behaves in this way is said to be a random coil. It has no well-defined shape or size. The onlymeaningfulparameter is the mean square end-to-end distance, This would typically be several orders of magnitude smaller than the molecule’s contour length (thedistance between the two ends when the molecule is stretched out to its fullest extent). In reality, however, interactions between adjacent units limit the freedom of

+

+

+

Rotational angles between adjacent monosaccharide units. Relative entations of adjacent monosaccharide units joined by glycosidic a linkageare defined by the two rotational angles 9 and I$.

-

180 -

90 -

60 -

0 -

I

l

I

0

4 Conformational mobilityof polysaccharide chains. Energetically feasible conformations of adjacent residues are confined to a narrow range of conformations illustrated here for cellulose. Shaded areas show conformations whose computed potential energy lies within 20 kJ/mole of the potential energy minimum (M). The probability of adoption of any higher energy conformation is less than 1 in 3000 at 25°C.(From Ref. 6.)

rotation, as we saw earlier for disaccharides. In this case, some conformations are energetically preferred over others. This is illustrated in Fig. which shows the structures of three glucose polymerswithverydifferentphysicalproperties.Cellulose, with linkages, is the major structural material of land plants, familiar in almost pure form as cotton. Its insolubility and its ability to form fibers are consequences of its regular structure, which enables the molecules to adopt ordered crystalline arrays of polysaccharide chains held together by hydrogen bonds (Fig. In contrast, amylose, with linkages, is water soluble and adopts a helical structure, forming gels (discussed in more detail below). Curdlan, linked is another gel-forming polysaccharide that forms helical structures. The mixed linkage “~-glucans”(present in barley and oat bran) have and linkages. This irregular structure prevents the formation of ordered crystalline arrays, in contrast to cellulose (also a ‘p-glucan’), these polysaccharides tend to be water soluble. Branching is introduced by disubstitution of a single residue. Branched structures, as in the arabinoxylans in wheat, are unable to adopt ordered

”\

C””

-c

P

r----=\

0

0

Three glucosepolymers. (1) cellulose linked); curdlan 1,3 linked); (3) amylose (a-1,4 linked). Cellulose is typical “type A” polysaccharides, which have a “trans” bonding arrangement across the sugar ring. Curdlan and amylose have “cis” bondingarrangementsandaretypical “type B” (see text).

4 l

l I

Hydrogen-bonded crystalline structure cellulose. Schematic diagram of the unit cell showing three the five cellobiose residues.

crystalline structures. Such compounds are also water soluble, as are polysaccharides with charged groups (COO- or SO;), such as the pectins and carrageenans. When charged groups are abundant along the polysaccharide chain, electrostatic repulsion prevents the molecules from packing close together in ordered structures Polysaccharide structures frequently show asimple repeating sequence. The carrageenans are based on a disaccharide unit of 3-linked P-D-galactose and 4-linked om-galactose, but with the different carrageenans having different degrees of sulfation of the monosaccharide residues and different extents of conversion of the 3-linked residue to the anhydride ring-form (Fig. 15). Similarly, pectin is essentially a homopolymer of cr(l+4)-linked galacturonic acid,but pectins of different origin showdifferent extents of methyl esterification. In general, structurally regular polysaccharides adopt sterically regular crystal-like conformations in the solid state that can be characterized by X-ray fiber diffraction.These structures correspond to fixed values of the rotational angles between adjacent residues (4 and $) rather than a statistical distribution of orientations, as for random coils in solution. Because the ring geometry is essentially fixed, it is convenient to treat the

SO3-

H

Structure and chain flexibility of the carrageenans. (1) (R = H) and L-carrageenan (R = SO;); Linkage of the anhydrogalactose residue (which is held in the ‘C.,chair form) is through equatorial bonds that allow considerable conformational freedom. These carrageenans form helical structures that crosslink to form gel networks. (2) X-carrageenan; in the absence the anhydride bridge, the preferred ring in 4C, and the linkage is then through axial bonds, imposing greater steric restriction on the chain geometry. X-Carrageenan does not form gels.

entire sugar ring as a single “virtual” bond [7]. classes of structure can be formed, again depending on the linkage geometry: Polysaccharideswith a “trans” bonding arrangement across the monosaccharide ring, termed “type A’, by Rees and Scott [8] (Fig. 13). These structures adopt ribbonlike conformations, of which cellulose is a typical example. Polysaccharides with a “cis” bonding arrangement across the ring, termed “type B” (Fig. 13). These structures from hollow helices, usually multistranded. and L-carrageenan andagarose form double helices; curdlan forms triple helices. These ordered conformations are stabilized by noncovalent interactions, particularly hydrogen bonds and sometimes electrostatic forces. It is important to emphasize that not all the molecules are in the ordered conformation. The system is always one of dynamic equilibrium, where the most energetically favored structure is statistically the most prob-

able, but other,less energetically favoredstructures are also present. Ordered conformations are destabilized by the loss of conformational entropy (that accompanies their formation), by structural irregularities, by branching, and by electrostatic repulsionbetweenchargedgroups. Opposing molecular forces are often delicately balanced, andsmall perturbations cancause abrupt change from an ordered toadisordered state (or vice versa). Figure 16 illustrates this effect for the case of K-carrageenan. In solution in water, inthe cold and in the presence of potassium-ions, K-carrageenan forms double helical structures. On heating, these structures “melt.” The molecules undergo a helix-to-coil transition, passing from an ordered state to the random-coil state at a well-defined temperature (Tc). This transition is accompanied by a change in optical rotation, as shown in Fig. 17. The process is reversible. On cooling, the K-carrageenan reverts to the helical structure, again at a well-defined transition temperature (Th).Qpically in such systems there is hysteresis, with a significant difference between T, and Th. Figure 17 also shows the effect of temperature on optical rotation for the same concentration of K-carrageenan in the absence of added potassiumchloride.In this case, there is nocoil-to-helixtransition. The added electrolyte is required to shield the negatively charged sulfate groups of the carrageenan as to reduce electrostatic repulsion enough to allow helix formation. If potassium chloride is added to a solution ofK-carrageenan at below the helix-to-coil transition temperature, the polysaccharide rapidly folds into the helical conformation. Norton and his colleagues used stopped-flow polarimetry to study this reaction and showed it to be second order, implying that the structure being formed is indeed a double helix, formed from a union of two molecules of K-carrageenan. Ordered structures are not necessarily continuous along the full length of the polysaccharide molecule. Often, odd monosaccharide residues occur along the chain that disrupt the ordered structure. For example, the carrageenan double helix is interrupted by the presence of 4-linked-a-~-galactose residues. For pectins,the ordered structure is interrupted by rhamnose,which introduces a “rhamnose kink” into the chain.

The familiar gelation properties of certain polysaccharides, such as pectin, agarose, and carrageenan (discussed in more detail in Chapter depend on the formation of a quaternary structure. In gels, the polymer molecules are aggregated into one immense, three-dimensional structure that “fills” the liquid. This continuous network gives a gel its unique rheological char-

Polysaccharide Molecular Structures

-

([I

.g a

20

30

60

70

temperature ("C) Change of optical rotation with temperature for K-carrageenan (2 gkg) with and without added potassium chloride (0.02 M). increasing temperature; A decreasing temperature; A no added salt.

acteristics, with both solidlike and liquidlike flow properties. This level of structure is best illustrated by looking at the aggregation behavior or three representative gel-forming polysaccharides: K-carrageenan-apolysaccharidewithcharged(sulfate) groups that forms helices Agarose-an unchanged polysaccharide that forms helices Alginate-a charged polysaccharide (carboxylate groups) that forms pleated sheets

A. Carrageenans As mentioned earlier, the carrageenans are sulfated polysaccharides containing essentially alternating copolymers of 19-linked P-D-galactose and 1,4linked 3,6-anhydro-a-~-galactose (Fig. 15). The linkage pattern introduces a twist into the molecule, giving rise to helical structures, as shown in Fig. 16. Gelation involves the formation of these double helices [lo]. (Singlestranded helices analogous to the protein a-helix are not stable.) Becauseof the ionic nature of the polymer, gelation is strongly influenced by the presence of electrolytes. The presence of a suitable cation, typically potassium or calcium, is an absolute requirement for gelation to proceed [ll].For both and K-carrageenan, the alkali metal ions pi', Na+, K+,Rb', CS') are all capable of inducing gelation, but K ' and Rb+ are considerably more effective than the other ions in inducing gelation at much lower concentrations of both the cation and the carrageenan [12-141. Anions also influence ge-

cturesMolecular Polysaccharide

33

lation [15], suggesting that at least part of the effect of these different salts is general and is mediated by the effects of different ions on the solvent properties of water. However, it is becoming increasingly clear that specific ion effects are also very important. Potassium ions, and possibly those of rubidium, because of theirsize, directly promote association of carrageenan chains.Other ions influence chain conformationandassociationthrough their effects on the solvent properties of water.

Like the carrageenans, this polysaccharide has a disaccharide repeat unit, in this case of alternating 1,3-linked p-D-galactopyranose and 1,4-linked 3,6anhydro-a-L-galactopyranose.Gelation occurs when hot agarose solutions are cooled to below about 40°C. The gels show a high thermal hysteresis, not melting until the temperature is above about 90”. As is the case for the carrageenans, gelation involves doublehelix formation [16].However, it seems that the agarose network involves much more than double helix formation. The high hysteresis, coupled with evidence from light-scattering, fluorescence depolarization studies and various other studies, suggests that junction zones are formed from substantial bundles of agarose chains [17].

Alginates have a more complex structure than the carrageenans and agar. They are, in effect, block copolymers consisting of a linear polyuronic acid backbone with three types of block structure-lY4-linked p-D-mannuronic acid(M), lY4-linkeda-L-guluronic acid (G),andmixedMG blocks containing both uronic acids [17]. Full characterization of the polymer would therefore require knowledge of the M/G ratio, the ratio of M, G, and MG blocks, the detailed composition of the MG blocks, and the length distributions and relative positions of the blocks. Divalent cations are required for gelation, and, unlike the carrageenans, the gels are not thermoreversible. The gel strength depends on the nature of the divalent cation with the order Ba2+>S?+>Ca2’>>Mg2‘. The junction zones probably have the “egg box” structure (Fig. 18) proposed by Grant andcoworkers [l81 based on evidence from circular dichroism and conformation studies. Linkage occurs via G blocks, since in experiments with isolated G, M, and MG segments, only G segments interact significantly with calcium ions, and this process is highly cooperative if the guluronate block exceeds about residues [17]. Guluronate sequences appear to associate into matched crystalline aggregates in a twofold ribbonlike form, with the ions cooperatively bound during the

.

,

.

18 The “egg-box” model for junction zones in gels alginate or lowmethoxyl pectin. The linkage geometry introduces a buckled sheet structure to the polysaccharide chains, andthe junction zones are stabilized by calcium ions The upper shows an approximation to the chelation a calcium ion by a single chain.

process, sitting inside the electonegative cavities like eggs in an egg box. However, more recent evidence seems to indicate dimeric association rather than the multiple aggregation suggested in the original model [19].

Polysaccharides have the deceptively simple (approximate) formula (C6H,,05)n, but they have the extraordinary ability of being able to form a vast range of structures. It is therefore not surprising that, after water, they are the commonest component ofliving things with numerous differentfunctions in the life process. These encompass functions ranging from cell recognition to that of being major structural components plants and insects (cellulose and chitin). Nature uses the ability of polysaccharides to form highly hydrated network structures (gels) in the synovial fluid of joints and the vitreous humor of the eye (hyaluronic acid). Polysaccharide structures are also of great importance in food science and nutrition as, increasingly, we turn our attention of food structure and texture. Polysaccharides as gelling agents and thickeners are essential in the development of food products low in fat and energy. Polysaccharides as dietary fiber are essential to good health becauseof structural characteristics that influence the flow behavior of the digesta [20,21].

1. Dictionary of Science and Technology. W. R. Chambers, London (1971). 2. Schulz, G. E., and Schirmer, R. H. Principles of Protein Structure. SpringerVerlag, New York (1979). Protein structure and enzyme 3. Linderstrom-Lang, K U.,and Schellman, J. activity. In: Boyer, P. D., ed., The Enzymes, Vol. 1, 2nd ed. Academic Press, New York, pp. 443-510 (1959). 4. Binkley, R.W. Modern Carbohydrate Chemistry. Marcel Dekker, New York (1988). and Smith, P. J. C. Polysaccharide conformation. Part IX. Monte 5. Rees, D. Carlo calculation of conformational energiesfor disaccharides and comparison with experiment. J. Chem. Soc. Perkin ZI: 836-840 (1975). 6. Moms, E. R. Polysaccharide structure and conformation in solutions and gels. In: Blanshard, J. M.V., and Mitchell, J. R.,eds., Polysaccharides in Food. Butterworths, London, pp. 15-31 (1979). Polysaccharide Shapes. Chapman and Hall, London (1977). 7. Rees, D. and Scott, W. E. Polysaccharide conformation. Part VI. Computer 8. Rees, D. model-building for linear and branched pyranoglycans. Correlations and biological function. Preliminary assessment of interresidue forces in aqueous lution. Further interpretationof optical rotation in terms of chain conformation. J. Chem. Soc. B: 469-479 (1971). Equilibrium and 9. Norton, I. T., Goodall, D. M., Moms, E. R., and Rees, D. dynamic studies of the disorder-order transitionof kappacarrageenan. J. Chem. Soc. Faraday Trans. 79: 2489-2500 (1983). 10. Rees, D. Structure conformation and mechanism in the formation of polysaccharide gels andnetworks. Adv.Carbohydr.Chem. Biochen 267-332 (1969). 11. Oakenfull, D., and Scott, A. The role of the cation in the gelation of kappacarrageenan. In: Phillips, G. O., Williams, P., and Wedlock, D. J., e&., G u m and Stabilizers for the Food Zndustry-5. IRL Press, Oxford, England, pp. 507-510 (1990). 12. Belton, P. S., Chilvers, G. R., Moms, V. J., and Tanner, S. F. Effects of Group I cations on the gelation of iota-carrageenan. Znt. J. Biol. MacromoE. 6 303308 (1984). 13. Watase, M., and Nishinari, K. Effect of alkali metal ions on the rheoIoaca1 properties of K-carrageenan and agarose gels. J. Tature Stud. 427z4.45 (1981). 14. Moms, V. J., and Chilvers, G. R. Rheological studies of specific cation forms of kappa-carrageenan gels. Carbohydrate Polym. 129-141 (1983). solutes on the 15. Rey, D. K., and Labuza, T. P. Characterization of the effect water-bindingand gel strength properties of carrageenan. J. Food Sci. 4 6 786-789 (1981). 16. Moms, E.R., and Norton, I. T. Polysaccharide aggregation in solutions and gels. In: Wyn-Jones, E., and Gormally, J., eds., Aggregation Process in Solution. Elsevier, New York, pp. 549-593 (1983).

Oakenfull, D. Gelationmechanisms, Food Ingredients J, Jpn. Grant, G. T.,Moms, E. R., Rees, D. Smith, P. J. C., and Thom, D. Biological interactions between polysaccharides and divalent cations:The egg-box model. FEBS Lett. 32: Moms, E. R., Rees, D. Thom, D., and Boyd, J. Chiroptical and stoichiometric evidence of a specific dimerization process in alginate gelation. Carbohydr. Res. Eastwood, M. and Moms, E. R. Physical properties ofdietaryfiber that influence physiological function: model for polymers along thegastrointestinal tract. Am. J. Clin. Nu& Oakenfull, D.Physical properties of dietary fiber. In: Samman, S., and Annison, G., eds., Dietaly Fiber Beyond Australian Perspectives. Nutrition Society of Australia Occasional Publications, Vol. pp.

3 Gel Formation andUltrastructure in Food Polysaccharides Tokuya Haradat and Akira Harada Osaka University Osaka, Japan

I. Food polysaccharides, dissolved, dispersed, or suspended in water, are generally classifiedas gelling or nongelling. The former class is ordinarily transformed to gels by heat and/or special solutions, e.g., by alkali neutralization and electrolyte additions, such treatments having the effect of initially decomposing mostly hydrogen bonds in the solid solute, followed by its solvation (hydration), dissolution, and reformation as a gel, upon the cooling or neutralizing of the sol. Gelation is a function of other variables, e.g., the degree of polymerization (DP), solute concentration, and cosolute additions; but for most gums, temperature is the most important variable. Changes promoted by heating can be followed by differential scanning calorimetry (DSC). A DSC of curdlan(Fig. 1) showed a sharp endothermic peak at 50-64"C7 possibly related to swelling and hydrogen-bond breakage; a broad endothermic peak between 70 and 120"C, and an exothermic peak in the range of 140-170°C. The swelling temperature decreased with increases in heating temperature. Heating (70-120°C) caused irreversible changes. Temperature hysteresis was observed between 60°C and 40°C. No exothermic peakatabout40°Cdeveloped on cooling after heating to 170°C. The strength of a gel depends on the temperature (Fig. 2). 'Deceased. In memory of Tokuya Harada.

Harada and Harada

38

20

40

72.5

137.5

105

Temperature

'C

Figure DSC curves of curdlan in aqueous suspension. Curve was obtained by heating an aqueous suspension of curdlan from 20°C 170°C. Curve B was obtained by cooling after heating the suspension to 90°C. (From Refs. 7, 32, 36, with permission.)

The development of ultrastructures in the sols of gelling polysaccharides is an important stage in the sol-gel transition. Those listed in Table 1 are curdlan, K-carrageenan, agar, pectin, alginic acid, konjac mannan, and gellan Curdlan is insoluble in water, but swells in it to form the two gel types [l-41.K-Carrageenan gels in the presence of Na+. We found that K-carrageenan/Na+remaineda gel after dialysis Agar develops a gel when its aqueous suspensions are heated to 90°C andabove and then cooled. High-methoxyl pectin gels with 65% sugar and acid; low-methoxyl pectin gels also with Ca2+but less sugar. Alginic acid gels with C a 2 + and no sugar. Gellan gum requires heating to no less than 90°C in the presence of Caz+, preceding cooling to about 30°C. Konjac glucomannan gels by heating and cooling and by neutralizing an alkaline sol. Screloglucan, xanthan, dextran, pullulan, A-carrageenan, locust bean, and succinoglycanare nongelling poly-

-

h

<

-

-5

M $ .

VI

500 -

IP

ot

50

I

70 80 Heating temperature

l

90

100

Effect of heating temperature on gel strength of curdlan. Heating time, min; concentration curdlan, 3%. (From Ref. 33, with permission,)

saccharides that nevertheless develop very weak structures, contributing to a high viscosity. The neutralization of some polysaccharide sols, e.g., curdlan leads to gelation. We examined the effect of alkalization on konjac glucomannan, curdlan, agar, and K- and L-carrageenan gels (Table 2) and discovered that konjac glucomannan gelled on neutralization of its alkaline solution but not on heating at neutrality. The strengths of the gels obtained by neutralization alone were much lower than those of the gels obtained by heating, except in the case of konjac glucomannan. The concentration NaOH required for solubilizationofagar,K-carrageenan;and konjac glucomannan was higher than the 0.02 N required for curdlan, although the latter's xerogel powder did requirea concentration of 1.0 N and heating at 90°C [6]. A very high NaOH concentration was required to solubilize agar. The order of gel strengths of those gels induced by heating and cooling was agar > K-carrageenan > curdlan > konjac glucomannan/Ca2+> L-carrageenan; for those inducedby alkali neutralization,K-carrageenan > konjac glucomannan > curdlan > konjac glucomannan/Ca*+agar.

Harada and Harada

3 'F W

8

Bn

W

h

P

W

Harada and Harada

We examined the X-ray diffraction patterns of powders of curdlan dehydrated with acetoneafter heating atvarious temperatures Figure shows the X-ray diffraction patterns of both the alkali-labile fraction of powdered preparations of the gel formed on neutralization andthe alkali-resistant fraction. The peak at about 2 = became sharper with increases in temperature, especially above 120°C: 3-5 peaks between 15 and 25" appeared at

3 Micrographs of gel-formingpolysaccharides. A, C, E, G, and I: gels heatedat 95°C for min in aqueous suspension, B, D, F, H, and J: gels formed by neutralization an alkaline solution.A and B, curdlan; C and D, agar; E and F, K-carrageenan; G and H, konjac glucomannan; I, konjac glucomannan with calcium chloride; J, L-carrageenan. Bars represent m.(From Ref. 6, with permission.)

Harada

and

Harada

3 Continued

80°C and higher, which may have been due to the formation of triple helices followed by hydrophobic interaction, judging from comparison with previously reported X-ray data [8-111 and 13CNMR data (Saito et al.) [12]. Ogawa et al. [l31 proposed that dilute NaOH solution induces an ordered conformation in curdlan, and high concentrations (0.2 cause it to behave as a random coil. This order-disorder transition in alkali was confirmed by Saito et al. [14], who later reported about 10% of an untreated curdlan powder in triple helical conformation to be involved incrosslinking with flexible single helices. The proportion of the triple helices increased to about 50% on heating the gel to 150°C and then to 100% on slow cooling. Native curdlan was observed to exist as a 6:l random coil in the dry state, but as a single helix in a dispersion of 85% moisture, after alkali neutralization [lo]. Takeda et al. [8] claimed that the single helices convert totriple helices upon neutralizationas well as heating. The existence of triple helices in curdlan gels was proved by X-raycrystallography [9,10,15-171. Thus the effect of alkali on a heated and cooled curdlan sol is largely a single-totriple helix conformation. A curdlan gel was immersed in 32% H2S04, and after reheating at 32°C a stable residue remained that was resistant to the usual p-1,3 glucanases [l81 but thatwas hydrolyzed bythe enzymes of Bacillus curdlanolyticus and Bacillus kobensis [19,20].

While helical conformations may be a prerequisite for gelation [21], singleand double-stranded fibrils were also seen in xanthan [22,23]. Stokke et al. [23] showed this gum to exist as single- and double-stranded fibers 2 m and nm wide,respectively. Inasmuch as nongelling schizophyllan and scleroglucan are known to exist as triple helices in water [24], conditions additional to those creating helices are necessary. The possibility of microfibrillar differences between nongelling and gelling polysaccharidesunder conditions normallyexpectedto induce a to examine photomicrographs of samples from both phase change led classes (Figs. There were differences. The nongelling polysaccharides

Micrographs of mucous polysaccharides (sols): A-carrageenan (A), screloglucan (B), succinoglycan (C), xanthan gum (D), pullulan (E), anddextran (F). (From Ref. 6, with permission.)

Harada and Harada

Figure Micrographs of dialyzed K-carrageenan and its derived preparations. Dialyzed K-carrageenan H-form (B), Na-form (C), Na-form with potassium chloride (D). (From Ref. 34, with permission.)

yielding viscous fluids contained microfibrils 1-2 nm wide, and the gels, 5-25 nm wide. Previously we reported that curdlan gave about 25 nm in a 120°C-set gel and 15 nm in a neutral gel [B]. Agar andK-carrageenan showing 7- and 8-nm widths, respectively, were similar in the heated and neutralized gels. Interestingly, the gelling K- and L-carrageenan showed microfibrils approximating 8-nm and 5-nm widths, respectively, whereas the nongelling X-carrageenan approximated 1.5-nm widths. The neutralized gel of konjac glucomannan had long, wide microfibrils, whereas heated Ca2+/ konjac-glucomannan contained none, only globules. The microfibrils ofsuccinoglycan and xanthan gum were wider, compared with those of other viscous polysaccharides, apparently depending on whether or not the solution contains single, double, triple, or multiple helices and branches. Triple helices from the curdlan H,SO,-, P-1,3-glucanase-resistant, 120°C gel were 75 nm long and 30 nm wide (Fig. 9). Heating at55°C released many considerably thinner elementary microfibrils 1-3 nm wide (Fig. 10). Implicit in Fig. 10, and throughout this chapter, is the inference that dissociation and association of helical conformations are solation-gelation phenomena.

Gel Formation and Ultrastructure

Figure Micrographs of Na-form alginate (A), Na-form gellan gum (B,Bf), and Na-form low-methoxyl pectin(c)with calcium ion (2) and without calcium ion (l), obtained by negative staining (A, B, C) or shadowing (Bf). Bars represent 100 nm. (From Ref. 34, with permission.)

Harada

Harada

Micrographs of locust bean gum alone (A) and with K-carrageenan and potassium chloride (B)or xanthan gum (C,D), obtained by negative staining. C, formed by cooling the heated mixture; D, formed by dialyzing a solution in dimethyl sulfoxide. Bars represent nm. (From Ref. 31,with permission.)

In the example of curdlan, gelation is effected by a DP 2 (Fig. 9). Judging from its electron micrographs (Fig.lo), this gum is self-ordering as microfibrils. Unheated DP-450 curdlan showed elementary fibrils about 100 nm long, those of DP-131 curdlan about 40-nm long, andthose of DP49 curdlan about 25 nm long. The microfibrils of the DP-49 curdlan were

Dependence of the specific rotation of curdlan at 439 nm on the degree of polymerization in 0.1 M sodium hydroxide at 30°C. (0) gel-fonning polymer; insoluble fractions; soluble fractions. (From Ref. 35,with permission.)

ructureandGel Formation

49

Figure Electron micrographs of the original curdlan @P B) and lowmolecular-mass (1-3)-p-D-glucanS DP 131 (C,D) and DP (E,F)before (A, C, E) heating at 120°C for 30 minand after heating (B, D, F). (From Ref. 7, with permission.)

parallel. Heated DP-131 and -49 curdlan gave 40-50 nm lengths of what could be argued to be pseudocrystalline structures It is also arguable that the DP-131 and DP-450 curdlan primary structures may have resulted in shorter microfibrils than those of DP-49 curdlan containing more than 50% fewer monomers, because of entanglement. When peptization of the polysaccharide solutes was effected by dimethyl sulfoxide, urea, or formic acid, instead of by NaOH, and the sols dialyzed, they have gels similar to the gels obtained from alkali neutralization.

Harada and Harada

X-raydiffractionpatterns of curdlanpowderwith and withoutresistance to acid hydrolysis. Neutralized preparation (A); part resistant (B) to acid hydrolysis in the preparation heated at 120°C for 4 h. (From Ref. 18, with permission.)

K' causes aggregation ofK-carrageenan molecules into superstrands and enhances the gel strength [26]. Stokke and Elgsaeter [27] observed much wider gellan microfibrils in 0.1 M KC1 than without. Thus, the gel-forming ability of K-carrageenan differs with the cation present. We studied gelation in the presence of certain cations (Table 3), using a shadowing technique insteadof negative staining withuranylacetate, which would have complicated the electron microscopy of the polyanions [26].We dialyzed a K-carrageenan gel against distilled water and noticed a marked lowering of gel strength, to the stage where it was unable to gel when heated; subsequentlyadding K+ caused the initial gel strength to return

Gel Formation and Ultrastructure

11 Micrographs of gels treated with 32% sulfuric acid at 32°C for 30 days after heating at 120°C for 4 h in water Micrographs of curdlan gels sonicated (B) after neutralization of the solution in 0.3 N NaOH with HC1 and then heated at 55°C (C).Bars represent nm. (From Ref. 2,with permission.)

to its initial value, and enhanced it, after the concentration required to neutralize the polyanions was exceeded. Next, on a suitable Amberlite IR-120 resin, we converted the dialyzed sol to each of the H+-,K+-, and Na+-forms, and discovered that the gel made therefrom had the same low gel strength. The dialyzate and its H- and Na-forms showed similarly short, narrow microfibrils,but the addition of K' to the Na-form gave long microfibrils, suggesting that K' increased the length of the Na-form microfibrils in carrageenan gels (Fig.

Harada 52 Table

and Gel Formation Dependent on the Presence

Harada Certain Cations Gel strength (g/cm')

K-Carrageenan K-Carrageenan plus KC1 (0.5%) Alginate (1%) %) aClz plus (1%) Alginate Gellan gum Gellan (0.2%) CaCl, plus (0.005%) pectin Low-methoxyl (2%) Low-methoxyl CaCl, (0.4%) pectin plus (2%)

140 2300

500 420

'No gel was formed. Source: Ref. 34, with permission.

The effect of Ca" on the Na+ forms of alginate [21], gellan [28], and low methoxyl pectin gels [29] was examined, only to find much longer microfibrils in the presence of C a z + than in its absence; the gellan microfibrils were about nm wide with Ca2+and about 5 nm wide without (Fig.

The strength. ofK-carrageenan/K+ gels is known to be enhanced by the addition of nongelling locust bean gum [21], which, moreover,is also known to form a gel with other nongelling gums, e.g., xanthan gum [30]. These synergistic pairs have similar glycoside linkages (Table It is the unsubstituted regions in the primary structure that are implicated in associative bonding (junction-zone formation). Again by electron microscopy,weexaminedpairedpolysaccharide Locust bean gum alone showed microgels for synergism (Table Fig. fibrils of less than l-nm width, and the mixed locust beangum/K-carrageenan gel revealed irregular rodlike microfibrils of 20-30-nm width. The locust bean and xanthan sols, heated separately at 95°C for min, mixed, and then cooled, showed many globularforms of various sizes (Fig. 7C).A dialyzed locust beadxanthan sol in dimethylsulfoxidegave fibers 20-30 nm these latter forms were similar to those from mixtures wide (Fig. locust bean gum and K-carrageenan with K+.

The way the microfibrils develop in a gel can have a significant impact on syneresis. It was shown that the 95°C-set curdlan gel was more syneretic

mation

Gel Table

Gel Formation by the Synergistic Actions of Ttvo Polysaccharides Gel strength

(g/cm’> Locust xanthan (1.5%) Locust gum plus bean gum Locustbeangum(1.5%)plusK-Carrageenan K-Carrageenan KC1 (0.5%)plus (0.5%) Source: Ref.

0

(0.5%) (0.5) andKCl(1.5%)

140 30

permission.

than the gel developed from alkali neutralization, a difference ascribed to the different gelation mechanisms [31].

VII. Generally, an aqueous solution of a nongelling polysaccharide contains narrower microfibrils than those of a gelling polysaccharide. Curdlan gels, unheated or originating from neutralization of alkaline sols, consist principally of single helices, whereas heated curdlan gels consist largely of triple helices. Curdlan pseudocrystallitesdevelop in sols as a result of heating to high temperatures. Arguably, therefore, under gelation conditions relatively short microfibrillar subunits associated in the axial direction, and normal to it, to produce microfibrillar lengths to nm and widths to nm; the evidence has been derived from examination of curdlan suprastructures by electron microscopy. It could be argued that these associating microfibrils accelerate gelation, because they are less mobile than single microfibrils. An aqueous nongelling polysaccharide has been illustrated on electron micrographs to contain narrower microfibrils than a gelling polysaccharide.

1. Harada,T.,Terasaki, M.,andHarada,A.In:Whistler, R. L., ed., Curdlan in “Industrial ” 3rd ed. Academic Press, New York, p. 427 (1993). 2. Harada, T., Kanzawa, Y., Kanenaga, K., Koreeda, A., and Harada, A. Electron

microscopic studies on the ultrastructure of curdlan and other polysaccharides in gels used in foods. Food Structures. 10: 1 (1991). 3. Harada, T., Production, properties and application of curdlan. In: P.A. Sandford, andLaskin, A., eds.Extracellular Microbial Polysaccharides.M C Symp. Ser. 265-283 (1977).

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Harada, T., Curdlan: A gel-forming p-1,3-glucan. In: Blanshard, J. M. V., and Mitchell, J. R.,eds., Polysaccharides in Food. Butterworths, London (1979). 5. Kanzawa, Y., Takahashi, F., Harada, T., and Harada, A. Ability curdlan gel 38: 363 (1987).In toholdhydrophobic substance. Kasei-gakuZasshi. Japanese. 6. Kanzawa, Y., Koreeda, A., Harada, T., and Harada, Electron microscopy of the gel-forming ability of polysaccharide in food additives. Agric. Biol. Chem. 53: 979 (1989). 7. Harada, T., Okuyama, K., Konno, A., Koreeda, A., and Harada, A. Effect of heating on formation of curdlan gels. Carbohydr. Polym. 24: 10 (1994). 8. Takeda, T., Yasuoka, N., Kasai, N., and Harada, T. X-ray structure studies of (1-3)-P-~-glucan(curdlan). Polym. J. 10: 365 (1978). 9. Fulton, W. S., and Atkins, E. D.T. The gelling mechanism and relationship to molecular structure of microbial polysaccharide curdlan. In “Fiber Diffraction Methods, French, A. D., and Gardner, F., eds., ACS Symp. Ser. 141, 385-410 (1980). 10. Chuah, T., Sarko,A.,Deslandes, Y., andMarchessault, R. H. Triple helical crystal curdlan and paramylon hydrates. Macromolecules. 1 6 1375 (1983). 11. Okuyama, K., Otsubo, A., Fukazawa, Y., Ozawa, Y., Harada, T., and Kasai, N. Sin of native curdlan. Carbohydr. Chem. 10: 645 (1991). 12. Saito, H., Yoshioka, Y., Yokoi, M., and Yamada, J. Distinct gelation mechanism between linear and branched (1-3)-p-D-glUCanS as revealed by high-resolution solid-state 13CNMR. Biopolymers. 19: 1689 (1991). 13. Ogawa, K, Tsurugi, J., Watanabe, T., and Ono, S. Conformational behavior of gel-forming (1-3)-P-D-glUCan in alkaline solution. Carbohydr.Res. 23: 399 (1972). 14. Saito, H., Ohki, T., and Sasaki, T. A nuclearmagneticresonanceof single helical conformation in a resilient gel of a curdlan-type polysaccharide 13140 from Alcaligenes faecalis var. myxogenes I F 0 13140. Biochemistry 16: 908 (1977). 15. Marchessault, R. H., Deslandes, Y., Ogawa,K.,and Sundarajan, P. R. X-ray diffraction data on p-(l-3)-~-glucan. Can. J. Chein. 25: 300 (1977). 16. Sarko, A., Wu, H. C., and Chuah, C. T. Multiple-helical glucans. Biochem. Soc. Transact. 11: 139 (1983). 17.Deslandes, Y., Marchessault, R, H., andSarko,A. Triple-helical structure of (1-3)-P-~-glucan.Macromolecules 13: 1466 (1980). 18. Kanzawa, Y., Harada, T., Koreeda, A., Harada, A., and Okuyama, K. Difference of molecular association in two types of curdlan gel. Carbohydr. Polym. 1 0 299 (1984). 19. Kanzawa, Y., Kurasawa, T., Kanegae, Y., Harada, and Harada, T. Purification and properties a newexo-(l-3)-P-~-glucanase from Bacillus circulans YK9 capable of hydrolyzing resistant curdlan with formationof only laminarbiosse. Microbiology 140: 637 (1994). 20. Kanzawa, Y., Harada, A., Takeuchi, M.,Yokota, A., and Harada, T. Bacillus curdlanolyticus sp. nov. and Bacillus kobensis sp. nov., which hydrolyze resistant curdlan. Int. J. System. Bacteriol. 45: 515 (1995). 4.

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Rees, D. A. Shapely polysaccharides. Biochem J. 126 Holzworth, G., and Presridge, E. B. Multistranded helix in xanthan polysaccharide. Science 19: Stokke, B. T., Smidsrod, O., and Elgsaester, Electron microscopy of native xanthan and xanthan exposed to low ionic strength. Biopolymers 28: Norisue, T., Yanaki, T., and Fujita, H. Triple helix of a Schizophyllum commune polysaccharide in aqueous solution. J. Polym. Sci., Part D. Macromol. Rev.18: Takahashi, F., Harada, T., Koreeda, A., and Harada, A. Structure of curdlan that is resistant to P-1,3-glucanase. Carbohydr. Polym. Hermansson, A. M. Rheological and microstructural evidence for transient states during gelation of kappa-carrageenan in the presence of potassium ion. Carbohydr. Polym. 10: Stokke, B. T., and Elgsaester, A. Conformation, order-disorder conformational transitions and gelation of non-crystalline polysaccharides studied using electron microscopy. Micron. 25: Grasdalen, H., and Smidsrod, 0. Gelation of gellan gum. Carbohydr. Polym. and Rooks, J. K Algin gel compositions, U.S. Patent In Chem. Abstr. Dea, I. C. M., and Moms, E. R. Synergistic xanthan gels. In: Sandford, P. A. and Laskin, A., eds., Exocellular Microbial Polysaccharides. ACS Symp. Ser. Miller,

45:

Kanzawa, Y., Harada, T., Koreeda, A., and Harada, T. Curdlan gel formed by neutralizing its alkaline solution. Agric. Biol. Chem. 51: Konno, A., Oluyama, K., Koreeda, A., Harada, A., Kanzawa, Y., and Harada, T. Molecular association and dissociation in formation of curdlan gels.In: Nishinari, K., and Doi, E., eds., Food Hydrocolloids: Structure,Properties and Functions. Plenum Press, New York, pp. Maeda, Y., Saito, H., Masada, M., Misaki, A., and Harada, T. Properties of gels formed by heat treatment of curdlan, a bacterial glucan Agric. Bid. Chem. 31: Kanzawa, Y., Koreeda, Y., Harada, A., Okuyama, K., and Harada, T. Significance of molecular association for polysaccharide gels in food. J. Home Econ. Jpn. 41: Ogawa, K., Tkurugi, J., and Watanabe, T. The dependence of the conformation of a (1-3)-p-D-@UCan on chain-length in alkaline solution. Carbohydr. Res. 29: K o ~ oA., , and Harada,T. Thermal properties of curdlan in aqueous suspension and curdlan gel. Food Hydrocolloids 5:

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4 Structures and Phase Transitions of Starch Polymers G. Biliaderis Aristotle University Thessaloniki, Greece

INTRODUCTION In addition to being a major component of the human diet, starch constitutes an excellent raw material for modifying the texture and the consistency of foods because of its ability to form viscoelastic pastes or gels when heated in water. Furthermore, starch-derivedproducts have long served in numerous nonfood applications, such as pharmaceuticals, textiles, alcohol-basedfuels, and adhesives. New uses of starch are steadily emerging, including lowcalorie fat substitutes, “biodegradable” packaging materials, thin films, and thermoplastic materials with improved thermal and mechanical properties. Many of these food and industrial applications starch rely largely on the colloidal properties of its two structurally distinct a-D-glucan components, amylose and amylopectin. Starches from various plant origins exhibit characteristic functional properties, dependingon a number of integratedfactors that include polymer composition, molecular structure, interchain organization, and minor constituents (e.g., lipids, proteins, and phosphateester groups). Moreover, chemical, enzymic, and physical modificationof starch structure, with either preservation or destruction of the native granular form, offer alternative routes of imparting certain functionality for this biopolymer. Starch modification is a common feature many processed foods, having both textural and nutritional implications. Recent advances in the physical chemistry and structure of starch also reveal many similarities with synthetic semicrystalline

Biliaderis

polymers [l-41.Despite the inherent difficulty in providing a complete description of starch morphology in terms of size, shape, amount, and orientation of ordered chain assemblies, as well as the exact nature of the amorphous domains, there is a growing recognition of the relationship between internal structure (molecular and supermolecular) and macroscopic properties of starch. Information derived withthe use of sensitive analytical probes of polymer structure (e.g., calorimetry, dynamic mechanical testing, electron andneutron scattering, andX-raydiffraction, electron microscopy, solidstate I3C-NMR, Fourier transform infrared spectroscopy) provides evidence for various levels of chain organization, not only withinthe granule but also in aging aqueous starch dispersions andprocessed foods. Such measurements further point to the nonequilibrium natureof starch structures (various metastable states) and the need to treat the processes of gelatinization, annealing,gelation,andretrogradation of starch in the context of solventmediated structural transitions of polymers. In the following discussion some aspects of starch structure are considered,particularly as theypertainto the function, physicalproperties, metastability, and transformations of this polysaccharide in model systems andin actual food products. The phase-transition behavior of starch and related materials is examined by drawing attention to the interactions of starch with water, lipids, low-molecular-weight solutes (salts and sugars), and other hydrocolloids. These properties are relevant to processing and enduse quality of starch-based materials.

II. It has long been recognized that starch materials differ in composition, structure, and supermolecular order of the polysaccharide constituents, which could account for differences in their functionality. Native starch occurs in the form of discretegranularparticlesthat are partiallycrystallineandbirefringent when viewed under polarized light.The cold water-insoluble granules, found in practically every plant tissue, represent an efficient means ofstoring energy, for the a-D-glucan molecules are densely packed into quasicrystalline aggregates following their biosynthesis in the amyloplasts. The granule structural organization also imposes some constraints on the action of a-amylases, resulting in a slow release of glucose upon mobilization of reserve starch in plant tissues and during the in vivo digestionof native starch, particularlyof tuber starches (banana, potato), in the human small intestine. The size, distribution, shape, and morphology of starch granules are e.g., cereal starches are markedly dependent on the botanical source

generally small and polyhedric, whereas tuber starches are large and ellipsoid or spherical. Some cereal starches (e.g., amaranth, cow cockle) have uniform, very small granules (0.5-2.0 p), whereas those of canna starch have diameters up to 175 p. An extensive study of the morphological features and shapes of starch granules from different botanical sources using scanning electron microscopy was recently presented [lo]. Contrary to most starches, whichshow single size distribution of granules, wheat, rye, barley, and triticale starches exhibit bimodal distributions (sphericalB-granules, 1-10 p, and lenticular A-granules, 15-35 p). In wheat, B-starch constitutes about 25-30% of the total starch volume but about 90% of starch granules in number. A low baking potential has been noted for small granules [ll]. The proportion of small granules is also important for the baking performance of wheat flours. The specific volume of loaves prepared by remixing A- and B-type granules in various ratios was maximum whenthe proportion of the small granules was 25-35% by weight [12]. Granule size and morphology have recently received much attention, because small granular starches are useful in determining taste and mouth feel of some starch-based fat mimetics [13,14], tensile properties of biodegradable plastic films [15], and surface-coating properties in paper production processes [9] and as laundry-stiffening agents. Besides the exploitation of novel raw materials, which requires the development of efficient wetmilling processes to produce small-granule starches, methods have been developed to break down granules of cereal starches into small particles by heterogeneous acid hydrolysis [16,17]. Hydrolysis is conducted at temperatures between 55°C and the gelatinization temperature of starch. The acidresistant residues consist of aggregated starch crystallites, which upon homogenization in a continuous water phase give a stable suspension with a smooth, creamy, fatlike mouth feel. Granule composition,morphology,andsupermolecularorganization are, to a certain degree, under genetic control [7,18]. The exact mechanism by which plant genetic information is translated into specific granule compositionandorganization is obscure.Granule-bound starch synthases are linkages of amylopectin. The ~ ( 1 - 6 ) branch responsible for points are formed by branching enzymes in a transferase reaction involving no new synthesis. However, the exact role and the in vivo regulation of the multiple forms of starch biosynthetic enzymes (phosphorylase, starch synthases, branching enzymes) are not fully understood [19-221. Only when this information is available might it be possible to manipulate the quantity and fine structure of starch. Plantbiotechnology offers newmolecularapproaches to modulate starch quality and quantity [23].A major breakthroughwith respect to starch composition has been the inactivation of granule-bound starch synthase in

potato tubers using antisense RNA mediated inhibition; starch of the transgenic plants had very low amylose content ( ~ 5 % [23]. ) It would appear that selective inactivation of genes encoding certain isoforms of starch synthases or branching enzymes may lead to starch polymers with altered molecular size and shape or different branching patterns. In addition to being able to modify the biosynthesis routes by controlling expression of endogenous genes in transgenic plants, genetic engineering may offer answersto more fundamental questions concerning the in vivo role of starch synthesis enzymes. For example, Muller-Rober et al. [24] were able to create potato plants in which expression of ADP-glucose pyrophosphorylase (AGPase) was inhibited via antisense RNA. This resulted in abolition of starch formationintubers, thus strongly favoring theADPglucose route as a predominant pathwayfor starch synthesis in higher plants. Production of novel starches in transgenic plants offers new possibilities to replace much of the postharvest chemical modifications, which are environmentally hazardous, expensive, and time-consuming. It may become possible biotechnologically to synthesize anionic starches containing carefully controlled levels of phosphate or sulfate groups to render certain functionality. For fat-replacement applications,the insertion of new typesof glycosyl linkages, e.g., ~ ( 1 - 2 ) or a-(1+3), would be useful for improving resistance to breakdown in the digestive tract or for other desired functions.

A.

Starch Macromolecules: Structure, Solution Conformation, and Properties

Starch granules are composed essentially of amylose and amylopectin, as evidenced by dispersion of the granular material and separation of these polymeric constituents [7]; i.e.,inmolecular terms granules are polymer composites. As is the case for most polysaccharides, amylose and amylopectin are heterogeneous, exhibiting a wide distribution of molecular structures and sizes. Most starches contain between 20% and35% amylose. However,plant breeders havedeveloped mutants that contain starch with essentially no amylose (waxy type) or of much higher amylose content (e.g., amylomaize with 50-85% amylose). Starch owes much of its functionality to the fine structure and relative proportion of these polysaccharidesas well as to the physical organization of starch polymers into larger structural domains in the solid state. However, the minor noncarbohydrate components of granular starches (less than 1.5%), and particularly lipids, proteins, and phosphorus, maywell be of significance in determining the processing quality and functional properties of commercially important starches [25,26].

Starch

1. Amylose consists mainly of ol-(l+4)-linked D-glucopyranosyl residues, thus often referred to as the linear starch fraction. However, it is now accepted that this polymer is slightly branched, having occasional a-(1+6) branch points; according to Hizukuri et al. [27] there are 9-20 branch points per moleculeand the side chains range in chain length from 4 to over 100 glucosyl units. The extent of branching depends on the origin of amylose [28] and increases with the molecular size of amylose from a particular source [7,29]. The extent of branching is reflected in the susceptibility of amylose to hydrolysis by the exoenzymeP-amylase;P-amylolysis varies between 73% and 95%, depending on the extraction procedure and the botanical origin of amylose [7,30]. When amylose is subjected to the concurrent action of pullulanase and P-amylase, conversion to maltose is essentially quantitative (97-100%) [31]. For laboratory-isolated amyloses,the reported range of M, varies between 2.0 X lo5 and 1.2 X lo6, with polydispersity indices (M,.,/M,) between 1.3 and 5.8 [7,30,32,33]. Despite its slight branching, amylose behaves essentially like a linear polymer, forming films and complexes with ligands. amylose has long been the subject of The solution conformation extensive research [7] and is still a matter of controversy. Early investigations of the hydrodynamic properties of amylose solutions (viscosity and light-scattering measurements) indicated that the conformation of anylose depends on the solvent employed. According toBanksandGreenwood [7,34], in neutral solutions as well as in good solvents (DMSO, formamide, and aqueous alkali), amylose behaves like a random coil, whereas a helical conformation is assumed in neutral or alkaline solutions in the presence of a complexing agent. Polymer chain stiffness in solution can be expressed by the dimensionless quantity C,, known as characteristicratio (C, = 6(Rg'),,,/(n)Z:,where @g'), is ,, the weight-average mean-square radius of gyration, (n) is the number of residues, and Z, is the average projection length a monomer on the chain axis), and by the persistence length, 4,defined as the average projection an infinitely long chain on the initial tangent of the chain. Estimates of these parameters for amylose (C, 5-6, 4 = 1.51.7 nm) [7,35,36] suggest that amylose chains in aqueous KOH (0.1 M) or KCL (0.1-0.3 M) solutions are more flexible than cellulose derivatives (C, 30-40, 4 4.6-16.0 nm), pectin (4 30-34 nm), and xanthan (4 255 m),but more rigid than pullulan (4 1.2-1.9 nm) [37]. Also, the C, of amylose in DMSO is -5 1 nm [38], close to the reported values for aqueousamylose [7,35,36,39]; i.e., for high-molecular-weight amylose (>los), the conformations in water and DMSO are essentially the same. The overall random-coil conformation of amylose in solution does not preclude

some local helical character in some segments of the chain. This is supported by small-angle X-ray scattering data [40]. Thus, the average conformation of amylose in aqueous solution may be viewed as a highly disordered coil involving many discernible sequences of short-range helical structures that are irregular and labile. The extent and direction of local conformational order change dynamically in solution, resulting in a sequence of conformational states that determine the macroscopic properties of amylose. There are two features of amylose in solution that are of special interest to the functionality of this polysaccharide in starch-based products.The first is the ability to form helical inclusion complexes when an appropriate ligand is present. The formation of a complex between amylose and iodine gives rise to the typical deep blue color (Amm = 640 m)of starch dispersions stained with iodine and forms the basis for quantitative assessment of amylose;in IJKI solution, the guestmolecules are polyiodideions,mostly I; or I; 171. By amperometric or potentiometric titration, the stoichiometry of the complexation gives a binding capacity of amylose some 20% of its own weight of iodine, whereas amylopectin binds none. Besides iodine, a variety of polar and nonpolar compounds induce coil helix transitions in an aqueous amylose solution. It is the ability of these materials to satisfy nm in the solvation requirements of the hydrophobic helical cavity (ca. diameter) that enables the polysaccharide chain to adopt a regular conformation (V-helix), where the ligand molecule resideswithin the helix [7].The interactions that stabilize the helix are intrachain hydrogen bonds between 0-3(2), interturn H-bonds adjacent glucosyl residues, 0-2 and numerous intra- and intermolecular van der Waals contacts [41,42]. Computermodeling of a fatty acid dockedinside a Vh-helixsuggests that only the H-5 atoms of each glucopyranose residue participate in short van der Waals contacts with the nearest CH, group of the aliphatic chain [43]. W Ostable conformations of the total fatty acid residue were determined with the polar group located near the entrance of the helix cavity. Steric and electrostatic repulsions prohibit the polar groups of fatty acids and monoacylglycerides from entering the helix [43,44]. The second feature of interest with respect to the solution behavior of amylose is related to its propensity to interchain associations mediated by local ordering of the polysaccharide chains; i.e., interchain double helix formation occurs over chain segments of less than 100 units. Thus, amylose solutions are not very stable, compared to amylopectin. of the most important variables that affect the solubility, molecular association, and/or crystallization of amylose are polymer concentration and chain length [45471. Amylose tends to precipitate (retrograde) in dilute solutions at concentrations CC* (critical or coil overlap concentration) or form gels in concentrated solutions. The instability is more marked for molecules of DP (degree

-

-

of polymerization) -80 units; smaller and larger molecules appear to be more stable [45,48]. The solubility also shows similar trends, with amyloses of DP 80-100 being very sparingly soluble, and solubility increasing in the direction of shorter and longer chains [48]. For nearly monodisperse amyloses, it was found that precipitation (and not gelation) occurred for DP < 110 at all concentrations up to 5% wlv, whereas gelation occurred for amyloses of DP > 250 at concentrations >1.0% wlv [47]. Precipitates of shortchain amylose are essentially fully double helical and crystalline (side-byside stacked helices), while the structure of amylose gels is generally rationalized as a continuous network of rigid doublehelical crosslinks, acting as junction zones, interconnected by more mobile amorphous single chain segments. Strong evidence for chain aggregation of amylose, even in dilute (0.1% w/v) solutions, is provided by static light-scatteringexperiments; such behavior gives rise to high-molar-mass estimates for the linear starch fraction [49,50].

2. Amylopectin with a M,., of the order of 107-109 [7,50] is one of the largest known naturally occurring polymers. Its fine structure has been the subject of extensive investigations for over 50 years. Early methylation and periodate oxidation studies indicated that amylopectin possesses some 4-5% of interchain linkages, a-(1+6), leading to a highly branched, compact structure. Because of their branched nature, amylopectins have relatively low intrinsic viscosities (120-200 mL-g”) despite their high molecular weight [7]. While the average size of the unit chains is 20-25 [51,52], the amylopectin molecule contains several distributions of chains differing in their chainlength[52,53]. There are A-chains, which are unbranchedandare linked to the molecule through their reducing end-group; B-chains (B1-B4), which are joined to the molecule in the same way but carry one or more Achains; and one C-chain, which has the reducing end-group of the molecule (Fig. 1). The shortest chains (A and B,) have a chain length (CL) of 1418; the longer ones @-B.,) have a CL of 45-55. The molar ratio of short to long chains varies between 3:l and12:1, depending on the botanical origin of the starch [51]. Cereal starches generally have shorter chains in both long- and short-chain fractions and larger amounts of the short-chain fractions, compared with those of tuber starches [51,52]. Various enzymic methods for structural analysis of amylopectin have been devised [7,54-57, 57al. The polymodal chain distributionin amylopectins, as revealed by chromatography of their enzymic digests using debranching enzymes as well as other relevant structural analysis data on acid-resistant amylodextrins [58621, is consistent with the cluster-type model of French [58] and Robin et

70

100 Retention time (min)

4

"_

110

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"_

er

1 Chain distribution profile (waxyrice starch) and model for amylopectin structure according to Hizukuri. (Reprinted with permission from Ref. 52.)

Starch

al. [59]. The general features of these models are that amylopectin is composed of compact parts of oriented chains (crystalline clusters) andthat branching points are not randomly distributed throughout the macromolecule. While the constituent chains are drawn for simplicity as straight lines in Fig. 1, they in fact exist as double helices in the native state or in aged starch gels. Conformational analysis and molecular modeling of the branching point of amylopectin revealed thatside chains can remain parallelto the main backbone strand, allowing formation of double helices and the development of dense three-dimensional structures during deposition of the growing polymer in the amyloplast [63]. Such molecules may have a diameter of about 100 and a length of several thousand [18,59,64]. The clustertype structural model provides the basis for explaining why amylopectin is the dominant partially crystalline polymer (alternating crystalline arrays of double-stranded helices and amorphous zones of dense branching and cluster interconnecting long-chain segments) for most granularstarches. Amylopectins, particularly those from root and tuber starches,contain phosphate ester groups, mainly at C6 and C3, which confer on the macromolecule the properties of a polyelectrolyte [7]; e.g., for potato starch, the total phosphorus (-1 phosphate ester/200-500 anhydroglucose units, AGUs) is practically associated with the amylopectin fraction, with the C6-phosphate/C3-phosphate at aratio of ca. 2/1 [65]. The biosynthetic route of the phosphorylation is not yet fully known, but it is an integrated part of starch synthesis [66]. Physical and biochemical methods indicate that theC3 phosphorylation level is almost constant, whereas phosphorylation at C6 varies among different potato varieties [67,68]. The C6 phosphates seem to interfere with the tallization of amylopectin during starch synthesis; i.e., crystallinity and enthalpy of gelatinization decrease with the level of C6 phosphorylation but not with the level of C3 phosphorylation [69]. The extensive branching in amylopectin restricts the degree of hydrolysis by P-amylase, the P-amylolysis limits achieved (55-60%) are significantly less than those observed with amylose. Also, iodine is unable to form stable complexes with amylopectin because of the short length of the unit chains, only small amounts of iodine are bound (~0.6%);a red-brown complex is formed with iodine (h,,, 530-540 nm). As the external chain length increases, the h,,, of iodine-amylopectin complex also increases [7]. Althoughamylopectin does noteffectivelycomplexwithlipids, there is indirect evidence for such interaction by calorimetry [70,71]. The ratio of M,/M, for amylopectin has been reportedto be about 300, indicating a wide distribution of molecular sizes. In a good solvent, e.g., DMSO, amylopectin molecules have a highly planar, disclike conformation and behave as oblate elipsoids, with an estimated ratio of major to minor axes, p = a/b, of about 17 (using pulsed-field gradient NMR) [72] or even

higher (p = 37, using ultracentrifugal techniques) [73]. In water, aggregates are formed that are somewhat more spherical particles and give estimates of molecular mass some 400 larger than single molecules of amylopectin [72]. Early studies on the hydrodynamic behavior of amylopectin have sugis synthesized in the plastids as atwogestedthatthemacromolecule dimensional entity [74]. Amylopectin solutions appear to be more stable than those of amylose. However, solutions with concentration aboveC* (-0.9%) exhibit slowly progressing aggregation, as evidenced by turbidity development [75] or formation of a continuous gel network [76], presumably due to interchain associations in the form of double helical structures. Granules of some starches (e.g., amylomaize, wrinkled peastarch) may also contain asubstantial amount of an intermediate material that is, in itself, heterogeneous with respect to structure and molecular size. According to Banks and Greenwood [7],the intermediate polysaccharide fraction consists of linear chains with 50 < DP < 200 and lightly branched low-molecularweightmoleculeswith greater chain length thannormalamylopectin. Branched amyloses with up to 20 or more branches may also constitute a part of the intermediate material [57a]. Because of its structure and heterogeneity, the intermediate material cannot easily be fractionated and characterized. Nevertheless, this fraction is highly unstable in solution, leading to excessive retrogradation.

B. Supermolecular Structures of Starch 1. Molecular Probes of Starch Structure Many physical techniques have been applied to monitor changes in starch structures, but porarizing microscopy, electron microscopy, wide-angle Xray scattering and diffraction (WAXD), small-angleX-ray scattering (SAXS), solid-stateI3C-NMR, various viscometric techniques, and differential scanning calorimetry ( D X ) are the most widely used. Each of these methods is sensitive to a different level of structure, and over a range of distances, present in a starch system. For example, WAXD is sensitive to crystalline order over distances from about 0.3 to 2 nm, although the minimum dimensions for significant diffraction effects to be observed are probablyof the order of5-10nm.Cross-polarizationmagic-angle spinning NMR gives characteristic detailed spectra of A- and B-type crystal polymorphs of starch as well as of V-amylose complexes. In contrast to X-ray diffraction, NMR is considered a short-distance range probe, measuring order at the level of individual helices; i.e., it distinguishes helices either in or out of crystalline registry. Information about starch structures with shortrange order can be also obtained by calorimetry, whereas electron micros-

copy is important for studying structures ranging from single macromolecules up to larger macromolecular assemblies in the range of0.1-1.0 Also, some of these methods require dilute solutions or dispersions, while others are applicable to more concentrated systems or even dry specimens. There is often a need to provide complementary information using different physical techniques to characterize the structure of starch and improve our understanding about structure-function relationships of this polysaccharide.

Molecular order in native starch is evident from the optical birefringence pattern (“Maltese cross77)shown by granules when viewed under polarized light. This is a typical feature of spherulitic type of organization and is also exhibited by acid-treated granular starches [l81 and short-chain amyloses crystallized from solution [77]. The fact that starch is birefringent implies that there is a high degree of molecular orientation in the granule; however, crystallinity is not a prerequisite of birefringence. Since the refractive index is larger in the radial direction, starch macromolecules are disposed perpendicular to the granule surface [58]. Both birefringence and small-anglelightscattering measurements have been useful infollowing the kinetics of granule structure loss during gelatinization and the influence ofvarious cosolutes to this process, as extensively reviewed elsewhere [1,2,18]. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) have largelycontributed to the elucidation ofthe fine structure and morphological features of granular starch as well as of supramolecular assemblies involving starch polymers (e.g., gels, highly crystalline amylose specimens). Both TEM and SEM have given information relatedto the external and internal structure of granules following their degradation by acids or enzymes [18,78-83,83a]. For granular starch, the general picture that has emerged from ultrastructural studies is that growth rings (120-400 nm thick) consist of alternating crystalline (ca. 50 A thick and several hundred angstroms broad, representing the double-stranded helices of short DP chains of amylopectin) and amorphous lamellae (ca. 20 A) [M]. According to all current modelsof amylopectin [18,52,59,64], parallel helices assemble into radially oriented clusters. The 7-nm periodicity deduced from electron microscopy data is lower than the 9.0-10.0-nm figure determined by smallangle X-ray and optical diffraction analysis of electron microscopic images [84-861; the electron microscopy data are somewhat low, most likely due to shrinkage occurring during sample preparation. The 9.0-10.0-nm repeat distance is in close agreement with the value reported in an earlier cornmunication using small-angle neutron scattering ( S A N S ) measurements [87].

Oostergeteland van Bruggen [88] haverecentlyproposeda superhelical lamellar structure for amylopectin in potato starch based on TEM and electrondiffraction data of nondisrupted granule fragments. The crystalline regions (5 nm thick) consist of double helices (i.e., short DP chains of amylopectin) that interpenetrate each other, yielding on larger scale a continsuperhelical network. The arrangement of crystalline domains in a helical fashion leaves voids of -8 nm in diameter. theoretical model for the structure of native starch was also presented by Cameron and Donald [89] based on a rigorous treatment of SAXS data. The growth rings consist of alternating shells of partially crystalline regions (identified with the dense shells of growth rings, and having thicknesses of -140 nm) and largely amorphous material (background amorphous materialwith thickness at least as large as that of the dense shells). The partially crystalline regions were considered as being the lamellar stacks (clusters) of short DP chains of amylopectin, with thicknesses for the alternating crystalline and amorphous lamellae of 6.64 nm and 2.21 nm, respectively, i.e., overall an repeat distance of -8.85 nm. total number of lamellae (repeats) in each dense shell of -16 (corresponding to a distance of 140 nm) was calculated according to the fit. The electron density of the background amorphous material of a growth ring was found in between thoseof the crystalline and the amorphous lamellae of the dense shells. In this respect, the model implies the existence of three types of material in the native granule. A new level of starch granule structural organization (between that of lamellae and the growth rings), the “blocklet concept,” was recently proposed by Gallant et al. [83a] by reviewing old and new microscopic results obtained with different techniques. By analogy with the “superhelical amylopectin structure” of Oostergeteland van Bruggen[88],these authors claimed that the crystalline and amorphous lamellae of amylopectin are organized into larger, more or less spherical “blocklets.” The blocklets range in diameter between 20 and 500 nm,depending on their location in the granule and the botanical source of starch, and contain between 5 and 50 amylopectin short DP chain clusters. With thistype of granule organization, amorphousmaterialmay exist in differentregions: (1) in each lamella (branching zones of the amylopectin); (2) between clusters of side chains within each lamella; (3) around each blockletof side-chain clusters; and in radially arranged channels in granules through which amylose can exit during gelatinization. According to Gallant et al. [83a], the blocklets may be viewed as the structural elements of the alternating crystalline and partially crystalline shells of the starch granules often seen in SEM micrographs of granular starches corroded by a-amylases. They have further suggested that the size of blocklets and their arrangement in native starch may be important determinants of the granule resistance to enzymic attack.

Starch

Starch granules exhibit distinct WAXD patterns that can be classified into three categories: B (Fig. 2), and C. The A form is typical of cereal starches (e.g., wheat, corn); the B pattern is given by tuber starches (e.g., potato) as well as by high-amylose starches (e.g., amylomaize) and some genotype [90]. The C pattern is believed to be waxy starches with the a superposition of the A and B patterns. An interesting aspect of granule structure is the apparentrelationshipbetween the chain length of the branched molecules and the crystalline pattern. Starches with amylopectins residues) exhibit A-type crystallinity; those with of short chain length amylopectins of longer average chain length show the B pattern [52,91]. The notion that chain length is a major determinant of crystalline polymorphism of starch was further supported by crystallization studies of maltooligosaccharides used as model compounds [92,93]. According to Gidley and coworkers [93,94], the A polymorph (the most thermodynamicallystable form) is favored over the B structure (kinetically preferred form) under conditions of: (1) shorter a-D-glucan chain length, (2) highercrystallization temperature, and (3) higher polymer concentration,(4) slower crystallization conditions, and the presence of alcohol. The reverse is true for the B polymorph, which crystallizes readily from pure water. Interpretation of diffraction diagrams of native granular starches has been difficult due to imperfections, small size, and multiple orientation of starch crystallites [B]. The most detailed analysis of the A and B polymorphs has been based on X-ray and electron diffraction data from crystalline amylose and modeling studies [18,95-991. Both structures consist of double helices in which the two chains fit compactly; the hydrophobic parts of the opposed AGUs (anhydroglucose units)being in close contact, and the hydroxyls allowingfor strong interchain H-bonding [M]. The double helical nature of starch chains in the crystallites explains their insolubility in cold water and their resistance to acid treatment (glycosidic oxygens are buried in the interior of the helix and thus are less accessible to hydrolysis by H,O'); within the doublehelix,interstrand stabilization occurs through 0-2 0-6 H-bonding between the two chains as well through numervan der Waals interactions.The inner diameter of the double helix cavity is -0.35 nm (i.e., it does not allow any water molecule to pack inside the helix), while the outer diameter is -1.03 nm. The two polymorphs differ in the packing arrangement of the double helices and in the water content. The three-dimensional structural models of Fig. 2, proposed by Imberty and coworkers [98,99], are based on left-handed, parallel-stranded sixfold double helices packed parallel inthe crystalline lattice and containing 12 AGUs per unit cell; each strand contributes three out ofsix residues per helix turn. For the Astructure, chains are crystallized in a monoclinic lattice (unit cell parameters = 21.24 & b = 11.72 & c = 10.69 & and a = = 90°, =

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Diffraction angle (28)

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m i c a 1 X-ray diffraction patterns of A-type granular starches (bottom) and B-type granular starches(top) and of ( U , b)-plane projections fortheir crystalline structure (models by Imberty et al.), showing the packing arrangements of double helices, water molecules, and hydrogen bonding; chain duplexes marked 0 and */2 indicate their relative translation along the c-axis. (Adapted with permission from Refs. 98 and 99.)

123.5') having the maltotriose as a repeating (asymmetric) unit and four water molecules per unit cell. The hexagonal subcell of the B pattern of tuber and retrograded starches'(u = b = 18.5 & = 10.4 & and (Y = = go', = 120') has a more "open" packing of double helices, a maltose moiety as an asymmetric unit, and 36 water molecules (at -27% w/w hydration) per unit cell. The water molecules fill the large central channel formed by the hexagonically packed double helices; half of the water is tightly bound to the chains, and the other half is connected only to other water molecules. In both structures, pairing of double helices corresponds to 1.1-nm distance between axes of the two double helices. There is also a shift of the relative position between neighboring double helices along the c-axis (translation by half the fiber repeat distance), which permits a very good fit between duplexes and allows for the occurrence of H-bonds. Thus, besides the van der Waals interactions, the cohesion between neighboring double helices in the crystalline lattice is strengthened through H-bonding between 0-2 0-6 and 0-3 . . . 0-3. Calculated densities for the crystalline regions of A and B structures are 1.48 and 1.41, respectively [98,99]. These structural models are consistent with the cluster-type molecularstructure of amylopectin and reconcileall known physical, biosynthetic, and morphological aspects of the starch granules. High-resolution solid-state 13C-NMRconfirmed the asymmetry assignments for the A andB structures [loo-1041. The characteristicmultiplicities observed for the C l signals reflect the difference in crystal symmetry between the two forms (Fig. 3). In the A-type starch, which has a maltotriose repeating unit (i.e., three nonidentical AGUs), the C l resonance appears as a cluster of three peaks (-99.3, 100.4, and 101.5 ppm). In contrast, the B structure with two nonidentical AGUs shows a twofold multiplicity for the C l peak (individual chemical shifts at -100.9 and 100.0 ppm). Only a small portion of the starch granule is believed to be crystalline. Kainuma and French[l051 have visualized the hydrated granule as an amorphous gel matrix in which the crystalline regions are embedded. Crystallite dimensions, estimated from line-broadening studies of the X-ray diffraction peaks, are of the order of 14.4-14.7 nm [18]. However, smaller crystallite sizes in the range of 40-50 A have been also reported. If the outer short DP (-14-16 AGUs) chains of amylopectin are taken as a factor in crystallite size, then an estimated length of the crystalline domains would be about (14-16) X 3.5 = 49-56 (repeat distance for six AGUs of double helices is 21 A). By integrating the X-ray scattering intensity separately Over the peaks and over the background (arising from the amorphous phase), estimates of the relative amounts of crystalline phase can be obtained. According to %bel [8], crystallinity values for granular starches range from 15% to 45%, with the A type showing values between 33% and45%.Waxy

.. .

Billaderis

U Me,Si

3 CF’/MAS I3C-NMR spectra of highly crystalline A-type amyloses (a) and B-type amyloses @), amorphous amylose (c), V.-amylose (d), and V,,-amylose (e). (Adapted with permission from Ref.

starches give good X-ray diffraction patterns, while high-amylose starches show low crystallinity,15-22%[8,106]. These observations support the viewthatamylopectin is theprincipal crystalline component native starches. Recent WAXS studies suggested the coexistence A-, B-, and Vtype polymorphs in some starches [106]; e.g., the crystalline regions of wheat and rye starch (A-type starches) granules were found to contain between 7% and 10% B-type structures, while the crystalline phase highamylose starches (amylomaize, wrinkled peastarch) consists of 74.6-84.6%

'

B type and 15.4-22.6% V type. It is likely that environmental conditions during seed development affect the proportion of B structures in A-type starches; high humidity and low temperatures would increase the amount of B crystallites present in granular starch. In considering the short-range order of a-D-glucans, including starch, an interesting approach in identifying ordered structures (A, B, and V) and quantifying the ratio of double helices to single chains has emerged from solid-state 13C-NMR studies [101,104,107,108]. Fora range of granular starches the double helical content was found between 40% and 50%, substantially higher than the respective crystallinity values, 25-35% [101,108]. With further development of the 13C-NMRmethodology, structure-function relationships and molecular mechanisms involvedin many technological applications of starch materials could be widely explored. Although it constitutes a major portion of granular starch, the amorphous material has received very little attention until recently. The amorphous phase is less dense and more susceptible to chemical and enzymic modification [18,109,110]. Diffusion of small water-soluble molecules (of
Bllladeris

of amylopectin and that most of amylose exists in a disordered state, it is not unreasonable to accept that there is no sharp demarcation between crystalline and amorphous domains in granular starch. Instead, a range of structures is expected between well-developed crystallites and fully disordered regions.Inthis type of supermolecularorganization, the amorphousand crystalline phases are interdependent. The arguments provided recently by Gallantetal.[83a] on the organization of lamellaeand their polymeric constituents in granular starch concur with such a view. The distribution and arrangement of amylose and amylopectin within the starch granule remains an intriguing question. As the amylose content increases with grain maturation [7]and the granule grows by apposition, it is reasonable to assume that the outer parts of the granules have a higher proportion of amylose. This was recently confirmed bygranule erosion (cold gelatinization, commencing at the outer layers of the granule and continuing toward the center) of potato starch using 4M CaCl, [115];amylose was more concentrated at the periphery and hadsmaller molecular size than that at the core of the granule. An interesting feature of amylose and amylopectin, when one attempts to depict the distribution of these polymers in the starch granule, is their incompatibility. When concentrated solutions of amylose and amylopectin are mixed, they exhibit immiscibility, with the formation of an amylose-rich and an amylopectin-rich phase [116].If amylose exists as a dispersed phase during biosynthesis, it may be incorporated as amorphous material filling the voids in the amylopectin lattice. Zobel [8]and Blanshard [l] presented several arguments to support the view thatamylose is separated from amylopectin in the granules of corn and wheat starch and is mixed (partly cocrystallized) with amylopectin in potato starch. Light crosslinking of granular starches (corn, potato) and characterization of the products by molecular sieve chromatographyindicatedthat amylose was crosslinked with amylopectin and that there was no crosslinking between amylose molecules [117,118].These datasuggestedthat, in granular starch, amylose molecules do not exist in the form of bundles at amorphous regions but rather are interspersed among the amylopectin molecules. Itis also plausible that some amylose molecules participate in double helices with amylopectin and thereby become less prone to aqueous leaching or complexation with iodine, particularly in potato starch.

3. Another crystallographically distinct structure of starch, typically observed when amylose forms complexes with various ligands, is the V-polymorph. In V-amylose, the chain conformation is a left-handed single helix with six residues per turn (V,) for complexes with aliphatic alcohols and monoacyl

Starch lipids; the rise per monomer residue is ca. 1.32-1.36 [41,42]. In most structural models proposed, based on X-ray analysis of crystalline fibers or electron crystallography data, helices are packed with either orthorhombic or hexagonal symmetry[119]. When the ligand is bulkier than a hydrocarbon chain, helices of seven or eight glucose residues per turn are also feasible [95]; the diameter of the helix cavity is 4.8 with six AGU, 6.3 with seven AGU, and 7.8 with eight AGU per helical turn. Most amylose complexes can be obtained as hydrated (V,>)or anhydrous (V,) structures giving characteristic X-ray diffraction diagrams (Fig. 4). For amylose-lipid complexes the unit cell is orthorhombic, with dimensions = 13.7 A, b = 23.8 c = 8.05 for the v,, form, and = 13.0 A, b = 23.0 A, c = 8.05 for the V, form [41,120]; in the crystalline lattice, the amylose chains are organized in antiparallel pairs, with center-to-center distances between adjacent helices of 13.7 in v,,. A difference in helix rotation ("21') and a helix shift (translation) along the c-axis (-1.3 has also been claimed between the two structures [41]. The observed slight expansion in lattice constants on going from V, to V,, is due to water present within the crystalline lattice (interstitial together with intrahelical) [41,121,122]. The interstitial water (eight molecules per unit cell) is hydrogen-bonded to the hydroxyl groups on the amylose helices in a dense network forming bridges between the chains [41]. The aliphatic chain in the helix is rather locked in a solidlike state (i.e., with a fixed conformation that is prevented from rotation due to steric conflicts), as suggested by Raman spectroscopy [123], "C CP/MAS-NMR [124,125] and molecular modeling [43] studies. The 13C CP/MAS-NMR spectra ofV, and V,, amyloses (Fig. 3) also indicate that these structures differ from those of A and B polymorphs [103]. For comparison, the corresponding spectrum of amorphous amylose is also shown in Fig. 3. The spectrum of amorphous amylose seems to be similar to that of V, except for the shape of the Cl line; the former appears as a composite of a relatively sharp resonance and an upfield tailing, while the latter exhibits only a rather sharp, single line for Cl. X-ray diffraction of scattering diagrams of granular starches do not usually show the presence of V-structures, with the exception of wrinkled pea starch, amylomaize, and some other maize genotypes (dull, all with amylose contents greater than 30% [90,106,112]. The lack of V-type characteristic peaks upon X-ray analysis does not necessarily prove the absence of amylose-lipid complexes; it merely indicates the absence organized helices into well-developed three-dimensional structures (long-range order). The question of whether amylose inclusioncomplexes are present in normal cereal starches has been recently resolved, after a long debate. Besides all circumstantial evidence for some form of association of amylose with granular monoacyl lipids (lysophospholipids, free fatty acids) in situ [26], l3c

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4 X-ray diffraction patterns of V.- and V,-amyloses along with the b) plane projections of helices, showing the hydrogen bonding arrangements. (Adapted with permission from Refs. 41, 120, and 281.)

Polymers

CP/MAS-NMR studies provided the proof for the presence of V-conformation in granules of maize, oat, barley, rice, and wheat starches [124-1271. The two special features of nonwaxy cereal starches, indicative of single Vamylose helices, were: (1) the presence of a broad resonance peaking at 31 ppm (corresponding to midchain methylene carbons of monoacyl lipids), which reflects a near-solid-state structure of lipids due to steric constraints in the helical cavity; and (2) a signal of C l at 103-104 ppm attributed to V-conformation. These resonances are enhanced in acid-treated (lintnerized) starches [126]. X-ray scattering data of enzymatically degraded wheat starch (to increase the proportion of ordered domains in the enzyme-resistant residues) also revealed the V-structure, indicating the presence of amyloselipid complexes in native granules [106]. Although common granular starches do not show the V-structure, at least in the sense of three-dimensionally ordered domains, development of this polymorph canbe readily inducedby heat-moisture treatments of starch (18-45% moisture,90-130°C for 1-16 h) [90], by extrusion cooking [128,129], or simply by gelatinization and cooling of starch dispersions. Under such hydrothermal conditions there is increased mobility of amylose chains complexed with naturally occurring monoacyl lipids or externally added ligands (monoglycerides, fatty acids), leading to formation of larger assemblies detectable byX-rays.Extrusionof cereal starches [l281 and blends of cassava starch with 2-4% monoacyl lipids [129], at 22% initial watercontent,led to formationof two types of structure, depending on extrusion temperature. Below 170°C the X-ray diffractionpatterns of extrudates were typical of the V,, structure. In contrast, whenstarch was extruded at >185"C and the final moisture content was <13%,a new structure called "extruded," or E,-type, was observed. The latter was characterized by a slight displacement of all three diffraction peaks of V,, to lower angles; e.g., the main peak of E,, occurred at -9"03', compared to 9"54' for V,. Differences between the two structures were attributed to different interaxial distances of the helices. According to the X-ray data, the interaxial distances for the V, and E,,, rebetween adjacent helices were 13.8 and 15.0 spectively. It was also shown that the E,, is fairly labile and irreversibly transforms to the v, by reconditioning the extrudates to 30% moisture. This suggests that the Eh form, with the less well-packed chains, is a metastable structure. A lamellar morphology has been proposed for the solid-state organization of helices in isolated V-amylose crystals [130-1321; i.e., polysaccharide chains are folded and lie with their axes perpendicular to crystal surfaces. From the Bragg equation, the calculated long spacings were 75-100 [130,131], while heterogeneous hydrolysis of V-amylose complexes with a-amylases (to selectively remove the amorphousregions) gave enzyme-

Billaderis

resistant amylodextrinswith chain lengths corresponding to a lamellar thickness of -100 [133]. However, a detaileddescription of the supermolecular structure of amylose-lipid complexes in thermally processed foods is a difficult task, because helices can exist in various states of aggregation, depending on the thermomechanicalhistory of the product.Applicationof calorimetry and X-ray diffraction to studies on starch-lipid interactions has undoubtedlyadvanced our knowledge of the structure andpropertiesof amylose-lipid complexes [134-1421. With monoglycerides [136-1391, internal starch lipids [141], fatty acids [142], and linear alcohols [l431 as complexing ligands, at least two structurally distinct forms were identified. A morphological model for the solid-state organization of chains in these polymorphs (I,11) has been postulated [137]. Form I (of low T,), obtained under conditions favoring rapid nucleation, was described as an aggregated state where ordered (helical) chain segments have insufficient packing for crystalline order to be shown by X-ray diffraction (i.e., it gives an amorphous pattern). In contrast, formI1 (of high T,) appeared as a polycrystalline aggregate with well-developed long-range order,showing the typical reflection of the V-pattern. Understanding the supermolecular structure, stability, and transformations between the various forms of amylose-lipid complexes is of great fundamental and technological importance, considering the multifunctional role of lipids in starch-based products. For example, incorporationof monoglycerides in the dough is known to retard starch retrogradation and bread firming [144,145]. Similarly, monoglycerides added to dried potatogranules prevent stickiness [146]. Improvements in structural integrity of cereal kernels during cooking (e.g., parboiled rice) [141], as well as decreased swelling, solubilization, and thickening power of starch [26], have also been ascribed to starch-lipid interactions. The limiting swelling of cereal starch granules during heating, compared with that of the waxy lipid-free tato) starches, is believed to be due to the presence of amylose-lipid complexes that melt at temperatures higher than the gelatinization temperature of starch [134]. On the other hand, formation of V-complexes maybe somewhat troublesome in processing and end-product quality of starch hydrolyzates [l471 and in some nonfood applications of starch, such as paper sizing [148].

4.

RetrogradedStarchStructures-ResistantStarch

Whereas in granular starch amylopectin is the main crystalline component, both amylose and amylopectin are involved in molecular reassociations in aging starch gels and baked products. Following gelatinization and pasting, these systems are not in a state of equilibrium (metastable states) and, there-

Starch fore, undergo structure transformation via chain aggregation and recrystallization on storage. Crystallites, of B type, form eventually, and this is accompanied by gradual increases in rigidity and phase separation between polymer and solvent (syneresis). The postgelatinization changes accompanying these molecular processes, collectively described by the term retrogradation, are thought to have a major influence on texture, digestibility, starch-basedproducts.Retrogradation is a andconsumeracceptability complexphenomenonand depends onmanyfactors, such as source starch, starch concentration, cooking and cooling temperature regimes, pH, and the presence of solutes such as lipids, electrolytes, and sugars [149]; e.g., repeated freeze-thaw cycles are known to accelerate retrogradation and syneresis drastically [150]. Because retrogradation involves a recrystallization process, either calorimetry or X-ray diffraction can be used to follow it. With DSc, the development of structure can be monitoredfrom the magnitude of the melting endotherm of recrystallized amylopectin (T, = 4560°C), while with the X-ray diffraction total crystallinity for both starch polymers is followed by the evolution of the B pattern. The intensity or the area under the characteristic d-spacing at 15.8 of the B pattern, corresponding to the (100) reflection, is often used as an index crystallinity. However, it is important here to remember that the intensity of this reflection is highlydependenton the water content of the specimen[99,113,114]. Finally, macroscopic effects, observed as changes in the mechanical properties of an aging starch system, can be probed viavarious rheological methods. The molecular mechanisms and transformations that occur during gelation andretrogradation of starch and its components are described in greater extent later in this chapter. Structural studies have been carried out to establish the length and the nature of polymer chains involved in interchain associations or crystallites of retrograded amylose and amylopectin [75,151,152]. In this respect, gels were submitted to heterogeneous acid hydrolysis to erode the amorphous regions. The acid-resistant amylodextrins were characterized by chromatographyand enzymic analysis. Crystallites inretrogradedamylopectin gels were found to consist single or multiple branched amylodextrins originating from the outer short DP chain clusters of the molecule. Because of the relatively low DP segments (-15) involved in the crystallites of amylopectin, their melting transition starts about 37-4OoC, and this explains the susceptibility of retrograded amylopectinto a-amylolysis in vitro and invivo [153-1551. In contrast, the chain length over which associations between amylose chains occurs in retrograded amylose is relativelylonger, with DP 30-50 [75,133,156]. High-resolution solid-state l 3 C - m confirmed the presence of aggregated double helices of B type in amylose gels [157,158], while calorimetry has shown that these structures are thermally

-

resistant (melting temperatures -130-170°C) [134]. Another consequence of conformational ordering and aggregationof double helicalchain segments in retrograded amylose is their resistance to acid and enzymic hydrolysis [133,154]. The amylase-resistant linear amylodextrin (originating from retrograded amylose) constitutes a fraction of the so-called “resistant starch,” i.e., the starch resisting digestion in the small intestine and becoming available for fermentation in the large intestine of man [159,160]. This type of resistant starch (RS) forms during thermal processing (heat-moisture treatments) and cooling of amylose-containing starchy foods as a result of amylose retrogradation (e.g., bread, breakfast cereals, cooked potatoes, canned beans). There is presently considerable interest in the nutritional implications of resistant forms of starch in foods, for they seem to have an influence on reduced plasma glucose and insulin levels [161], increased feacal bulking [162], and the potential for beneficial effects on the health of colonic epithelial cells [163]. The amount and structure of enzyme-resistant retrograded starch in autoclaved starch pastes and actual food products depend on several factors. Formation of RS is enhancedwithincreased amylose content of starch [164-1671, increasing number of autoclaving-cooling cycles [164,166], a starcywater ratio of k3.5 [166], increasing autoclaving temperatures (up to 134°C) [164,166], and low storage temperatures [165,168]. Monoacyl lipids have a negative influence on RS yield, because they can form inclusion complexes with amylose and thereby competitively decrease the extent of interchain associations between linear starch chains [169-1721. The main techniquesused to characterize the molecularand physical structure of RS are size exclusion chromatography [133,154,169,173,174], DSC [166,167,170,171,175], X-ray diffraction and, recently, solid-stateI3C-NMR[158,178]. Depending on source, sample preparation, and enzyme hydrolysis conditions, the reported average degree of polymerization (DP) of linear dextrins of RS ranges between 20 and 65 Moreover, it has been demonstrated that the isolated linear fractions of RS formed in in vitro and in vivo studies are of comparable size (DP, 35) [179,180]. With most chromatographicsystems employed, there seems to be a rather broad distribution of chain lengths in enzyme-treated retrograded starch. Gidley et al. [178], using high-performanceanion exchange chromatography,havereported chain distribution profiles with DP between 10 and -100. The wide range of chain lengths is consistent with the broad melting endotherm (spanning from about to 170”C, with a T,,, of 150-160°C) observed for most preparations of RS heated in excess water [134,166,167,175,178]. The resistant portionsof acidand enzyme-resistant retrograded starches give diffraction patterns charac5 presents DSC andX-ray teristic of B-typecrystals.Forexample,Fig.

-

Starch Polymers

t -

-

resistant Yield: 18%

Acid resistant

I

.

.

.

.

l

.

.

.

*

'

*

8

24

Diffmctlon

(20)

---_

0.1

l

ob

ab

lio lio

Temperature CC)

I&

4b

ob

Temperature ("C)

Figure DSC solids) and X-ray diffraction diagrams of acid- and enzymeresistant amylodextrins derived from retrograded amylomaize starch gels.

diffraction patterns of acid- and enzyme-resistantforms of retrograded amylomaize-VI1 starch.The diagrams clearlyindicate that the two residues differ substantially in the level of crystalline and molecular order. The enzymeresistant amylodextrin, although it had a greater content of double helical segments 27 J-g" vs. 16 J-g"),showeda weak X-raypattern, compared to its acid counterpart. However, the DSC profile of the enzymetreated residue is indicative a more homogeneous distribution of ordered segments than that of the acid amylodextrin, which gave multiple melting transitions. The level of crystallinity for RS, estimated by X-ray diffraction, is dependent on the conditions employed for sample preparation (starch con-

a2

Biliaderis

centration, heating-cooling regime, acid or enzyme hydrolysis conditions, etc.). Estimates of crystallinity for enzyme-resistant residues range between 25% and 60% [176,178,181], while quantitative analysis of the molecular order (double-helixcontent), as assessedby solid-state 13C-NMR, gives much higher values (60-95%) [158,178]. The high level of double-helical order in RS is consistent with the resistance of such material to enzymic hydrolysis, as was first suggested by Jane and Robyt [133]. The average size of crystallites in retrograded amylose, calculatedfrom X-ray line broadening analysis, ranged from 7.3 nm to 9.3 nm [181]. This is consistent with the crystallite size of -10 nm corresponding to an average chain length of -32 AGU found for acid-resistant fragments of retrograded amylose [133]. Several models depicting chain packing in the partially crystalline structure of retrograded amylose and RS have been proposed [133,156,174]. Based on X-ray diffraction, I3C CP/MAS-NMR, and chromatographic data, Gidley et al. [l781 have recently presented a structural model for enzyme-resistant retrograded starch from wheat and amylomaize starches. Accordingto their description,there is a substantial amount of double-helical order (-6070%) in RS, with the chains (DP:10-100) arrangedintoaggregates of limited crystalline packing (-25-30% crystallinity). The disordered nondouble-helicalmaterial,interspersedbetweenaggregates, may consist of folding regions, chain ends, andsingle chains trapped within the aggregates. There is also a small fraction (5-8%) of amylose-lipid inclusion complexes in the solid-state matrix. It was further suggested that the broad distribution of linear chains observed in RS is a consequence of the highly aggregated structure of gelled amylose (high double-helix content,low crystallinity, and a broad range in DP of chain segments at junction zones), and the reduced accessibility of a-amylase to the domains of aggregated double helices due to steric reasons.

Over the past two decades, the state and phase transitions of starch, related to changes in molecular mobility as well as the ordering-disordering processes starch polymers undergo during heating, cooling, and storage, have been intensively investigated. By analogy with synthetic polymers, gelatinization and retrogradation are currently viewed as melting and recrystallization of starch in the presence of water. A variety of chemical, enzymic, andphysicalmethodshavebeenemployedtostudythesephenomena [2,182]. Among them, X-ray diffraction, rheological methods, and thermal analysis haveattracted the mostinterestin studying phasetransitions of aqueous starch systems. Usingthesetechniques, a great deal hasbeen

Starch

learned about the effects of water content, annealing effects, and the influence of various cosolutes or other constituents on the thermal and mechanical properties of starch dispersions [l-4,134,149,182-1961. Thermal analysis, in particular, has provided invaluable insights into the critical role of water and other low-molecular-weight solutes as plasticizers of starch, as well as the relevance of water plasticization to the state and phase transition behavior of this biopolymer during processing and storage. The subject has been extensively discussed in several reports [2-4,191,194,197-2001 that addressed the nonequilibrium (metastable) character of aqueous starch systems andthetemperature-,moisture-,andtime-dependence of structural, mechanical, and functional properties of starch. This section will focus on the description of phase transitions of starch polymers in relation to their supermolecular structure and its response to environmental conditions (temperature, solvent, other solutes, etc.), as schematically illustrated in Fig.

A.

The Glass-Rubber Transition of Starch

The glass-rubber transition, although somewhat foreign to starch chemists, is a phenomenon well known to polymer and material scientists. It is considered the most important property of synthetic polymers, affecting their mechanical properties, processing behavior, and stability [201]. Despite the existing controversy with regardto the fundamental nature the glass transition [202], glass formation (at a characteristic temperature, T,) is generally viewed as “freezing out” of large-scale molecular motions (rotational and translational), i.e., for polymers, the wriggling motion of conformationally disordered chains ceases. Thus, amorphous materials mayexistin either brittle,glassy(viscosityapproaching 1012-1014Pass), or mobile,rubbery state, depending on whether they are below or above their T,. The glass transition is accompanied by a gradual change (discontinuity) in the thermodynamic properties, such as thermal expansivity andheatcapacity, cp [203]. Also, a dramatic drop in modulus or viscosity at TR(a change of approximately three orders of magnitude for a typical amorphous synthetic polymer [201]) reflects the onset of segmental motion of the polymer chains as the material passes from a glassy to a rubberlike state. Analysis of this transitionalbehavior of a polymer by various thermal analysis methods [DSC, dynamic mechanical-thermal analysis (DMTA),thermomechanical analysis (TMA)] is in fact based on these T,-inherent property changes. In pure amorphous macromolecules, the glass transition entails a change over a temperature range of about5-20°C. However, insemicrystallinematerials, the presence of microcrystals and strained chain segments between crystallites tends to increase the temperature and range over which this transition occurs [204]. Each amorphous or partially crystalline material will have its

I

Starch own T, (often specified as the temperature at which the polymer is midway between the immobile glass and the rubbery liquid), depending on several factors, such as molecular size, side-branching, degree of crosslinking (inFor the latter, it has cluding crystallinity), and diluent content long been known that the addition of a small-molecular-weight diluent, or plasticizer, to a glassy polymer lowers its Tr Water, for example, is a very effective plasticizer of manysynthetic and natural polymers, causing a large depression in their TB The addition of small quantities of water (plasticization) produces an effectsimilar to an increase in temperature; i.e., there is an increase in segmental mobility of the polymer, compared with the nonplasticized material, at any given temperature above the T,. This is the result of an increase in free volume in the polymer matrix and/or the breaking or weakeningof some intermolecular bondsby the plasticizer The ability of a diluent to depress T8 decreases as its molar mass increases. The increased mobility of chains in the amorphous regions has, in turn, implications on processing quality, mechanical properties, and storage stability of the plasticized polymer. Because glasses are nonequilibrium states, the glass transition is also affected by kinetic factors, annealing treatments, and the previous thermal history of the sample e.g., slower cooling rates give rise to lower values ofTn whereas annealing at sub-T, temperatures shifts the transition to a higher temperature. Following the pioneering theoretical and experimental works of van and Slade and co-workers there is a den Berg growing realization of the importance of glass transition as a physicochemical event (change of state but not a change of phase) controlling the phase transitions and properties of starch. Some of the first evidence consistent with the existence of a glass transition for heated starch specimens came from heat capacity measurements with DSc. An incremental change in c, J.g"-K") prior to the melting endotherm of starch crystallites (Fig. and a minor baseline shift upon superposition of initial scan (granular starch) and rescan of the same sample were attributed to glass transition of the amorphous domains of starch granules. However,it appears is not due to that the Ac, accompanying the major endothermic peak a glass transition, as recently argued by Lelievre For native wheat starch, Zeleznak and Hoseney have reported a Ac, typical of a glass transition at lower temperatures than just prior to the melting peak. The discontinuity in c, at T, is small and not easily detectable calorimetrically for most granular starches.Despite the relatively low crystallinity of these materials the difficulty of identifying and assigning a definite T, most likely originates in the nature of the amorphous granular phase. For partially crystalline polymers, the reduction in Ac, due to increasing crystallinity does not obey a simple additive rule of a two-phase system (fringe-

ii (II

I

5 U

40

80

160

Temperature ("C) DSC thermal curves a granular starch (at two different weight fractions) showing a glass transition (TB)and melting transitions.

micelle model), where crystallites and amorphous matrix behave independent of each other [213,214]. The crystalline regions, acting as physical crosslinks, cause changes in the thermal properties of the amorphous material and decrease Ac, more than would be anticipated assuming an ideal two-phase model structure; e.g., for polycarbonate with a degree of crystallinity as low as 23%, there is no detectable Ac, at T, [215]. To account for such thermal behavior of granular starch, a simplified "three-microphase" model was proposed [191]. In addition to starch crystallites,thismodel incorporates twotypes of noncrystalline material:(1) bulk, mobile-amorphous regions giving rise to a heat capacity change (e.g., amylose); (2) intercrystalline rigid-amorphous material that does not contribute to the Ac, observed at T, as it is motionally restricted byits proximity to crystallites (e.g., regions of dense branching in amylopectin). The sequence of thermal transitions in such a system would be Tg(mobileamorpho Tg("gid amorphous)

Tm("gid crysyslaUine).

Although this polymeric view of starch granule structure is convenient to explain the lack of a discernible glass transition on many DSC thermograms, it is likely that, instead of two distinct amorphous microphases, a range of nonordered structural domains exist in granular starch. Each of

Starch these regions could manifest its own T, and exhibit a different plasticization behavior by water. This, in turn, would spread the glass transition over a broad range of temperatures and account for the difficulty to detect it by calorimetry. In contrast to native starch, the glass-rubber transition is more readily detected for gelatinized materials [212,216,217] or isolated starch polymers[218-2241bythermal analysis techniques. The change in heat capacity at Tg for dry amorphous amylopectin was reported to be 0.47J.g" -K" [221]. It has been also noted that Tgincreases with increasing crystallinity [212,223], which is in agreement with observations made on synthetic polymers [214], indicating the stiffening action of crystallites on molecular mobility ofchains in the amorphous domains. Moreover,the temperature location of glass transition of starch is dependent on the thermal history of the sample as well as the time scale of the experiment, i.e., the heating rate (DSC) and/or the frequency used in a relaxation type of experiment (DMTA or dielectric measurements). Thus, for amorphous starch and amylopectin, the T, values determined from the midpoint of heat capacity change at a rate of 10°C/min, usually fall between the drop in modulus and the tan peak obtained with DMTA at a heating rate of 2OClrnin and a frequency of 1Hz [217,223]. For all the reasons already stated, and in view of the strong dependence of TBon moisture content, it is not surprising that there is considerable variation in the literature among the reported values of T, for starch materials. The most important feature of amorphous starch, having many implications on processing and product quality, is its plasticization by water and other low-molar-mass polyhydroxy compounds.A detailed atomistic model for amorphous starch with water was recently presented using computer simulation [225]. In the dry amorphous starch model structure, a high cohesive energy density was found, proportional to the density of interchain hydrogen bonding [226]; the strongly hydrogen-bonded network can account for the high Tg of amorphous starch. With increasing water content of the amorphous starch matrix there has been a decrease in the interaction energy and an increase of the distance between chains [225]. These effects are manifestations of the plasticizing role of water onstarch at a molecular level. The dependence of T, on water content of starch samples is shown in Fig. 8 using DSC datacompiled from several reports. It appears thatwater strongly depresses the Tgof starch, particularly between0 and 20% moisture content (m.c.); at higher m.c., T, drops below the freezing point of water [3,208,211]. For granular starch, it has been shown thatabove 30% moisture content, TBis constant (i.e., amorphous material is fully plasticized) due to the formation of a separate water phase outside the granules [191]. The drop in modulus at T, as assessed by DMTA, is also dependent on the moisture content of starch and is usually smaller than that expectedfor typical amor-

"1

4

Arvanitoyannis (1994) [ref. Arvanitoyannis et (1994) [ref. 2771 Kalichevsky (1992) [ref. 223) Zeleznak Hoseney (1987) [ref. 2721

5

Zeleznak & Hoseney (1987) [ref. 2721

1

2

3 A

120

A

100

80

"

60

--

40

--

20

"

I

5

15

20

25

. The variation in glass transition temperature starch (DSc data) as a function of water content: 1, 2, and correspond togelatinizedpotato,rice,and wheatstarches,respectively;3and 5 correspondtoamorphousamylopectinand granular wheat starch, respectively. (Data compiled from Refs. 212, 217, and 223.)

phous polymers (-lo3 Pa) [196]. The Tg of dry amorphous starch is experimentally inaccessible because of thermal degradation of starch polymers at indirect procedures have been employed to eselevated temperatures. timate the Tg of dry starch: (1) study of the plasticizing effect of water and extrapolation to zero moisture content [191]; and (2) study of TBfor dry malto-oligosaccharides and extrapolation to a high-molecular-weight limit [221]. Applying these methods, we find that the estimated TRof dry starch is in the range 230-240°C. It has been possible to fit T, data of starch-water systems and make predictions using the classic thermodynamic theory of Couchman and Karasz [227], adapted for polymer/diluent mixtures [228]:

Starch

where T, is the glass transition temperature ofthe polymer-diluent mixture, wl,W, are the weight fractions of diluent and polymer, respectively, and Acpl, Acp2 are the corresponding changes in heat capacity atTgl(pure diluent) and Tgz(unplasticized polymer). Application of this equation requires the use of published values for T, (136 K) and Ac, (1.94J-g")ofwater [228,229]. Predictions of glass transition behavior for binary mixtures of starch-water systems using this fundamental approach had a reasonable degree of success [221,223,230-2321. The Couchman-Karasz equation has also been used to fit DSC data of binary blends of amylopectin with gluten or casein (1:l) [233], while an extended three-component version of this equation was employed to predict the T, of ternary systems, amylopectinwater-sugar [234,235]. The DSC and DMTA data of gluten-amylopectin blends (l:l), at watercontents between 10% and 20%, indicated the presence of two distinct Tg, (defined from the shifts of c,, the change in slope of modulus or the tan peak temperatures of DMTA curves) corresponding to the two polymeric components of the blend [233].This behavior is indicative of polymer incompatibility. The T, of both components decreased, and the lower transition, arising from the gluten-rich phase, came closer to that of amylopectinwith increasing watercontent[233].Foramylopectin-sugar blends, a high degree of polymer plasticization by the sugar was observed at reduced watercontents (7-10%) [196,234,235]. The plasticizing effect of various sugars increased with the decrease of the glass transition of sugar [234]; i.e., among sucrose (T,= 70"C), glucose (T,= 38OC), xylose (Tg= 13"C), and fructose (T, = 7"C), the degree of plasticization of amylopectin followed the order sucrose < glucose < xylose fructose. On increasing sugar content (amy1opectin:sugar from 4:l to 1:1, moisture 7-lo%), a second transition occurred at about 10°C, which increased in magnitude with the sugar level andseemedto dominate the mechanical behavior of the blend, as attested by DMTA and the three-point bend test [196,235]. This transition, attributed to the sugar, does not necessarily imply phase separationbetween amylopectin andsugar, as pointed out byKalichevskyand Blanshard [235]. It may simply be a manifestation of the less free volume required for diffusion and mobility of the low-molecular-weight plasticizer molecule in the blend; i.e., although sugar and amylopectin experience the same free volume, mobility of the smaller plasticizer molecule commences at a lower temperature than the T, of the polymer. Another empirical expression, known as the Gordon-Taylor equation [236], has also been employed to calculate the T, of amorphous starch or sugars at various water contents [234,237,238]:

Biliaderls

where TR1and Tg2are the glass transition temperatures the polymer and diluent, respectively, and W, are the corresponding weight fractions of the polymer and diluent, respectively, and K is a constant related to the strength of polymer-diluent interaction (thelarger the value of K,the greater the plasticization effect). The impact of variation in structure on T, has recently been examined by Bizot et al. [224] using several om-glucans differing in the degree of branching, molecular size, and type of glycosidic linkages. The T,-water content curves, modeled by the Couchman-Karasz equation, were almost parallel within the range 5-25% (w/w) water content. The glass transition temperatures followed the following order: amylose (mol. X lo') > potato starch > amylopectin > phytoglycogen > short DP (-15) amylose. The effect branching as T, depressor is well known and is often described as internal plasticization [201]. The presence of o~(1-6) linkages in &-Dglucans would increase total free volume and chain mobility, not only by introducing additional chain ends, but also because of the three rotational degrees freedom of this linkage; for low levels of branching, these effects cause depression of the T, [220,224]. distinct chain length dependence of Tg for the homologous series malto-oligosaccharides (maltose-maltoheptaose) has been realized [221]; there was a linear relationship between Ti1 and DP" for the oligomers. The relationshipbetween T, andmolecular weight (M) is generally described by the Fox and Flory equation:

where Tirn)is the limiting glass transitiontemperature of thepolymerat infinite molecular weight and K is a constant characteristic of the polymer. The increaseof T, withmolecular weight levels offathighmolecular weights due to entanglement coupling of polymer chains [202]. Treatment of T, for substituted amyloses with this relationship was only partially successful [220]. An endothermic peak near or below the glass transition temperature of starch at low mixture contents (9-20% w/w) has been observed by DSC [216,223,239-2411. This endothermic event, also shown for a wide range of polysaccharides at similar levels moisture, increases in magnitudewith water content andagingtimeat sub-T, temperatures [216,239,240]. The development of a sub-T, heat capacity peak is a general feature of physical aging of many synthetic amorphous polymers and blends [242,242a] and is aconsequenceof the glass transitionkinetics. Glasses always exist ina

Starch

nonequilibrium state and relax toward a lower-energy state upon annealing at sub-T, temperatures [201]. The short-time portions of structural relaxations in the glass reach equilibrium rapidly, even far below TB,while longer relaxations continue for very long times. A direct consequence of annealing at sub-T, is a shift of the relaxation time distribution to longer times. The rate of relaxation during annealing is markedly dependent on the state of the polymer when annealing commences, e.g., cooling rate and thermal history of the sample prior to annealing. The endothermic effect can be manifested either as a distinct sub-T, peak, when aging occurs at temperatures far below T,, or as an enthalpy overshoot at TRY whenaging takes place at temperatures close to the glass transition [242,242a]. Overall, the magnitude of the sub-T, endothermic peak provides a quantitative measure of the enthalpy relaxation that has occurred during annealing of the glass at temperatures below T,. For amorphous starch-water systems, the appearance of the sub-T, endotherm is more pronouncedwith higher moisture contents and annealing temperatures [216,240,241]. Further to the plasticizing action of water (entropic mechanism), specific interactions (energetic associations) between water andthe hydroxyl groups of the amorphous carbohydrate matrix (i.e., establishment of a long-range hydrogen bonding network upon aging) were suggested as contributing to the sub-T, endothermic event [240,241]. However, in the recent DSC study of Thiewes and Steeneken [243] on low moisture (16%)potato starch it was conclusively demonstrated thatthe subTB endotherm of amorphousandnative starch has its origin in enthalpy relaxation rather than in the melting of carbohydrate-water interactions. It was noted that the T, of potato starch granules is partly obscured by the sub-T, endotherm. M e r erasing the thermal history of the granules (at 16% moisture) by a veryshort heat treatment at 90°C (temperature slightly above the sub-T, endotherm) and rapid cooling of the sample, a distinctglass transition for the granules was observed on rescan, with an onset at 80°C. This T, was much higher than that for amorphous starch (T, 40°C) of the same moisture content, presumably due to motional restrictions imposed by the crystallites in native starch. In addition to water and sugars, plasticization of starch can be also effected with other low-molecular-mass compounds. A number of studies have been carried out with the aim of producing thermoplastic materials; i.e., starches have undergone thermomechanical transformation in the presence of plasticizers other than water (e.g., glycerol), leading to destructurization and frequently depolymerization [244-2461. The mechanical properties [216,231,232], fracture behavior [245,247-2491, and other physical [232,244,250] of starch extruproperties (density, textural attributes, etc.) dates are dependent on processing parameters (amount of plasticizer, ternperature, shear), cooling regime (i.e., how fast the polymer matrix reaches

-

its TRas it cools down), andthe state of the end product; thislast is controlled by the extent of starch plasticization with water or other polyhydroxy mmpounds. Thus, with increasing moisture content, the fracture mechanism of starch extrudates changes from a rapid, brittle fracture to slow, plastic fracture (tearing) [247-249,2511; fracture stress also decreases with increasing water content (8-20%) [247,248,251]. Coplasticizing solutes (sugars, poly01s) also decrease the flexural modulus and fracture stress of thermoplastic starch at any given water content [231,245,251]. At high temperatures and low water content during extrusion, conditions leading to a high T, for the starch extrudate, the material (foam) solidifies more quickly as it emerges from the extruder's die, thus yielding a low-density product [232]. In this respect, it has been shown that a rapid setting of the foam also favors the formation of nonequilibrium structures; e.g., for the V-amylose complexes, The amylose comthe Eh form is preferredcomparedto[128,129,232]. ponent at a level of 5-20% provides elasticity and crispness to extruded products [252], whereas maximum expansion of starch extrudates is observed at50%amylose content [253].Moreover, the tensile strength of starch films decreases and elongation at break increases with the amount of plasticizer [254,255], while the strength of water- or water/glycerol-plasticized films increases with the amylose content [255]. Gas permeability data for starch films indicate that incorporation of water and sugars results in extensive plasticization of the polymer matrix and enhanced diffusion rates of gases 12171. Inflections in the gas permeability and diffusivityplots versus 1/T (K) provide an estimate ofthe TRfor water-plasticized films made from starch [217] or blends of starch with other biopolymers [217a7217b]. Thermomechanical analysis (TMA) [256] and DMTA [257] have been used to study the viscoelastic properties of bread, a composite two-phase system. The moisture dependence of TRwas similar to that of pure amorphous starch and gluten [212,249,258,259]. The depression of T, upon hydration was about lO"C/% moisturebetween and moistureand 5"C/% moisture between and 20% moisture content [256]. At the moisture content of bread, water exerts its full plasticization effect on the composite polymer matrix, bringing the effective T, at about -10" to -12°C [256,257]. Over the past decade, research on the glassy behavior of starch and starch-based products has undoubtedly increased our understanding about the behavior and stability of this polysaccharide during processing and storage. The phenomenon of glass transition has been receiving considerable attention because of its pertinence to the structure, mechanical properties, and kinetics of physicochemical changes this polymer undergoesas it passes from the glassy to the rubbery state. Even more important is the recognition that water andother low-molecular-weight solutes (e.g., sugars) are effective

plasticizers of starch, whereby lowering its T,. As has been advocated in several reviews by Slade and Levine [199,200,206,210,260], this has many implications on the structure, physical properties, andother quality attributes of the end product. For example, baked wheat dough products, immediately after baking and during cooling-dehydration,undergotransformations through a continuum of nonequilibrium rubberystates (starch and gluten) to leathery and possibly to a glassy state, depending on the composition of the product (sugar and/or moisture content). Such transitions are accompanied by drastic changes in the mechanical properties (texture) and stability of the composite network. Moreover, many processed-cereal foods (e.g., breakfast cereals, biscuits, and wafers) have a moisture contentin the range where the glass transition is close to storage temperatures. Thus, small deviations in moisture content of the product or its temperature could bring about the transformation of the metastable, solidlike amorphous matrixto the unstable rubberlike state where major changes in the rheological properties of the product occur. The result is an undesirable texture that maybe perceived as a loss in crispness or crunchy texture. It has been also shown that changes in local viscosity with temperature or diluent-plasticization have an effect on the rate of diffusion of small molecules through the polymer network structure [217]. The importance of recognizingthe Tgdepression by diluents and the need to identify an operative (effective) glass transition temperature of starch-based products lies in the control by T, over processes such as annealing, gelatinization, and (re)crystallization of starch [l-4,191, 194,197,199,2001. All these phenomena can proceed only within the temperature range defined by the T, and T,,,;below T, crystal growth practically ceases, while above T,,, the melting rate is greater than the crystallization rate.From the perspective of stability andshelf-lifeextension, it is also important to note that the temperature dependence of the rate of any diffusion-limited relaxation processin the rubbery zone does not obey the typical Arrhenius kinetics, but instead follows the Williams-Landel-Ferry (WLF) [261] equation [206,210,238,260,262]:

where uT is a shift factor, T is the test temperature, Ts is a reference temperature, and C , and C , are constants. If the glass transition temperature is selected as the reference temperature, and viscosityis chosen as the material property the WLF equation becomes

in the WLF kinetic domain(attemperatures,

T, between TB and

-TB

+

Blliaderls

100°C), the log q or log k (where k is the rate of any relaxation process) depends on AT (=T - T,). According to this relationship, relaxation times or rates could change by a factor of 10 for every 3"C, whereas the Arrhenius behavior (for a typical kinetic response deteriorative processes in food materials, Qlo = 2) requires, for the same change inrate,atemperature change of -33°C [260,263]. Manipulation of T, (via control of moisture or product formulation) in relation to storage conditions (temperature, relative humidity environment) has been suggested as means of maintaining quality and extending the shelf life of food products [206,260,263-2651. This is found in practice for pasta, extruded and other thermally processed cereal products (low-moisture systems having an elevated T,), and frozen items where freezer temperatures are kept below the effective T, of the product.

B. Annealing, Solid-state Transformations and Melting Transitionsof Native Starch Starch granules, being partially crystalline entities, are amenable to reorganization (annealing) during heating at temperatures above T, and below the equilibrium T, of the crystallites. Heating granular starch under these conditions, in excess water, enhances chain mobility and thereby may cause changes in the crystalline phase toward more organized structures of lower free energy (larger and more perfected crystallites). The first observations of the effects of annealing (72 h/SO"C) on wheat starch [266], using the Kofler hot-stage microscope, were an increase in gelatinization temperature and a narrowing inthe gelatinization temperature range.These findings were confirmed later by DSC experiments [186,209,267-2721. Depending on the annealing temperature and time, there might also be an increase in the gelatinization enthalpy of the annealed starch granules [186,267-269,2722761, implying a net increase in the level of short-range molecular order. However, if the temperature annealing exceeds a certain limit, there is reduction in the apparent AH values, presumably due to partial melting of the less thermostable crystallites or granules [209,268,271,277]. Long-term annealing is also accompanied by aloss the intercluster distance (-9 nm) observed with S A X S [89,271], a substantial loss in long-range radial order (birefringence) [271], and no apparent change [209,271] or a slight increase [278] in crystallinity as assessed by WAXD. The unchanged positions the d-spacings of annealed potato starches indicate that the B structure remains the same during the annealing process [276]. The dynamic responsesof structure to various temperature-time-solventconcentrationregimesto which granular starch is exposed prior to gelatinization may be of industrial significance. For example, wet milling corn involves steeping of kernels for 24 h in water (containing 0.2% SO,) at 40-50°C; these conditions are

adequate to modify the structural domains of the granule by annealing. Air or vacuum-oven drying of starch suspensions may also improve granular order, as shown for wheat starch by DSC and X-ray diffraction [278]. VIScoamylograph curves for annealed starches usually show increased viscosities upon cooling of the paste. However,the observed changes in the pasting properties and leaching behavior of amylose, as a result of annealing, vary with the source of starch and the hydrothermaltreatmentemployed [272,279]. A related hydrothermal process for structural modification of granular starches is that involving heat treatment at lower water contents (18-27%) and higher temperatures (95-110°Cfor 12-16 h). Such heat-moisture treatment induces major structural alterations and modification in the physicochemical properties of granular starches [280,281]. In cereal andtuber starches, heat-moisture treatment increases the gelatinization temperature, water-binding capacity, stability of starch pastes to heat and shear, and, in most cases, granule susceptibility to enzyme attack [280-2851. The same process also decreases the swelling power and, for most starches, the hotpaste consistency and solubility,due to reduced amyloseleaching [281,284]. These effects are dependent on the moisture content (greater at 27% moisture) during thermal treatment and are particularly pronounced with tuber starches. Electron microscopy revealed a large, hollow region at the center of the heat/moisture-treated granules of potato and corn starches [285], implying major structural rearrangements of the granular material. At a molecular level, X-ray diffraction diagrams indicate a transformation of the Btype crystalline lattice of tuber starches towards the A-typepolymorph, whereas cereal starches show the development of well-defined V structures On the other hand, the characteristic d-spacings of A-type crystalline domains in heat/moisture-treatedcereal starches do not show any change, suggesting that chain packing remains the same in their granules on hydrothermal treatment. Donovan et al. [l851 have examined heat-treated (100°Cfor 16 h at 18, 21, 24, and 27% moisture content) potato and wheat starches by calorimetry. Withexcess amount of added water during DSc, the granules showed a progressive shift of the melting endotherm toward higher temperatures, compared to the untreated samples, with increasing moisture content. There was also a change from a single cooperative melting endotherm to a broader biphasic transition (at 27% moisture), indicating chain reorientation in starch crystallites on hydrothermal treatment. Defatting before the heat treatment resulted in sharper peaks and a narrower temperature range for the biphasic melting transition [MS]. The A” or “A type” was polymorphic transformation of “B type” to “B recently studied by calorimetry and X-ray diffraction [286] using short amylose chains (potato starch lintners with DP 15). At a water content above

+

20% (d.b.), the B-type potato lintner showed a small endotherm (T,’ Fig. 9) preceding the main melting peak (‘M,). This small endotherm (at 90100°C, AH J/g) was absent in similarly heated A-type lintners. The structural origin of the ‘T’ endotherm was identified as corresponding to the polymorphic transition “B-” to “A-type,” which involves a change in the packing arrangements of helices in the crystal lattice. This interpretationwas corroborated by X-ray diffraction measurements performed onsamples subjected to heat treatments at successively increasing temperatures (e.g., 35% H,O d.b.,Fig. 9). Below20% water content, the ‘T’ endotherm was no longer observed in the DSC curves, and there was no evolution of the Btype &spacings in the X-ray diffraction patterns of the thermally treated lintners up to temperatures where thermal degradation occurred. Obviously, a minimum water content of -20% is required to facilitate the structural transition from the hexagonal E-typelattice to the more compact,monoclinic A-type lattice. model for the solid-state transition of B starch to A starch, involving the progressive removal of interhelical water and a unidirectional slipping of the chain duplexes (Fig. g), was proposed earlier by Perez et al. [287]. The changes in the physical properties of heat-treated starches under limited water conditions have not been exploited commercially to a large degree. A heat treatment process of soft wheat flours (12O0C/1O min at 10% moisture) has long been known to improve cake volumes and crumb scores, similar to the effects brought about by chlorination of cake flours [288-2901. Although the exact mechanism for the improvement in cakebaking performance of heat-treated flours has not been yet elucidated, it is likely that this behavior is related to structural alterations of the starch granules and to changes in their surface chemistry (i.e., modification of surfacebound proteins andlipids). As pointed out byColonnaetal.[281],an increase in the gelatinization temperature, obtained by hydrothermal treatments, could be of advantage in thermal processing of some foods. For example, starch swelling and solubilization would be slightly restricted, thus affecting the competition for available water with the denaturation process of proteins in a composite food matrix; this in turn may have implications on the structure development and textural attributes of the end product. On heating aqueous suspensions of starch granules, the optical properties of starch (e.g., birefringence) and its X-raydiffractionpattern are retained, until the gelatinization temperature is reached. Above this temperature, there is disruption of starch granule structure and loss of molecular order (loss of birefringence,crystallinity,and periodicities observedby S A X S and WAXD), irreversible swelling of the granules, amylose leaching, and increase in viscosity. All the irreversible physical changes accompanying the disordering process are collectively known as gelatinization. The most

-

Polymers

c

-

a

6VC IOO'C 112'C d 135'C e 152'C

The "B-"to"A-type"transition for B-type potato starch lintner @P 15) at 35% water content (d.b.). The mows on the DSC thermal curve specify the temperatures at which the samples were heated prior to X-ray analysis; the model is also Perez et al. [287] for the solid-state polymorphic transition ''B''+"A'' given, illustrating the progressive removal water molecules (small dots) and the change in packing of chain duplexes. (Adapted with permission from Refs. 286 and 287.)

widelyused technique in studies of starch gelatinization is DSc, which reveals several endothermic events during heating of aqueous suspensions of granular starch. Figure 10 shows a typical thermal profile of rice starch (22% amylose) heated at 50% (w/w) moisture content. Although there is a diversity of opinion regarding the origin of the low-temperature (
l

40

I

90 Temperature ("C)

-.

A typical DSC thermal curve solids) of a cereal starch showing the different melting transitionsand the corresponding structural domains undergoing a phase change.

Starch

The melting behavior of starch crystallites, manifested by the M , and

M 2endotherms, has been followed as a function of water content for many granular starches of different botanical origin [183-1857188,191,209,2917 m i c a 1 DSC thermal for a waxy rice starch of varying water content are shown in Fig. 11. For water contents greater than single endotherms are usually observed, indicative of cooperative melting. However, as the water content is reduced, the thermal profiles reveal multiple

" ""

T,

T,"

Volume

40

.

80

AHu

V,

H20

v1 I

1 .200 Temperature ("C)

11 DSC melting profiles a waxy rice starch at various weight k t i o n s starch solids; inset shows a plot the melting data according to Flory theory.

endotherms, implying that the granular structures respond differently in lowmoisture environments.The development of the M 2transition at intermediate water levels and its progressive shift to higher temperatures as the moisture content decreases have led to several hypotheses for the structural origin of the observed peak multiplicity in the thermal profiles [2,4,198]. First, Donovan [l831 proposed that, in excess moisture, crystallites melt cooperatively as a result of the tensions exerted by the neighboring amorphous regions on extensive hydration and swelling; this yields the MI endotherm (frequently referred to in the literature as the G endotherm). He has graphically described the disordering process as "stripping" and unfolding ofstarch chains from the crystallites due to the swelling of the amorphous chain segments. As water becomes limiting, only partof the crystallites destabilize with this mechanism, while the remaining give rise to the second endotherm, M2,in accordance with the Flory-Huggins theory of polymer-diluent interactions [300]. This theory, which is based on equilibrium thermodynamics, describes the depression of the equilibrium melting point, p, of a polymer to a new value, T,, as a function of the volume fraction of the diluent (solvent), U,:

where V, and V, are the molar volumes of the polymer repeating unit and diluent, respectively, AH,, is the enthalpy of fusion per repeating unit, and x is the Flory-Huggins interaction parameter, whose value is determined by the solubility of polymer and diluent in one another. Since this theory applies only to equilibrium crystals, its application to phase transitions of starch-water systems has met only with limitedsuccess. As with other partially crystalline polymers, moststarch structures exist as metastable states, which are amenabletoperfection(annealing)upon plasticization with water and thermal treatments [4,191,209]. According to Lelievre [301,302], equilibrium conditions in heated aqueous starch systems can be approached only under very slow heating rates (-1"C/3 h), far below those commonly employed in DSC experiments. Slade and Levine [3,208,211] have questioned the Flory-Huggins treatment of DSC melting data of starch on the grounds that true equilibrium conditions are not met during gelatinization and that melting of starch crystallites is controlled by the glass-rubber transition ofthe amorphous regions. Even with highly crystalline preparations of A- or B-type starch lintners @P 15), Whittam et al. [303] suggestedthat estimates of T: for starch crystallites, using the Flory-Huggins approach, may be somewhat low; with granular starch, the amorphous material would have a greater depressing effect on crystallite perfection and the melting temperatures. Despite its theoretical limitations, the Flory analysis (as illustrated in Fig. 11, inset) is useful in comparing the

-

Starch Polymers

thermal stability of different starch materials and in allowing estimation of their apparent Tmunder dynamic heating at various moisture conditions. An alternative explanation for the biphasic endothermic events at intermediatewatercontents was given by Evans andHaisman who suggested that crystalline zones in different granules vary in their thermal stabilities. Granules with the least stable crystallites melt cooperatively first (Ml), and the disorderedpolysaccharide chains absorbmorewater. This means that the remaining crystallites and granules have less water available (effective volume fraction of diluent is reduced) and, therefore, will melt at a higher temperature (M2). They have also applied the Flory theory and, by making a series of substitutions and simplifying assumptions for aqueous solutions of small-molecular-weight solutes, derived a relationship between T,,,, a,, and v1 for ternary starch-solute-water systems:

Although this relationship can generally predict the direction and relative magnitude of the effect of simple solutes (NaC1, CaCl,, glycerol, sugars), it cannot account for specific solute effects, e.g., interactions between starch and certain ions. This view has subsequently been supported by Liu who claimed that the positions of melting endoand coworkers therms in DSC thermal curves are governed by the melting of granules with different thermal stabilities, heated at continuously changing volume fractions of available diluent. According to Slade and Levine at the leading edge of the first transition (Ml) there is a change in heat capacity, attributed to the glass transition of the amorphous phase of the granules; completion of the glass transition permits the crystalline domains to undergo a nonequilibrium melting process,giving rise to the second peak (Mz). This postulate has been challenged on the grounds that the observed heat capacity shift occurs throughout the temperature span of both endotherms Other interpretations for starch structure loss in limited water systems invoked crystallite disruption followed, at a higher temperature, by doubleor disordering of double helices associatedwith helixmelting short-range order (Ml) and followed by melting of crystallites (M2) These latter models, however, are not consistent with the findings that both double-helical order (assessed by solid-state 13C-NMRor DSc) and crystalline structure (X-ray dieaction) are lost concurrently during heating for Recently, mostgranular starch systems examined Svensson and Eliasson attributed the biphasic endotherm to a slow plasticization of the amorphous granular regions under restricted water conditions,which forces the melting of crystallites tohighertemperatures.

Tomka [W] has examined the structural transitions potato starch in the range of 10-20% moisture. At 18% water content, the DSC tracing showed four endothermic transitions, denoted G (at "60-70°C), S, (at llO°C), S, (at lSS"C), and H (in the range 165-200°C). According to the work of Thiewes and Steeneken [243], the G transition corresponds to the sub-T, endotherm as a result of enthalpy relaxation of the amorphous material. On the other hand, the melting process of the crystalline domains was attributed to both S, and S, (i.e., M,and M,) transitions [244]. Although in all of the preceding models gelatinization was treated as a solvent-assistedmelting of crystallites, the fact that gelatinization is subject to kinetic limitations, due to the complex morphology the partially crystalline starch granules, has often beenignored.MarchantandBlanshard [307], using a small-angle light-scattering device, have considered the kinetic constraints of gelatinization from the viewpoint of three consecutive processes: (1) diffusion of water into the granule; (2) melting of crystallites; and(3) swelling of the granule due to increasedhydrationof the disentangled polymer chains. Temperature jumps of variable size, in conjunction with birefringence measurements, revealed that only melting and swelling exhibit time dependency. It was also suggested that within partially gelatinized granules, rearrangements the polymer chains in the amorphous phase and the remaining crystallites might occur. In view the inherent molecular heterogeneity and nonequilibrium nature of crystallites in starch granades[3,4,191,207-2091, the interdependence between amorphous and crystalline domains [4,191,194],and the thermalbehavior ofV-amylose complexes .[134-137,139,140], we have proposed that the multiple melting profiles observed during heating of starch in limiting water contents may reflect melting and reorganization processes occurring concurrently in a dynamic DSC experiment [4,191,197]. In this respect, the MI and M2transitions (Fig. 10) arethe direct manifestation composite thermal effects due to partialmelting(endothermic)andchainreorganization (exothermic) events and, therefore, the observed gelatinization behavior is governed by nonequilibrium factors (crystallite size, perfection, timescale of the process, etc.). Upon water plasticization of the amorphous granular regions, at temperatures .above Te where chain mobility is greatly enhanced, andfollowing partial melting of small or more imperfect ordered domains, reorganization of chains would be favored, because the temperature is still below the melting point of equilibrium crystals.Such structural reordering is rather shortrange scale, undetectable by X-ray diffraction. Certainly, this interpretation does not preclude any possibility for development water gradients in the sample during heating, which could also affect the melting process of the more resistant granules or crystallites due to changesin the effective diluent conoentration.

Starch

There is some experimental evidence to substantiate this latter view on the origin of the Ml and M2 transitions for granular starch. First, removal of the intercrystallite amorphous material of granular starches by acid etching (lintnerization) eliminates the biphasic melting behavior and broadens the transition [184,294,308,309]. The acid-resistant granular residues exhibit a broad, single melting endotherm at intermediate water contents, despite the heterogeneity in crystallite size. Such response would be expected, conbe verylittle sidering that after removalof the amorphous material there chance for reorganization ofthe remaining ordered structures during heating. The calormetric findings of Donovan [294,309], on purified and recrystallized waxy amylodextrin fractions, are also consistent with this notion. At evireduced water conditions (solvent volume fraction range dence for multiple melting transitions was seen only for multiple branched chains in which a substantial amount of nonordered material wouldbe pressingly branched dextrins ent. In contrast, crystallized fractions of linear of short DP, presumablyhavingvery little portion of disorderedchains, exhibited only single, broad endotherms. Second, there is variation in the melting profilesas a function of heating rate [191]; the Ml and M2 transitions (Fig. lo), seen at heating rates between 3" and 20"C/min, mergeinto a single endotherm at higher heating rates. As the heating rate increases, and because reorganization is time limited, melting will occur as a single-stage process, Ml. However, changes in resolution the DSC signal with increasing heating rate as well as sample size maybe contributing to the shape of the thermal traces, as argued by Liu and Lelievre [198,310]. Finally, the very sharp peaks often seen at the concluding end of the thermal curves of granular starches low moisture content [198,209,244,311] (e.g., Fig. 11, starch weight fraction of 0.6-0.8) provide additional evidence for melting of annealed structures. An appropriate and crucial experiment to test this hypothesis further would be to "freeze in" the preexisting morphology ofthe starch granule by mild crosslinking, hence restricting chain mobility, which is a prerequisite for reorganization during heating. The temperature interval over which loss in structure occurs during heating of starch granule suspensions in excess water has been the subject of several investigations. For waxy maize starch, residual birefringence was consistently at a lower level than extent of crystallinity (X-ray diffraction) or short-range order (DSC, solid-state l3C-MMR) [312], whereas for corn starch decreases in crystallinity commenced before birefringence began to disappear andcontinued after all birefringence was lost [304]. With the possible exception of potato starch, other granular starches exhibit a parallel loss of crystalline and molecular order [108]; for potato starch granules, complete loss in crystallinity occurs at a lower temperature than that for disappearance of short-range order (DSc, 13C-NMR) [108,149].

Biliaderis

Attempts tocompile

DSC gelatinization dataofnative starches from the same or different botanical origin, are of questionable significance if one considers that, besides the granule structure organization effects, the melting transition parameters (T,,,, AH) are influenced by heating rate, moisture content, mechanical damage, thermal history of the sample (annealing effects), extraction conditions of starch, etc. According to the calorimetric studies of Shiotsubo and Takahashi [319] on potato starch, the gelatinization peak temperature increases with increasing heating rate (superheating effect). It was also shown that only at heating rates below O.S"C/min (well below those found in conventional DSC studies), the melting peak temperature remains constant, thus approaching equilibrium conditions. As to the structural origin of the DSC endothermic enthalpy values, the general consensus is that they reflect primarily loss of double-helical order rather than loss of crystalline register [108,303,320]. This implies that the forces responsible for structural stability of starch granules are largely at the double-helical level and that chain packing energy contributions are very small. For highly crystalline preparations of A- and B-type spherulites, obtained by crystallization of aqueous solutions of potato starch lintners (DP 15) underdifferentconditions, the DSC melting data indicated a surprisingly similar enthalpy valuefor the two polymorphs (-35J/g) and a difference in melting temperatures of 15-20°C at a fixed water content [303]. The more closely packed A crystals, possessing a more stable structure [94], had a higher T,,, than the less densely organized B polymorph; in excess water, the B spherulites melted at 75°C. Despite the observed difference in T, between the model crystalline materials, melting temperatures of granular starches do not necessarily follow this rule; e.g., wheat starch granules (A type) usually exhibit lower melting temperatures Obviously, than those of potato starch (B type) crystallite size and perfection of ordered chains as well as the depressing influence of the amorphous domains have a major impact on the thermal stability of granularstarch.Reported gelatinization enthalpies of native starches are generally in therange 5-20 J/g. It is perhaps worthcommenting that the apparent gelatinization enthalpy of granular starch represents net thermodynamic quantities of different events: granule swelling and crystallite melting(endothermic),hydrationand chain reordering(exothermic) [185,191]. There is generallyagoodagreementbetween DSC transition temperaturesandbirefringenceend-pointtemperatures of native starches [184,191,315,317]. Large variations in DSC melting parameters (AH, transition temperatures) among different genotypes of corn [186,314,316,317, 322-3261, rice [269,315], barley [124], amaranth [327], sorghum [328], legume [184,296,329], and potato [330] starches have been also reported.

-

Polymers

For starches of similar genetic origin, handled under identical processing protocols to minimize contributions from annealing effects, amylose content does not seem to correlate with T, or melting enthalpies of starch tallites [315].Most studies involving thermalproperties of corn starches have concentrated on the effect of mutant endosperm genes, such as waxy amylose-extender dull (du), and sugary-2 (S&). Forexample, among three wx-containing genotypes and du the had the highest AH and gelatinization temperature [325]. The amylopectin fraction of the wx genotype was found to contain a high proportion of longer chains, which can account for the B-type crystallinity this starch as well as its elevated T,. Moreover, starches from corn mutants with the gene frequently show broad endothermic peaks or even a double melting transition for starch crystallites in excess water, indicative of heterogeneity ordered structures inside their granules [325,326].Incontrast, the allele in corn results in starches with lower gelatinization temperature and melting enthalpy than normal starch [323,324]. Environmental factors may also influence the thermal properties of starch from corn [331], wheat [332], and potato [330] genotypes. Finally, the loss of structural integrity when starch [333] or flours [313,333,334] are subjected to shear or compressive forces (e.g., milling) results in a reduction in transitiontemperatureand melting enthalpy of the mechanically damaged granules; the overall loss in order, as measured by X-ray diffraction, DSc, and solid-state "C-NMR, is dependent on the severity of milling conditions and milling time [333,334]. Starch gelatinization is also influenced by the presence of small-molecular-weight solutes (nonionic and electrolytes) and hydrocolloids. Understanding these effects is important for better process control and for improving the texture and other quality attributes of starch-based products. In general, increasing concentrations of sugars and other polyhydroxy compounds are known to impede granule swelling, raise the temperature of gelatinization (Fig. 12), and elevate the temperature viscosity rise (pasting) [187,188,335-3411. For example, at the concentration used in cakes (55-60%), sucrose delays gelatinization wheat starch from 57" to 92°C [335,339]. Furthermore,SEM of granules isolated from high-sucrose-content baked products indicates decreased granule deformation, compared to products of low sugar level [342,343]. There might be also restrictive effects on the extent of conformational disordering upon gelatinizationof wheat starch as impliedby the reducedenthalpy values in the case of sucrose [187,336,339], glucose, or fructose [335,339]. However, in other studies the AH has been reported to be unaffected [l881 or even increased [340,344]. There have been several interpretations for the influence of sugars in delaying starch gelatinization. These include competition for water between starch and sugar (lowering of [345], sugar-starch interactions [302,338],

Billaderis

350 -

370

sucrose 2 glycerol

3 ribose

330

m 20

--

Concentration (%

C

.L

al

r

350 rn

330

I

2

3

4

Molarity (M) 12 The effect of hydroxycompoundsandneutralsaltsonthemelting transition of potato starch (10%wlw in water). (Adapted with permission from Ref. 188.)

and more recently an “antiplasticization” mechanism by the sugar-water cosolvent [3,199,211]. The antiplasticizing effect of sugars has been associated with a decrease in free volume, relative to the plasticizing action of water alone, i.e., the plasticizing cosolvent with a greater molecular weight than water itself is less effective in increasing the free volume and in depressing the T, of the amorphous material in starch granules;itthereby increases the gelatinization temperature. For ternary starch-sugar-water systems, Slade and Levine [3] presented linear relationships for the dependence of gelatinization temperature on the amount of sucrose and the inverse molecular weight of the cosolvent (water + sugar) using a series of sugars as cosolvents. The N M R data of Chinachoti et al. [344] also showed that water mobility in a starch-water system, as measured by ”O relaxation times (T2),decreased drastically by the addition of sugar. It was then suggested that such reduced mobility of

water in the presence of the sugar may play a role in lowering the effectiveness of plasticization of starch by the cosolvent. Attempts have been even made to explain and predict the thermal responses of granular starch on purely thermodynamicgrounds by using an extension of the Nory equationtoathree-componentsystem(water-starch-sugar)[2,188,302]. Although this approach appeared to provide a convenient framework for analyzing starch-melting data, its development is based on equilibrium melting and on making simplifying assumptions regarding the magnitude of interaction parameters among solvent, solute, and polymer. However, in view of the nonequilibrium nature of starch gelatinization, as discussed earlier, as well as the uncertainty concerning the role of solute on processes like annealing, such treatment of gelatinization data would be inappropriate. As to the qualitative aspects of starch gelatinization in limited-water systems, the double melting endotherm of starch crystallites (Ml and M,) is changed into a single, narrower transition with increasing sugar concentration, implying a more cooperative melting process [340]. Hydrocolloids at low concentrations (0.5-2.0%), despite a markedincrease in the viscosity of the composite gel matrix[346,347],causevery little change in the DSC gelatinization parameters (AH, T,) of starch [347-3491; at high concentrations, the effects of hydrocolloids on phase transition behavior of starch are similar to those of sugars at equivalent concentration [350]. Electrolytes exhibit more complex behavior than nonionic solutes, as illustrated in Fig. 12 [188]. With increasing salt concentration, NaSO, and Na,HF'O, progressivelyincrease the gelatinization temperature, whereas NaCl and CH,COONa show different effects. For potato starch, the gelatinization temperature initially increases and then decreases as the concentration of NaCl and CH,COONa increase [188]. Granules of A-type starches are more resistant to the decline in their T, as the concentration of NaCl is raised above 4M, presumably becauseof their more compactstructures than tuber starches [311]. A similar trend was also shown for CaCl,, which at intermediate concentrations (-3 M) brings about swelling and gelatinization of starch granules at room temperature.Starch granule suspensions in >4 M CaCl,exhibit exothermic transitionsonheating,which suggests that the starch is dehydrated at these salt concentrations and that, on melting, the exothermic process of hydration obscures the endothermic event of crystallite structure disruption [188]. In general, the effect of neutral salts on starch gelatinization follows the order of the classical Hofmeister (lyotropic) series, particularly in the case of anions [188,351]; cations of different chloride salts exhibit a more complicatedbehavior, as evidenced by polarizing microscopy[352].According to Oosten [353], who viewed starch as a weak acidic ion-exchanger, anions are the actual gelatinizing agents, whereas cations exert a protective

Billaderis

effect on granule structure by generating a negative Donnan potential. Jane [354] has attributed the electrolyte effects on starch gelatinization to:(1) indirect effects of the ions on water structure (enhancement or disturbance of the hydrogen-bondednetwork of watermolecules,depending on the charge density and size of the ion); and (2)electrostatic interactionsbetween the ions and the polymer chains. Undoubtedly,additional information should be acquired to elucidate fully specific interactions between ions and starch and thus predict the direction andmagnitude of salt effects on starch gelatinization. A physical modification of starch, characterized by a change in molecular order while maintaining its granular form, is the production of granular cold-water-soluble (GCWS)starches, which give pastes in coldor warm water, comparable to those of “ c o ~ k - u p pregelatinized ,~~ starches; i.e., they provide an “instant” paste without heating. Further to the development of an injection (atomization) and nozzle spray drying process of starch slurries [355], GCWS starches can (15-50% w/v) at high temperatures (-150°C) be prepared by several other methods: (1) heating starch in aqueous monohydric alcohol solutions (75%alcohol,w/w)athightemperatures (150170°C) under pressure [356,357]; (2) heating starch in aqueous polyhydric alcohol (e.g., propan-l,2-diol) solutions at elevated temperatures (>115”C, atmospheric pressure) and exchanging the solvent with ethanol [358]; and (c) treating starch in a slurry with alcoholic alkali solutions at low temperatures (25-35°C) [359]. The transformation of native or chemically modified granular starch to a GCWS product upon heating in an alcohol/water mixture involves melting of crystallites and, depending on starch composition and the alcohol used, formation of helical V-type commolecular structure plexes [357,358]. Indeed, the X-ray diffraction diagrams of GCWS starches show the V-pattern, except for GCWS-waxy maize and GCWS-chemically modified starches,which give an amorphous profile [360-3621.The aqueous alcohol medium used during thermal treatment restricts swelling and molecular dispersion of the granules, although it facilitates melting of crystallites. There is also enough mobility in the system to foster complexation of starch chains with the alcohol (ligand) on cooling. The development of such single helical structures as well as chain entanglements between amylose andamylopectinmayhelp to maintain the granularform of the GCWS starch. Cold water solubility of these materials was explained by the conversion of the double-helical crystalline domains in nativegranules into single helices; single-helical amylose and amorphousstarch have been reported to be water soluble at 25°C [95]. Dispersibility in cold water, smoothness of gel, paste consistency,and stability tofreeze-thaw cycles of GCWS starches vary with the type of starch and the temperature-solvent conditions employed for their preparation [361,362].

Starch

Attempts have been made to monitor phase transitions of starch in complex food systems, where, due to the presence of various thermally responding components, the DSC curves may become complicated. A DSC study of the effect of cake ingredients on starch gelatinization was carried out by simulating the baking conditions of angel cake [363].It was shown that high sucrose levels are required to elevate the transition temperatures of starch and egg white proteins to the same temperature region and thereby to maximize the cake volume. The effects of sucrose, salt, and other bread and cookie dough ingredients on starch gelatinization were also reported [337,364-3671.With multicomponent systems, where the various constituents undergo thermal transitions in the same temperature zone, the assignment of endothermic transitions to individual components may require their thermal characterization separately; e.g., during thermal processing of gel-type seafood products (surimi), four melting transitions proceeding independently have been identified by DSC and attributed to the thermal denaturationoffish proteins (three)and the gelatinization of starch [368]. Wheat gluten, lacking any denaturation endotherm, is suitable to examine the influence of added protein to starch gelatinization; at a constant waterto-starch ratio, the gelatinization temperature of wheat starch increased with increasing amount of gluten [366].

C. Gelation and Retrogradation of Starch The ability of starch polymers to form ordered structures upon cooling of their aqueous dispersions, via interchain associations, is a critical step for the setting of hydrated three-dimensional gel networks and the development of texture for thermally processed products. Formationof tertiary structures is due to the structural regularity of &-(l-4) linear glucans and may involve both short- and long-range order, depending on the extent of lateral associations between ordered chain segments. These structures constitute what are known as crosslinks, or junction zones, in a gel network. Interconnecting single chain segments between the double-helical junction zones are relatively more mobile, whereas spaces not filled by the polymer chains are occupied by the solvent. In physically crosslinked systems, such as starch pastes and gels, the density and stability (related to the size and perfection of ordered chain microdomains) ofthe junction zones govern the mechanical properties of the network and its responses to applied stresses or to changes in temperature and solvent quality. Starch gels, in particular, are metastable, nonequilibrium states and undergo structural transformations (chain aggregation, crystallization) during storage. Thus, any examination of starch gel structures and physical properties must be considered in the context of temperature- and time-dependenceof gel-curing events. The crystallization pro-

cess of starch gels, expected to occur only between T, and T,, can also be viewed as following the classical three-step mechanism for crystallization of synthetic polymers: (1) nucleation, (2) propagationor crystal growth, and (3) maturation, i.e., continuing slow growth and crystal perfection [3].

and The mechanism of network self-organization from amylose solutions has been a topic of several investigations [47,151,152,369-3731, because the linear polymer of starch is largely contributing to its gelling properties. Kinetic experiments with amylose concentration between 0.3% (below this, amylose precipitates without gelation) and2.0% (above this, gelation is very fast to distinguish any sequential stages of chain organization) are usually conducted to resolve all steps leading to polymer self-assembly from the homogeneoussol.In this respect,many probes of structural order (e.g., turbidity, dilatometry,light scattering, X-ray scattering or diffraction, optical rotation, NMR, viscometry, andsmall strain dynamic rheometry), responding to different levels of macromolecular organization, have been applied. The time evolution of shear modulus (G') for amylose solutions (0.2 M KC1) in the concentration range 1.03-1.78% (at 25"C, 5-10% strain, 5 rad-s"), as shown in Fig. 13 [372], reveals two stages: an initially rapid rise in G', followed by a step of slower increases in modulus (pseudoplateau region). The initial rise in G' and attainment of the modulus plateau region occur over shorter times with increasing amylose concentration [370,372]. The G'@)function is also dependent on amylose chain length. For nearly monodisperseamyloses in the DP range of 250-1100 (at2%solutions, 25"C), Clark et al. [370] observed that aplateau G' value was attained faster with amyloses of shorter chain length. The rheological data also indicated that amyloses of very high DP (2550,2800) exhibit a veryslow development in G' under identical gelation conditions. For such long amylose chains, it is possible that initial formation of relatively few crosslinks significantly retards chain mobility and therebyslows down the gelation kinetics. Inother studies on amylose gels [374], it was shown that a gel rapidly quenched to 25°C (3.4%, DP3080, and acure time of 24 h) had alower limiting modulus of -3.5 X lo3 Pa than a slowly cooled (over 4 h) system that reached at a plateau value of -5.3 lo3 Pa. All these observations clearly point to the dynamic (nonequilibrium) nature of amylose gel structures; i.e., diffusioncontrolled aggregation processes govern the mechanical properties of the hydrated networks. Amylose gelation was noted to proceed even at concentrations below the critical overlap concentration of this polymer (C* 1.5%); a minimum, amylose concentration for gelation (C,) was found to be -l%, regardless

-

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Evolution of shear storage modulus (G') for amylose solutions varying (a) concentration @P 2400, in M KCl, 25°C) @) chain length (2%in water, 25°C). (Data adapted with permission from Refs. 370 and 372.)

-

of the molecular weight [370,372]. Thus, molecular entanglements do not necessarily determine the critical gelling concentration of amylose, as originally proposed byMiles et al. [l521 and often found in the case of synthetic polymers, for which overlap of macromolecules is a prerequisite for gel formation. Besides the formation of an elastic network, amylose gelation is also characterized by the development of opacity, which is generally attributed to chain aggregation [152,370,372]. For a polydisperse amylose preparation (DP3080,2.4%solution, quenchedto 32"C), the increasein turbidity slightly preceded the onset of G' development [152]; on the other hand, crystallization, as detected by X-ray diffraction (intensity of the 100 diffraction peak),was shown to be the slowest process.In similar experiments with

amylose @P 2400) solutions in 0.5 M KC1 at 25°C a fairly good correspondence between cloud time (onsetof turbidity) and gelation time over point of the G' and G'' curves) was observed [372].These findings supported the notion that networkformation in amylose solutions is initiated by phase separation (demixing) in the homogeneous sol, which yields polymer-rich regions interspersed with polymer-deficientregions. This brings about enhancement of local concentration of amylose in the polymer-rich regions and leads to interchain associations; crystallization within the separated polymer-rich phase occurs at a much slower rate than that of demixing. Depending on polymer concentration, molecular size, solvent quality, and quenching temperature, such liquid-liquid demixing may be a relatively slow process. Using both SAXS and WAXD measurements, with synchrotron and conventional X-ray radiation, 1'Anson et al. [369]have probed the gelation of a 6% amylose solution after quenching from 75" to 22°C over short and longer time scales. Their findings suggested if gelation is initiated by a phase separation, this is closely followed by crystallization in the polymer-rich phase. The SAXS data, from use of the high-density synchrotron beam, indicated that nucleation and limited growth of rod-shaped microcrystals occur during network formation, at short times after quenching. [152],the studies of Gidley and coworkers Contrary to Miles et al. [47,370,371]with monodisperse amyloses have shown that turbidity development either precedes or lags behind the increases in G', depending on the amylose chain length; e.g., for amylose of DP 250, turbidity commenced earlier than the rise in G', whereas for amyloses with DP > 1100 the reverse was true. Because gelation was noted even at concentrations significantly lower than thoseleading to molecular entanglements, Gidley [371]suggested that gelation is due to interchain associations in the form of double helices and aggregation of helices that act as junction zones. It is these cooperative noncovalent interchain interactions that bring about a phase separation in the developing network. Moreover, 13CC P M S and 'H T2measurements of amylose gels provided evidence for the existence of two conformational states having different mobilities [371];rigid double-helical chain segments (of B type) andmore mobile, amorphous single chains. Estimates of doublehelix content in 10% amylose gels were 67%, 67%,and 83% for potato (polydisperse), DP 2800 (synthetic, monodisperse), and DP 300 (synthetic, monodisperse) amyloses, respectively [157,371]. recent computer simulation study [373]of the structural features of amylose solutions and gels, using X-ray scattering, has favored the Gidley's gelation model, which assumes chain aggregation and crystallizationduring the phase separation, and confirmed the coexistence of single- and doublehelical segments during amylose gelation and aggregation. The scattering features of defatted amylose solutions (2%) cooled to 40°C indicated at first

Starch

the development of B- and V-type nanocrystallites. These crystallites were not includedin a phase-separated network,supporting the gelling mechanism of Gidley [371]. Following storage of the gel (5 weeks, 21"C), the V-nanocrystallites seemed to vanish and the number and size of B-crystallites grew compared to the freshly prepared gel at 40°C [373]; the B-crystallites most likely grow by dimerizationof extended helices or V-helices, which undergo an accompanying conformational change. The aged amylose networks would thus consist double helices, small aggregates of double helices (junction zones), nanocrystallites (B type), and their aggregates. In Fourier a transform infrared (FTIR) studyof amylose gelation [375], changes in spectra indicated that a fast short-range ordering takes place over a time scale similar to the development of the gel network. Thus, double helices form before or at the same time as phase separation, to create a gel network, with subsequent aggregation to yield crystalline domains. Based on the turbidimetric and rheological data of Clark et al. [370], Morris [376] has proposed that growth and coarsening of amylose gel networks may develop in different ways depending on molecular size, concentration, and gelation conditions. For high-molecular-weight amyloses, poor chain matching and associations over relatively short chain segment lengths occur, favoring the formation of a fine network that coarsens thereafter. In contrast, shorter chains may initially foster lateral interchain associations due to a better matching upon double-helix formation, followed by interlinking of the coarse aggregates to form a network. Once formed, amylose gels are thermally stable networks up to 140-160°C [134]; indeed,the shear modulus of amylose gels could not be reversed by heating at 100°C [151]. The microstructural aspects of amylose gels, such as porosity [156,377], molecular order and crystallinity [176,371], and lengths of chain segments in the ordered regions of the network structure [133,156], have been probed. structural model for the organization of the polysaccharide chains in amylose gels, with crystallite lengths of 8-18 nm, was also presented [156]. The limiting G' value of amylose gels is strongly dependent on concentration. Depending on origin, molecular size, degree of polydispersity, concentration range, and gel-curing conditions, G' was found to vary with amylose concentration as C' (DP 3080, 1.5-7 [374], CY4-'.' @P 300-1100, 1-3 [370], and c" @P 1150, 1.9-8.8 [378]. These different exponent values suggest that a simple power law expression is not sufficient to describe the concentration dependence of gel modulus. masterplot of G' concentration was presented by Biliaderis [193], summarizing published data for amyloses of different origin and molecular size, which by extrapolation tothe concentration axis yielded acritical concentration for The modulus-concentration data for monodisperse gelation of-0.9 amyloses were analyzed with the method of Clark and Ross-Murphy [379],

Billaderis

114

which can give an accurate prediction of the critical gelling concentration, C, [370]. Amylopectin gelation is a much slower process than that observedfor amylose (Fig. 14), and it occurs at substantially higher polysaccharide concentrations (usually >lo%), well above the coil overlap concentration (C* 0.9%) of this biopolymer [75,378,380]. Moreover, the kinetics of amylopectin gelation is more sensitive to storage temperature (1-25°C) than that of amylose. A range of physical, spectroscopic, and chemical techniqueshas

-

(w/w) W-

maize gels

2

54 200 -6

z

zEi 8J

2

20

0

Time (h) Figure Temperaturedependenceofshearstoragemodulus (G’) development forwaxymaizeamylopectinandpotatoamylose gels. (Adaptedwithpermission from Ref. 378.)

been employed to monitor structure development in amylopectin networks [75,375,378,380-3821. For a 20% amylopectin solution (at l"C), turbidity data showed changes up to 4-5 days, whereas events monitored by dilatometry, shear modulus measurements, DSc, andX-ray diffraction followed similar parallel-time scales over longer periods (30-40 days) [75]. These data were interpreted as reflecting aggregation (increases in turbidity) of amylopectin molecules prior to gelation and as showing that the changes detected bythe other techniques involved crystallizationof amylopectin; i.e., gel network development was related to ordering and crystallization of the short-DP (10-20) chains of amylopectin. Similar time dependence in the evolution of G' and AH of the melting endotherm was also found for a 40% amylopectin dispersion during the first 3 h of storage at [381]; however, over a longer time scale (3-48 h), the G' development was progressing faster than that of retrogradation endotherm. The strong dependence of rigiditydevelopmentof40%amylopectin gels on storage temperature, as shown in Fig. 14, further supports the notion that gelation of amylopectin follows typical nucleation kinetics. Moreover, FTIR spectroscopic studies [375] revealed chain ordering at the very early stages (complete within 0.2 h for a 20% waxy maize solution), not detectable by any other physical methods, which was interpreted as involving coil + double-helix transitions of the short-DP chains of amylopectin. This phase is followed by a slower aggregation process (giving rise to turbidity) of these helices to produce crystallites. Small-strain rheological measurements of rather dilute amylopectin solutions (3-10% w/w) indeed provided evidence for a rapid establishment of network structure involving only short-range intermolecular associations, presumably via double-helix formation [76]. No visible inhomogeneities of these weak gel networks ( 4 0 N-m") were detected in the time scale of the experiments (i.e., the gels remained clear), implying a gelation mechanism other than long-range ordering (crystallization)of amylopectin chains. Studies on aging waxy maize starch gels using 'H crossrelaxation NMR and high-resolution N M R spectroscopy revealed the presence of three differentmotional states [382]: (1) ahighly mobile chain fraction; (2) highly rigid regions, corresponding to the crystalline domains; and (3) an intermediate in mobility fraction, which may include unassociated or partially associated chains in the vicinity of crystallites. Amylopectin gel stiffness does not show a strong dependence on concentration; for waxy maize amylopectin gels (10-25%), the modulus varied linearly with concentration [75]. The G'-concentration data of waxy maize amylopectin gels were recently fittedwith the Clark and Ross-Murphy model over the concentration range of 20-50% [381]. The fine structure of amylopectin also seems to influence its gelation kinetics. Cereal amylopectins (wheat, barley, maize) ofshorter chain length exhibited slower rates in mod-

Biliaderis

ulus development than those of tuber starches (potato, canna) [380]. The acceleration in retrogradation kinetics for amylopectins with a higher proportion of longer chains has also been reported by other authors [325,383,384]. The gelationhetrogradation of amylopectin is thermoreversible, since the gel structure (G’), retrogradationendotherm (4, and X-ray crystallinity are abolished by heating at temperatures below 100°C (T,,,of amylopectin crystallites -50-60°C) [151,381]. Amylopectin gelation and crystallization are influenced bythe presence of small-molecular-weight solutes (salts, sugars, lipids). The retrogradation rate of amylopectins isolated from different starches was reduced with increasing concentration of NaCl [384]. The effects of polyhydroxycompounds (PHCs) on structure formation in aging waxy maize starches or amylopectin gels were investigated by DSC [384-3861 and dynamic rheometry [386]. At a weight ratio of starch:PHCwater of 1.0:0.5:1.5, some PHCs retarded the kinetics of chain ordering in the amylopectin network, while others had a structure-promoting effect [385] (Fig. 15); e.g., ribose, maltotriose, and talose suppressed the growth rate of G’ and (gel-melting endotherm), whereas fructose appeared to accelerate these processes. The magnitude of these effects is dependent on solute concentration. As shown in Fig. 15, gels containing ribose showed a descending level of structure withascendingriboseconcentration. For fructose, athigh concentrations w/w), chain ordering in amylopectin gels is accelerated, whereas this solute seems to exert an inhibitory effect at low concentrations. In the homologous series of glucose oligomers, maltotriose exhibited the largest inhibitory effect,whereas maltooctaosepromoted amylopectin retrogradation [385]. Reduced rates of firmness(large-deformationmeasurements)and crystallinity (attributedmainly to the amylopectin component) of aging wheat starch gels containing ribose, glucose, or sucrose at the nominal ratio of starch:sugar:water of 1:l:l were also reported by l ’ h s o n et al. [387]; at this level of sugar, the effectiveness of reducing firmness and crystallinity followed the order ribose > sucrose > glucose. In a subsequent study [388], the effect of sugar concentration on the crystallization rate of amylopectin in wheat starch gels was investigated by WAXD. The pentoses ribose and xylose were found to suppress the retrogradation of amylopectin with increasing level of sugar,whereas fructose promoted crystallinity development. The unusual behavior of fructose was first observed by Slade and Levine [3] using calorimetry to follow aging of wheat starch gels (starch: sugar:water at 1:l:l) at These authors reported that the extent of amylopectin recrystallization decreased in the order of fructose > mannose > water alone > galactose > glucose > maltose > sucrose > maltotriose > xylose. They have also suggested that sugars, in general, behave as antiplasticizing solutes (relative to the plasticizing action of water alone), thus

reducing chain mobility and crystallization of amylopectin. Obviously, the results obtained for fructose are not in accord with the antiplasticization theory. In a comprehensive study using a variety of PHCs [385], changes in the retrogradation kinetics of waxy maize starch gels were found to be solute specific. The DSC data were correlated with several physicochemical properties of the PHCs in solution (e.g., hydration characteristics). On the basis of the relationships found, it was proposed that the compatibility of the sugars with the water structure, as governed by the stereochemistry of their main conformers in solution, is an important determinant of amylopectin stability in a composite starch-solute gel matrix [385]. Sugars that fit well in the hydrogen-bonded network of water (e.g., ribose) are very effective in inhibiting amylopectin retrogradation. In contrast, PHCs that greatly disturb the “normal” water structure (e.g., fructose), forming strong hydration layers around them, promote chain ordering and crystallization of amylopectin. Of importance to the microstructureand viscoelastic properties of starch gels is the thermodynamic incompatibility between amylose and amylopectin in an aqueous medium [116]; this leads to a phase separation, one phase being enriched in amylose while the other contains mostly amylopectin. When phase separation occurs, the effective concentration of both polymers in their respective microdomains is raised, and this may lead to the establishment of a composite gel network at concentrations where neither one of the two polymers is capable of bringing about alone. The polymer composition of the continuous and dispersed phases are dependent on the ratio of amy1ose:amylopectin (r). At a particular value of this ratio (called the phase inversionpoint), the continuous phase becomes the discontinuous dispersed filler, and vice versa. For 8% mixed amylose-amylopectin gels, Leloup et al. [l531 have shown that the supermolecular organization, mechanical properties (using large-deformation testing), and a-amylase digestibility of the gel vary with the ratio, r; mixed gels exhibited behaviorsimilar to pure amylopectin for r 0.43 and to pure amylose for higher r values. A sharp transition in gel properties at r = 0.43 (corresponds to -30% amylose content) was assumed to reflect an inversion in the polymeric composition of the continuous and dispersed microphases, amylopectin being the continuous phase at r 0.43 and amylose above this value. Small-amplitude oscillatory shear measurements of mixed amylose-amylopectin systems (at a total polymer concentration of -4%) also confirmed that their rheological properties strongly depend on the weight ratio of the two polymers [389]. The viscoelastic behavior of the gels seemed to be governed by a phase separation process, yielding a composite system. Drastic changes in network properties, identified with the point of phase inversion, occurred atr = 0.17. Amylose also tends to separate from amylopectin during heating of aqueous dispersions of starch granules, as evidenced by light microscopy of starch

0

12

36

24

Time (h)

0

2

4

6

Time (days) 15 Effect of added sugars on retrogradation kinetics (G', AH) of amylopectin gels (rheological and calorimetric tests at 8°C and 6"C, respectively) for (a), (b) starch:sugar:water at 1.0:0.5:1.5w h , for (c), (d) starch:sugar:water at where X vaned between 0.1 and 0.9. (Adapted with permission from Refs. 385 and 386.)

Ribose

A

AH

Continued

pastes and gels stained with iodine solution the extent to which this process occurs woulddependon starch concentration,temperaturetime, shear, molecular size, and degree of branching of amylose, as well as on the structure of amylopectin crystalline networkand its interactions (chain entanglements) with the amylose chains. Thus, in low-shear cereal starch pastes, amylose seemed to diffuse outside the granules, forniing a continuous network whereas in potato starch pastes amylose was retained more in the peripheral layers of the swollen granules Even when gels were prepared under high shear, wheat starch amylopectin did not appear to solubilize (still localized in the granular fragments), compared with potato starch pastes, where the continuous phase was a mixture of This led to the suggestion cosolubilized amylose and amylopectin that cereal starches form stronger gels than potato starch because of the more efficient separation of starch molecules during heating, which promotes the It is also likely that gelatinized gelation of the linear starch fraction cereal starch granules (because of their lower swelling capacity and deformability) are stiffer than those of potato starch, thus increasing the firmness of the gel; amylose is believed to act as a restraint on granule swelling of cereal starches because of the formation of amylose-lipid complexes, which dissolve at temperatures well above the gelatinization temperature. According to Steeneken for concentrated suspensions of gelatinized starch granules, the thickening power is mainly governed by the rigidity of the closely packed granules. Overall, it is questionable whether one can draw a direct relation between the extent of macromolecular demixing occurring during pasting and storage and the changes observed in the rheological propertiesof starch gels. These systems are highly complex rheologically, as described next, andthere should be more fundamental work to relate structural aspects and physical properties of composite starch gel networks. The practicalaspect of the nature of amylose-amylopectin composite gels is that amylose offers better rigidity and thermostabilityto the network structure.Gels with amylose conwhere amylose forms the continuous phase (entraps tents greater than amylopectin in the form of droplets), exhibit increased cohesiveness, cooking stability, and resistance to a-amylolysis in vivo and in vitro. This has been demonstrated for pasta products made from mungbean starch and rice flour

2. Starch Gels Starch gels are readily formed when gelatinized starch dispersions of sufficient concentration (usually are cooled to room temperature. On cooling a starch paste, the exuded amylose from the swollen granules forms

Starch

an interpenetrating network (via interchain associations) that surrounds the gelatinized granules. In this respect, starch gels can be regarded as hydrated polymer composites where swollen, amylopectin-enriched granules (filler particles) are embedded in and reinforce a continuous matrix of entangled amylose molecules[395]. The mechanical properties of such amaterial would depend on the rheological characteristics of the amylose matrix, the volume fraction and the rigidity (deformability) of the gelatinized granules, and the interactions between the dispersed and the continuous phases [396]. Granule size and uniformity, amylose content, macromolecular organization of the granules, minor starch constituents (lipids, phosphate ester groups), the presence of other solutes (salts, sugars, lipids), pH, starch concentration, and shear-temperature-time regimes employed during gel preparation are all important to the viscoelasticity of starch dispersions and can account for the differences inrheologicalpropertiesamong starches or the variation often reported for starch gels of the same botanical origin [192,193,397]. Moreover, any transitions or interactions that affect the phase separation of amylose and amylopectin during pasting, the stiffness the amylose gel matrix, and the deformability of the gelatinized granules ultimately have an effect on gel mechanical properties. For cereal starches, where amylose leaches out of the granule during gelatinization and largely contributes to the viscoelasticity of the continuous phase, it is interesting to explore the relationship between amylose content and mechanical properties of starch gels. Rice starches, because of their uniform granules (shape, size, and size distributions) offer a unique model system in this respect. Figure 16 shows the dependence of G' andtan 8 (= G"/G')on amylose content for 25% gels of rice starches from 43 different varieties [315]. Isolation of these materials, gel casting under static conditions (no shear) in hermetically sealed disk dies [145], and testing protocols were identical, to minimize other contributions to the rheological properties of the gels; e.g., gel rigidity is highly dependenton aging effects. An inverse linear relationship between tan and amylose content was observed (r = 0.75, p .Ol). Also there was a general trend for increased G' with amylose content; G' (A%)'.'*, r = 0.76, p .01. However, the scatter of the data in these plots clearly points to the fact that in addition to amylose several other factors contribute to the mechanical properties such composite networks. The dependence of gel rigidity on starch concentration has also been examined. Linear relationships between the rigidity modulus (as determined with a Ward and Saunder's U-tube) and concentration (6-30%) were reported for potato, wheat, and maize starch gels [395]. A linear relationship of G' concentration for maize starch gels was also shown, using :mall-amplitude oscillatory shear testing [398], while a slightly stronger con-

I

0.4

.

-

20

0I--

I

50

20

(%) Storage modulus (G') and tan 8 of rice starch gels varying amylose content. (Reprinted with permission from Ref. 315.)

w/w)with

centration dependence ( C I . 3 was reported for wheat starch pastes (2-4%) using an oscillatory viscometer [399]. Over a broader concentration range (8-40%),much stronger power law relationships were found for wheat, legume,and rice starches with dynamic rheometry[145,315,378]: G' m C2,"2.9. The viscoelastic behavior of gelatinized starch dispersions is also dependent on the shear conditions used during their preparation. Svegmark and Hermansson [400,401] have shown that warm cereal starch pastes are less sensitive to shearing thansimilarlytreated potato starch pastes; the complex shear modulus of the sheared 10% pastes decreased to -50%, for wheat and maize starches, and to -2.5%, for potato starch, the respective modulus values of the low-shear control pastes. Moreover, the mechanical properties potato starch gels are sensitive to pH and ionic strength of the medium because of the polyelectrolyte nature of potato amylopectin [402]; i.e., phosphate ester groups present in some glycosyl residues greatly increase the swelling capacity of potato starch granules and thus affect the stiffness of the gel.

During storage of concentrated starch gels, the starch molecules rearrange (retrograde), forming larger chain assemblies, and thereby enhance the stiffness of the network. Similar changes also occur in the structure of most products with high starch content, and these are responsible, at least in part, for the decrease in eating quality of baked items, e.g., the staling of bread [149,403,404]. The time course of retrogradation for starch gels has been shown to consist ofat least two separate phases. The short-time (hours) increases in rigidity (modulus), short-range molecular order (AH), and crystallinity have been ascribed to gelation of the solubilized amylose in the intergranular spaces [151]. In contrast, changes that occur over longer periods (days) were attributed to reordering and crystallization of amylopectin [75,151,378]. On the basis of time-dependent FI'IR spectral changes of 10% potato and waxy maize starch gels, as well as other spectroscopic data presented in previous reports [375,405], Soest et al. [406] have proposed a multistage process for starch retrogradation. The first stage has been ascribed to conformational ordering (double helices) of amyloseandmayalso involve short-range ordering of amylopectin side chains. Amylose aggregation and crystallization occurs mostly in this period. The second stage was described as an induction timefor the onset of amylopectin helix aggregation. In stage 3, amylopectin aggregation and crystallization eventually proceeds; stage involves the phase separation of water (syneresis) due to excessive retrogradation of the polymer chains. The observation, with various probes of structure, that amylose retrogradation is almost completed within the very first few hours of storage [151,152,375,378,406], has recently been confirmed by isothermal microcalorimetricstudies on retrogradationof aqueous dispersions of amylose and starch [407]. A comparison of the initial rate of modulus development for 30% gelatinized starch dispersions (at 20°C) from various botanical sources revealed the order of pea > maize > wheat > potato and was related to the amount of amylose solubilized during gelatinization [408]. However, the rate of long-term modulus increases followed the order pea > potato > maize > wheat; the higher firming rates for pea and potato may reflect the lack of endogenous lipids from these starches, which are known to retard amylopectin recrystallization. The long-term changes in rigidity and crystallinity of starch gels, related to amylopectin crystallization, correspondkineticallywith staling events of aging baked items [403,404]. Thus, information concerning the rates of amylopectin crystallization has implications on the storage stability of bread and other starch-based products. The presence of microcrystalline junctions in aged products has a direct bearing on the retrogradation kinetics from the perspective of temperature-time-water content dependence of the crystallization process. The melting enthalpy increase for retrograded starch

shows that crystallization occurs at a faster rate and to a greater extent when a lower single storage temperature is employedbetween and Furthermore, in retrograded potato starch andcooked wheat grains storage at refrigeration temperatures resulted in the formation less perfect crystallites (lower T, and broader melting transitions) than those formed on storage at room temperature. This is in accord with classical polymer crystallization theory which predicts growth of more imperfect crystals with increasing degree of supercooling (T,-T). Crystallization follows the classical three-step mechanism of nucleation growth(propagation) + crystal ripening (perfection) Within the range T,-T,,,, both nucleation and propagation exhibit an exponential dependence on temperaturesuch that the nucleation rate increases with decreasing temperature, down to the T,, while the propagation rate increases with increasing temperature up to the T , Single-step or multistep temperature crystallization studies showed that the ratelimiting step is nucleation rather than propagation. The DSC kinetic data of Slade further indicated that the effective temperature range in which starch crystallization (in wlw gels) can takeplace is confined between -5°C (T, of fully water-plasticized amorphous starch) and (T, of B-type crystallites of amylopectin), as the theory predicts.Starch crystallization, as determined by DSc, is also affected by the water content. Crystallization in gels and bread does not occur below and above solids level; the overall response of enthalpy to moisture content shows a bell-shaped curve, with maximum values in the range of starch content. The acceleration of retrogradation with increasing water content from to merely reflects the plasticizing effect of water in depressing the Tg the gelatinized starch; i.e., it brings the effective T,-T, into a temperature region, relative to storage temperature, where nucleation and crystallite propagation proceed at high rates. On the other hand, the rate starch crystallization is reduced with increasing moisture content above presumably due to dilution the crystallizable component in the plasticized starch matrix. variety of physical [differential thermal analysis @TA) and DSC X-ray diffraction small-deformationmechanical tests and compression modulus measurements microscopic enzymic and spectroscopic (NMR FTIR and Raman spectroscopy) techniques have been employed to follow aging events in starch gels. These methods not monitor the same starch gel properties and are not sensitive to the same fractions or structural elements developing in the aging gel network. For example, thermal analysis is evidently sensitive to chain ordering of the amylopectin fraction; X-ray

diffraction measures crystalline order of both amylose and amylopectin combined; microscopy reveals spacial distribution and assemblies of starch macromolecules; FTIR and Raman spectroscopy probe conformation- and tallinity-dependent vibrational frequencies of chemical linkages; N M R monitors chain segmental motions, conformational-dependentchemical shifts, and short-range order; and mechanical tests are sensitive to chain entanglements with or without the formation of crystallites. As a result, the time course of retrogradation events in starch gels varies with the measuring technique. Kinetic data of starch gel retrogradation have often been analyzed uswhich ing the Avrami equation relates the fraction of the total change in the measured property (e.g., crystallinity, modulus, enthalpy of staling endotherm) still to occur (‘p) to time

Am - A, ‘p=-- exp(-kt”) Am - Ao or log [-h ‘p] = n log t

+ log k

where k is the rate constant and n is the Avrami exponent. The kinetic data of these studies were shown to fit reasonably well the Avrami model, with calculatedexponent values close to 1. However, the application of the Avrami analysis to concentrated polymer-solvent systems, such as starch gels, has been criticized, due to the inherent limitations in interpretation of the exponent value in terms of nucleation type and crystal morphology [3,432]. Crystallization in aqueous starch systems is known to proceed via a three-step mechanism(nucleation growth maturation), controlled by the amount of water present, and it involves two separate polymers. Despite these theoretical limitations, the Avramiequation provides aconvenient means for quantifying retrogradation kinetics andmaking comparisons among similar starch systems submitted to various processing treatments. In this context, Russell reported thatthe addition of glyceryl monostearate during dough mixing [430] or spraying bread loaves with ethanol after baking [431]led to a reduction in the rate constant for the development of the “staling endotherm,” while they had little effect on the limiting enthalpy different flour value. In a similar study [429], it was shown that the use grists had no effect on the staling kinetics of bread and that the rate constant was independent of the specific loaf volume. The acceleration in storage modulus (G’) development in aging potato starch pastes (12.5 W%) with increasing NaCl concentration (above M) [422] as well as the changes in the retrogradation kinetics (compression modulus) of concentrated (50%

- -

wlv) wheat starch gels caused by various ions have been also evaluated by the Avrami analysis [424]. The effects ofsmall-molecular-weight solutes and other structureforming hydrocolloids (biopolymers) on starch gelation and retrogradation are important in considering the complex nature of starch-based foods and the need to control their rheological and textural properties. For instance, lipids have long been known to affect the gelatinization, retrogradation, and texture of starch-thickened products. Monoacyl lipids, in particular, form inclusion complexes with starch molecules [70,144], and it is likely that texture modification is brought about by such interactions. There appears to be a reduction in the pasting temperature (viscoamylography) [433-4361 and an increase of swelling [306] of cereal starches following solvent extraction of their endogenous lipids; interestingly, this is not accompanied by changes in the characteristic melting temperatures of the granules, as assessed by DSC [306,435,436]. When present as adjuncts, monoacyl lipids reduce the gel volume (granule swelling) and amylose leaching [146,4374391. The extent and modeof interactions between granular starch and added lipids also depend on the chemical nature of the lipid. Thus, while swelling and gelatinization are generally restricted with fatty acids and most emulsifiers [437-4401, addition of some ionic surfactants (e.g., sodium dodecylsulfate, SDS) promotes swelling of the granules [439,441,442].These effects and the resultant rheological behavior are modulated by the type starch, the concentration of the various components, the pH, the presence of salts, and the heat-shear regime used during pasting and cooling. This explains the large variability in the published data concerning the influence of lipids on starch rheology. Moreover, most of the earlier studies were carried out using empirical rheological tests, where due to the temperature-shear cycles employed, it is difficult to separate the impact of mechanical damage of the gel network structure from contributions of the lipid additive itself. Consequently, a whole gamut of effects has been observed, ranging from large increases in gel consistency,noeffect,to large viscosity reductions Therefore, it is important to study the rheological properties of such systems under controlled conditions of thermal and mechanical treatments. The viscoelastic behaviorofheated starch dispersions (10% wlw, wheat, corn, potato, and waxy barley) in the presence of emulsifiers was first investigated by Eliasson [445]. Glycerol monostearate (GMS) and dium stearoyl lactylate (SSL) at l%(drystarch basis) delayed the onset of G’ and G” rise on gelatinization; this effect was less pronounced with the waxy barley starch.The G” of the hot starch pastes (except for wheat starch) was increased in the presence of GMS, SSL, and SDS; this resulted in a higher tan value, compared with free-lipid gels. In a subsequent study

a cationic surfactant (cetyltrimethylammonium bromide, CTAB)was found to increase the G' of native and modified corn starch pastes (7.5% w/w, lipid at2%w/w of starch, testing temperature 25°C); instead, saturated monoglycerides increased in G' for pastes of native corn starch, decreased the G' of crosslinked waxy maize, and had no effect on an acetylated highamylose maize starch derivative. Tm, and lysoInteractions between monoacyl lipids (SDS, GMS, C phosphatidylcholine, or LPC) at 2-4% (starch basis) were also investigated in thermoset gel networks at high starch concentrations (20-35% W/W)by dynamic rheometry and DSC [145]. Although rice and wheat starch gels (21-24% amylose) exhibited higher G' values when lipids were included, smaller changes in the viscoelastic properties were observed for legume starch gels (31-33% amylose); i.e., the higher amylose content of pea and garbanzobean starches seemed to dominate the gel networkproperties. Among the lipids tested, LPC (one of the main endogenous lipids of cereal starches) exerted the greatest effect in increasing the G' and lowering the tan of the cereal starch gels. Kinetic experiments on the development of G' and starch crystallization (AH of retrogradation endotherm)in aging rice and pea starch gels (35% w/w) indicated that lipids retard both processes [145]. The antistaling role of native granular lipids or added surfactants in starch gels or bread has been repeatedly confirmed by DSC [71, 297,404,416,428,447-4491 and mechanical measurements [404,430, 449,4501. The antifirming action of lipids may be caused by amylose complexation, which in turn weakens the cohesion of the amylopectin-enriched granules in the composite gel network [450]. However, the reduction in the rate of amylopectin staling (AH) during the aging of waxy maize starch gels [71] or gels made with blends of varying ratios of amylopectirdamylose [447] as well as other indirect evidence [70,451-4531 imply that amylopectin can also interact with lipids. Isothermal microcalorimetric studies have recently demonstrated that added monoglycerides or SDS inhibit the crystallization processes of both starch polymers during the first 24 h of aging starch gels [407]. The effect of sugars on starch retrogradation is also complex and not well understood. Overall, there appears considerable confusion in the published literature for the role of sugars on aggregation and crystallization of starch molecules; such discrepancies may arise from differences in the proportion of starch to sugar, the conditions for gel preparation and storage, and the various techniques employed to monitor the retrogradation process. Maxwell and &bel [454], using large-deformation mechanical tests, showed a marked increase in the rate of firming of starch gels by fructose, a slight increase byglucose,and essentially no effect for sucrose (wheatstarch: sugar:water at 1:l:l). Similar rheological studies by Germani et al. [425] indicated that maltose and sucrose were more effective than glucose in in-

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creasing the firming rate of corn starch gels. In both these studies, however, the elastic moduli of sugar containing gels were lower than for pure starch + sugar) of gels (starch:water at l:l), due to the greater plasticization the polymer matrix. In other studies using X-ray diffraction and firmness measurements [387,388] it was also concluded that sugars reduce firmness and crystallinity developmentinwheat starch gels (starch:sugar:waterat 1:l:l).The magnitude of these effects was dependent on the concentration and type of sugar used; e.g., while most sugars inhibited starch crystallization, fructose seemed to accelerate the process. The structure-promoting behavior of fructose in aging starch gels, compared to other polyhydroxy compounds, has been also confirmed by DSC at starch:sugar:water of 1:l:l [3], 1.0:0.5:1.5[385,386],and0.2:0.09:0.71[455].However,in other calorimetric studies on retrogradation of sweet potato starch gels (33% w/w) [456], fructose was found to prevent retrogradation, although itwas not as effective as glucose and sucrose. On the basis of viscoelastic responses (creep compliance) of starch gels (-33% w/w) containing sugars at -6.0% (w/w on starch basis), Katsuta et al. [457,458] and Miura et al. [449] have shown that the ability of sugars to impede retrogradation is related to the mean number of equatorial hydroxyl groups; solutes with large numbers of equatorial-OHS were more effective in stabilizing the amorphous and entangled matrix of gelatinized starch. The relative ranking of sugars in decreasing the rate of retrogradation was maltotriose > maltose > sucrose > glucose > fructose > xylose, ribose > control gel. Among monosaccharides, hexoses were more effective at inhibiting retrogradation than pentoses, which contradicts other findings [385 -3881. A comparative study of the effect of sugars on the thermal and mechanical properties of aging waxy maize, wheat, potato, and pea starch gels (starch:sugar:waterat1:0.5:1.5w/w)revealedthat sugars inhibited chain organization in the order ribose > sucrose > maltotriose > water alone, glucose > fructose [386]. The effects of sugars on the development of staling endotherm (AH) or gel rigidity (G') were more pronounced with starches of low amylose content; i.e., amylose in high amounts dominates the gel network properties. These findings and the calorimetric data of the effect of a large number of polyhydroxy compounds on the stability of waxy maize starch gels [385] have led to the hypothesis that starch retrogradation kinetics is controlled by the stereochemistry of the sugar. Solutes that seemed to cause very little perturbation in the hydrogen-bonded network of water also had a stabilizing effect on the polymer chains, thus retardingretrogradation[385,386].In designingasmall-carbohydrate solute as an effective antistaling agent, emphasis must therefore be placed on the stereochemistry of its main conformer(s) in solution, particularly in relation to water structure. 4

The effectiveness of n-alkyl glucosides in inhibiting retrogradation of waxy maize and wheat starch gels was recently studied [459]. The rheological and thermal propertiesof aged gels indicated that n-alkylglucosides are as efficient as other commonly used emulsifiersin retarding starch retrogradation; glucosides with intermediate chain lengths in their alkyl group 8 carbons) were the most effective in this respect. Corn [455] and wheat [378] starch gels exhibited greater rates in amylopectin crystallization (AH) in the presence of low dextrose equivalent (D.E.) starch hydrolyzates, although the G’ data suggested the weakening of all composite wheat starchstarch hydrolyzate gels [378]. These observations clearly point to the fact, often not appreciated by researchers in this field, that the development of ordered structures (as assessed by DSC and X-ray diffraction) andthe firmness of starch-based products do not necessarily follow similar trends. Rogers et al. [460] demonstrated bread crumb firming rate to be inversely proportional to its moisture content (within 22-37% m.c.). In breadsof low moisture content (e.g. 22%), starch did not retrograde noticeably, yet those breads firmed very rapidly. Obviously, starch retrogradation and crumb firming do not correlate with each other across varying moisture contents. Anyvariation in moisture content of the end product, as a result of changes in formulation, may therefore lead to different trends in crystallizationkinetics and sensory attributes.This has been shown for breads fortified with water-soluble arabinoxylans [461]. Breads supplementedwiththese cell wall polysaccharides (ata level of 0.3-0.9% w/w of flour) retained highermoisture content during storage and exhibited greater rates in starch retrogradation (AH) than control breads; yet the fortified bread crumbs, because of their higher water content, hadsofter a texture. Overall,the final firmnessof a product wouldbe dependent not only on the level of crystallinity of the starch fraction in the composite food matrix, but also on the extent of network plasticization with water. In the presence of other food hydrocolloids (gums, proteins),the pasting and gelling processes of starch and the rheological properties of the resulting gel networks undergo profound changes.These systems are highly complex to characterize rheologically, because the hydrocolloid may affect the phenomena of granule swelling and exudation of amylose during gelatinization, thereby influencing the deformability of the dispersed granules, as well as interact with the solubilized starch molecules in the continuous phase. The gelation process of corn starch dispersions (4%) with added galactomannans (0.1-0.5%) was examined by a constant-stress rheometer [462].Galactomannansaccelerated the gelation kinetics andresultedin higher G’ values for the final networks, compared to the starch alone. The mixed gels were also characterized by greater tan S values, implying a less elastic network, and their G‘ value showed a slightly greater dependence on

frequency. These findings were rationalized in terms of thermodynamic incompatibility between amylose and galactomannan in the continuous phase of the gel. In similar studies, the kinetics of gel formation in 7% mixed starch-hydrocolloid dispersions (6.65% corn starch gum) was shortenedconsiderablyin the case of guar gum, locust beangum,and K-carrageenan instead, warrageenan seemed to interfere with the network development. Addition of these hydrocolloids also led to an increase in the viscous component (G”) of the gel, raising the tan 6 values for the mixed gel, with the exception of L-carrageenan, which showed the opposite effect. Acceleration of gelation was interpreted as resulting from increasing concentration of starch and gum in their respective microdomains due to their phase separation, whereas the opposing behavior of L-carrageenan was attributed to intermolecular interactions between starch and this hydrocolloid. The influence of purified arabinoxylans (2% w/w of a-D-glucan) on the retrogradation kinetics of concentrated amylose (20% w/w), wheat, and waxy maizestarches w/w) was examined by dynamic rheometry, DSc, andX-raydiffraction Amylose-andwheatstarch-arabinoxylan mixtures exhibited higher G‘ and a greater rate of structural order development than pure amylose and starch gels. Similar kinetic responses were also shown for the arabinoxylan-amylopectin systems in terms of order (AH and crystallinity), but a markedly retarded gelation rate (G’) was evident. These observations suggested that arabinoxylans interfere with intermolecular processes responsible for the establishment of an extensivethreedimensional amylopectin gel network structure. The gelation kinetics and viscoelasticity of wheat starch (6%)-caseinate (7.5%) blends were recently studied by dynamic rheometry The addition of caseinate accelerated the gelation process but decreased the final plateau G’, compared to starch alone. The rheologicalbehavior of the protein-starch blends was interpreted in terms of two antagonistic effects. First, caseinate significantly reduces swelling and solubility of starch during pasting, which would lower the volume fraction of the dispersed phase and the concentration of amylose in the continuous phase; this should result in a substantial decrease of paste viscosity. On the other hand, because of the incompatibility of caseinate with starch components,aphase separation might occur in the continuous phase, where caseinate and solubilized starch molecules coexist, which would enhance gel viscosity. With the aim of texture modification and quality improvementof composite food formulations, there is certainly a need for more fundamental work on these mixed gelling systems to provide a sound basis for predicting and controlling their rheological responses.

+

From a practical viewpoint, several possibilities exist to retard or prevent starch retrogradation and staling events in thermally processed foods [197,466]:(1) storage at temperatures below T, (e.g., bread products promptly wrapped and stored at <-2O"C);(2) control of effective T, by moisture content and/or product formulation, relative to storage temperathat conditions for crystallization become less favorable (e.g., extures, truded cereals of low final moisture content); (3) modification starch of structure to reduce intermolecular associations and continuity of the gel network (e.g., by the addition of hydrolytic enzymes to cleave interconnecting chain segments between crystallites, lipids to complex with individual starch chains, and sugars to decrease the rate of chain aggregation); (4) incorporation of hydrocolloids for better moisture retention and asofter crumb texture. The various aspects of the staling of bread and other bakery foods as well as all the critical factors influencing the shelf life of these products have recently been reviewed [149,467].

D. Phase Transitions of V-Complexes Over the past fifteen years, X-ray diffraction and DSc studies on starchlipid interactions have contributed new knowledge aboutthe structure, thermal behavior, and transitions between the various polymorphic forms of amylose-lipid complexes in model systems and in actual foodproducts The presence of these complexes is revealed by endothermic transitions at temperatureswell above the melting endotherms of amylopectin crystallites in granular starch or in retrograded starch gels (M3 and M4, Fig. 10). Complex formation is thermoreversible, as evidencedbyanexothermonthe DSC cooling curves [134,468,471,472]. This transition displays marked thermal hysteresis (AT: 15-25"C, cooling rates 3-1OoC/min) [134,468,472], as anticipated for nucleation-controlled ordering processes of macromolecules. The thermal stability (T,) and melting enthalpies (AH) of V-amylose complexes are dependent on the chemical structure of the complexing ligand [134136,142,469-473,4761, amylose chain length [135,476], and the conditions (temperature-time-solvent) employed during complexation [134,137,139142,469,471-474,4761. When fatty acids were used as ligand mIecules, T, increased with the length of the saturated aliphatic chain [469,471,473], and it decreased with increasing level of cis unsaturation [142,469,472,473]. The T,,,of highly crystalline complexes of fatty acids with amyloses of different DP (20, 30, 40, 80, and 100) increased with amylose chain length [476]. Moreover, complexes crystallized from melt at high polymerconcentrations (10-40% w/w) exhibited broader melting transitions and lower melting enthalpies than their counterparts prepared by crystallization from dilute

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lution (0.8% w/v) [134], implying a lower degree of perfection and a wider distribution of ordered structures for the former.Apparent melting enthalpies of amylose-lipid complexes usually vary between 8 and 32 J/g. As with granular starch, the melting-transition temperature of complexes is influenced by water content [134,135,185,297]. Water also affects the melting behavior of the complexes, as illustrated in Fig. 17a for the amylose-glycerol monostearate complex. Melting is highly cooperative at high moisture levels (heatingrates5-1O0C/min), yielding a single endotherm;i.e.,zero-entropyproductionmelting conditions (melting without change in the metastability of the system during heating) are approached. However, as the water content is decreased below 70% w/w, the peak temperature is raised, and a second melting transition progressively evolves. These thermal responsesare typical of nonequilibrium melting of metastable structures. Additional evidence for reorganization is provided by the exothermic effect shown between the two endotherms in the DSC curves (Fig. 17a, weight fraction of complex 0.6-0.9) as well as by DSC experiments in which melting is examined as a function of heating rate. Increasing the heating rate from 3" to 60"C/min reduces the magnitude of the high-temperature transition (Fig. 17b). Overall,the effect of water on the phase transitions of thesesystems, following heat-moistureplasticization of their amorphous chain segments (i.e., at T > Tg),can be rationalized by considering the thermodynamic melting-point depression of the ordered domains as well as the structural reorganization during heating. The thermal behavior of amylose-lipid complexes should therefore be viewed in terms of the temperature dependence of metastable states [4,197]. Since the various processes are under kinetic control, their manifestation in the thermal curves will depend on all the parameters governing nonequilibrium phase changes; these include the supermolecular structure of the complex, heating rate vs. reorganization rate, moisture content, and the presence of other solutes. In a number of calorimetric studies 473,4741, multiple melting transitionswere reported, implying a relationship between thermal properties and supermolecular structure of the complex. The structural origin of the endotherm multiplicityhas been sorted outusing amylose-monoglyceride complexes prepared by varying the crystallization conditions [137,139,478]. Several supermolecular structures were thus obtained (Fig. 18, forms I, IIa, and IIb), which were characterized by calorimetry, X-ray diffraction, distribution of chain segments in the ordered domains of the complex following a-amylase etching, and gel permeation chromatography of the resistant amylodextrins. For any given monoglyceride used as ligand, forms I and IIa were characterized by constant melting temperatures, regardless of the degree of supercooling (AT = T,,, - T,) employed for their formation from dilute amylose solutions (0.25% w/v, amylose:li-

Starch

133

.l0 .20

40

120

80

MO

EO

Temperature

240

("C) -1

0

10

M

- Cl8

30

Heating rate (OClmin) 1 7 (a) DSC melting profiles of amylose-glycerol monostearate complex (form I) as a function of weight fraction the complex. (b) Effect heating rate on the melting-transition enthalpies (AH,and AH,,)of amylose complexes (50%W/ W in water) with the l-C,,, and l-C18saturated monoglycerides.

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T

II

k I

26

Diffraction angle (20)

Temperature CC)

DSC solids) and X-ray diffraction diagrams three amyloseglycerol momstearate complexes differing in the degree of chain organization in solid state. The inset depicts the relationships between Gibbs free energy and temperature for the structural forms I, 11%and IIb, the arrow indicates melt-mediated transformation (of metastable form I to IIa. (Reprinted with permission from Ref. 194.)

gand ratio 5:l wiw). Form I, obtained under conditions favoring rapid nucleation (at low Tc),had a low T,,, and in the hydrated state gave an amorphouslike X-ray diffraction pattern;i.e.,it has only short-range order. In contrast, form IIa (preferred polymorph under high Tc) appeared as a polycrystalline specimen, giving the typical reflections of the V,, complex. At intermediate crystallization temperatures the solid-state structures of the complexes were characterized by various ratios of forms I and Ha, and no intermediate form was detected. These observations were later confirmed by similar studies on amylose complexes with linear alcohols (4-6 carbon atoms) [l431 and fatty acids [142]. Differences in birefringence, density, andviscoelastic properties among different structural forms of the complexes were also detected [137,143]. Crystallizationandannealing studies for amylose-monoglyceridecom-

plexes, under controlled temperature-time storage protocols [137,139], further suggested that forms I (aggregated state with random distribution of helical chain segments) and II (more compact structure with well-defined crystalline order) represent two distinct free-energy domains, separated from each other by high-energy barriers. This implies that conversion of the kineticallypreferred form Ito the thermodynamically favored structure I1 (state of lower free energy) of the complex occurs only after partial melting of the former (Fig. 18, inset). Annealing of type IIa complex at high temperatures gives rise to more perfected V-crystals (IIb of higher T'), typical of metastable partially crystalline polymers [137,142]. The thermal stability and transitions between forms I and I1 were also examined in the presence of various solutes [139,140]. The observed conformational responses in stabilizing (Na,SO,, sucrose, CsCl) and destabilizing (urea, guanidine hydrochloride) solvent environments further suggested that interconversion betweenthe various structural forms of the complex can be explainedby considering changes attwo levels of ordered structures [139]: association * dissociation of aggregated helices, and V-helix* coil transitions. Interestingly, CsCl at high concentrations (>3.0 M) caused disruption of the crystallites of the I1 form without affecting the conformation of individual helices. The transition enthalpies of amylose-lipid complexes show very little dependence on the extent of long-range ordering of chains in the supermolecular structure [137,139,142,143]. This implies that measured enthalpic changes mainly represent contributions from the breaking of intrahelical H-bonds on conformational disordering of the V-helices. A small difference in melting enthalpies between IIa (AH 27.5 2 0.9 J/g) and IIb (AH 32.2 2 0.7 J/g) forms of the amylose-stearic acid complex was attributed to dissociation energyof the numerous van der Waals contacts between helices in the crystallites and limited interhelical H-bonding [142]. Ordering of amylose-lipidcomplexesupon heating of nonwaxy starches, involving either endogenous granular or added monoacyl lipids, occurs during gelatinization. The exothermic effect of this process is superimposed on the melting transition of starch crystallites and thus results in a reduced enthalpy of gelatinization [70,136,191,479]. However, changes in AH observed in the presenceof surfactants must be interpreted with caution. For example, SDS destabilizes the granular structure, depending on incubation temperature and time, that reduced transition temperatures and enthalpies are found [442]. It is also possible that enthalpy contributions from phase transitions of the surfactant itself, occurring in the same temperature region, are involved [480]. The melting enthalpy of amylose-lipid complexes, when determined at increasing amounts of lipid, reaches a limiting plateau value at a weight ratio of 1ipid:amylose 0.06-0.1 [70,481]. This ratio is consistent with the stoichiometry of the complex at saturating

-

concentrations of the ligand, based on viscosimetric measurements [482]. Rapid calorimetric methods for determining amylose content of starches have been developed basedon the magnitude of enthalpy changes observed on melting [481,483] or formation [484] of amylose-lysolecithin complex when amylose in a starch melt is complexed with saturating amounts of lipid; AH is proportional to the amount of amylose-lipid complex formed and consequently to the amount of amylose present in the starch sample. The complex-forming ability differs among various lipids [144]; e.g., the best ligands among monoacylglycerides are those with a saturated aliphatic chain of 14-16 carbon atoms. The complexing ability of lipids is also dependent on their physical state; e.g., for monoglycerides the best results are obtained when they are added as aqueous dispersions at elevated temperatures (60°C) (lamellar phase) or in the form of liposomes [485]. The resistance to enzymic degradation of amylose-lipid complexes follows the same order as their thermal stability [472];i.e., the enzymic hydrolysis rate of amylose-monoglyceride complexes with a-amylase plus amyloglucosidase increased in the order of monopalmitin, monostearin < monomyristin < monolaurin. The supermolecular structure of the complex also influences its in vitro digestibility, as observed for the amylose-glycerol monostearate complex [478]. The rate and extent of degradation with pancreatic and Bacillus subtilis a-amylases, in heterogeneous reaction mixtures, decreased in the order of forms I > IIa > IIb; i.e., this ranking corroborated with thatof the degree of long-range order of these V-polymorphs. However, unlike other forms of resistant starch, evenhighly crystalline V-amylose complexes are fully degraded under prolonged reaction time and high enzyme levels. Even more important to product quality attributes is the influence of structure of V-complexes on the postgelatinization behavior of starch. In the presence of glycerol monostearate, wheat flourextrudates showed an inverse relationship between water solubility index and the amount of polymorph IIa formed during extrusion [138]. Similarly, for parboiled rice kernels (varying in their amylose content between 10.9% and 25%),cooking losses due to solubilization of starch components were minimized when the parboiling conditions favored extensive interaction between amylose and endogenous lipids and development of the IIa polymorph (Fig. 19) [141]. A number of different structural domains can be found in parboiled rice, depending on the rice variety and the hydrothermal conditions employed during parboiling [141,486,487];these include ungelatinized/annealed A crystallites,retrograded amylopectin (B crystallites), and V-polymorphs. Ungelatinized starch and high-melting V-complexes are most likely responsible for the firmer texture and restricted swelling of this product during cooking. In high-am-

Polymers 4.0

3.2

-

--

X amylose

o”-o R 6 4 D--cI

2.4

IR24

15.8

I

0.0

.o Raw

1.o

1

.o

1

L

I 0.0 Raw

0

Parboiling Conditions (kg/cm2) Relationships between the amount of complex IIa formed in parboiled ricekernels (AH) varyingamylosecontentandthehot-water-solubleamylose leached during their cooking in excess water. (Adapted with permission from Ref. 141.)

ylose corn starch films, prepared by the drying of autoclaved starch dispersions (2%), different typesof structure (identified by X-raydifhaction) were found, depending on the drying temperature [488,489].B-type crystals were detected mainly in films dried at temperatures below 60"C,whereas a ture of B-, and V-crystallites was obtained when drying was carried out between 60" and 100°C.Finally, at lOO"C,crystallites of type only were present. It was shown that formation ofcrystalline amylose-lipid complexes reduces the maximum elongation obtained for the films in stressstrain experiments [489].In other studies, the addition of glycerol monostearate (1.0 dl00 gsemolina)inpasta formulation led to asignificant decrease in product stickiness upon cooking, due to the complexation of the available amylose in the form of the II polymorph [477].The results presented here clearly demonstrate that some physical properties of starch materials can be explained on a molecular and supermolecular scale. Future studies should focus on establishing such structure-property response profiles, which would be useful in modeling and optimizing processing parameters for desired product quality.

In this chapter, consideration was given to the thermal and physical properties of starch polymers in the solid state from the perspective of supermolecular organization, stability, and transformations of ordered chain domains, as well as interactions of starch with waterandothercosolutes. Experimental data drawn from various literature reports do indicate that the macromolecular organization of starch in food products exerts a major influence on texture and other sensory attributes, on nutritional value, and on shelf life (Fig. 20). These studies also pointed to the nonequilibrium nature (metastability) of various starch structures and processes.Starch in thermally treated foods may be present in different forms related to various levels of polymer organization in the solid state (molecular andsupermolecularorder, micro- and macrostructure, etc.), depending on processing and storage conditions, starch fine structure and composition, amount of water, and the presence of other ingredients. For example, at a supermolecular level, at least three distinct ordered chain domains have been identified, which have their own characteristic T,,, (in excess water) and exhibit different time scales for theirdevelopment: (1) retrograded amylose (T, = 14O-16O0C), which is formed rapidlybyassociation(doublehelical)and aggregation of linear starch molecules; (2) crystallites of retrogradedamylopectin (T, = 4560°C); and amylose-lipid helical inclusion complexes, forming rapidly upon cooling of starch melts containing monoacyl lipids, with varying de-

.

Gelation, Water binding 0 Flavor retention 0 Adhesive/binding power

STRUCTURE constituents (lipids,proteins small solutes)

0 Ordered

Chain Domains

long-range (crystallites) 0 Amorphous 0

Macro Structure

0 Crystallization

medium HzO-plasticization (TgGTmdepression)

20 Structure-property relationships

Chemical enzymic modification

Processing-Storage effects (temp., H20, shear, pressure) Storage conditions

starch materials in processed-food

grees chain organization in the solid state (T, = 1oO-12O0C). Moreover, a large variability in the partially crystalline organization, porosity and accessibility to digestive enzymes can be found, which spans from a compact macrostructure with ungelatinizedgranules (m biscuits and high-sugar cookies), to an anhydrous glassy foam (e.g., extruded products), and to a highly retrograded and fully plasticized (by water and other solutes) gel-like product (e.g., puddings).

Bllladerls

For complex products, inwhich starch is mixed with other ingredients, the leached amylose on gelatinization may interact with other components, either negatively or positively. This would affect the composition and distribution of the microphases, and therebyalter the rheological properties and stability of the composite matrix. If appropriate processes and storage conditions are carefully selected, technological control of physical properties, texture, digestibility, and stability of the final product could be possible by manipulation of granular starch dispersion during cooking, gelation and retrogradation events, and interactions of starch with other constituents (e.g., with lipids, proteins) in the composite food system. The importance of TB in this respect is pivotal (Fig. 20). By controlling the T, (via water management or ingredient formulation), textural parameters (e.g., brittleness, stiffness), crystallization rates, and the stability of the product can be altered. The complex interplay betweenvarious structural components andthe effect of solvent and other plasticizing solutes on the physicochemical properties of processed starch are not yet fully understood. As more information concerning structure-function relationships for starch materials becomes available, researchefforts will be directed towardthe modification of starch composition,fine structure, and supermolecular order as well as to the establishment of appropriate processing and storage protocols to impart certain functionality, to optimize quality, and to extend shelf life.

1. Blanshard, J. M. V. The significance of the structure and function of the starch granule in baked products. In: Blanshard, J. M. V., Frazier, P. J., and Galliard T.,eds., Chemistry and Physicsof Baking. Royal Society of Chemistry, London, pp. 1-13 (1986). 2. Blanshard, J. M. V. Starch granule structure and function: Aphysicochemical approach. In: Galliard, T.,ed., Starch: Properties and Potential. Wiley, New York, pp. 16-54 (1987). 3. Slade, L., and Levine, H. Recent advances in starch retrogradation. In: Stilva, S. S., Crescenzi, V., and Dea, I. C.M.,eds., IndustrialPolysaccharides. Gordon and Breach Science, New York, pp. 387-430 (1987). 4. Biliaderis, C. G. Nonequilibrium phase transitions of aqueous starch systems. In: Levine, H., and Slade, L., eds., Water Relationships in Foods. Plenum, New York, pp. 251-273 (1991). Snyder, E. M. Industrial microscopy of starches. In: Whistler, R. L., BeMiller, J. N., and Paschall, E. F., eds., Starch: Chemistry and Technology. Academic Press, New York, pp. 661-673 (1984). 6. Fitt, L. E., and Snyder, E. M. Photomicrographs of starches. In: Whistler, R. L., BeMiller, J. N., and Paschall, E. F., eds., Starch: Chemistry and Technology. Academic Press, New York, pp. 675-689 (1984).

Starch Banks, W., and Greenwood, C. T. Starch and Its Components. Edinburgh University Press, Edinburgh (1975). 8. Zobel, H. F. Molecules to granules: A comprehensive starch review. Starch

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Starch Hinrichs, W., Buther, G., Steifa, M.,Betzel, Ch., Zabel, V., Pfannemuller, B., and Saenger, W. An amylose antiparallel double helix at atomic resolution. Science 2 3 8 Godet, M. C., Tran, V., Delage, M.M.,and Buleon, A. Molecular modelling of the specific interactions involved in the amylose complexation by fatty acids. Int. J. Biol. Macromol. 44. Lagendijk, J., and Pennings, H. J. Relation between complex formation of starch with monoglycerides and the firmness of bread. Cereal Sci.Taiay Pfannemuller, B., Mayerhofer, H., and Schulz, R. C. Conformation of amylose in aqueous solution: Optical rotatory dispersion and circular dichroism of amylose-iodine complexes and dependence on chain length of retrogradation of amylose. Biopolymers Pfannemuller, B., and Ziegast, G. Properties of aqueous amylose and amylose-iodine solutions. In: Brant, D. A., ed., Solution Properties of Polysaccharides. American ChemicalSociety,Washington, D.C., pp. Gidley, M.J., and Bulpin, P. V. Aggregation amylose in aqueous systems: The effect of chain length on phase behavior and aggregation kinetics.Macromolecules 22: Pfannemuller, B. Models for the structure and properties of starch. Starch 38: Roger, P., and Colonna, P. Evidenceof the presence of large aggregates contaminating amylosesolutions. Carbohydr. Polym Aberle, Th., Burchard, W., Vorwerg, W., and Radosta, S. Conformation contributions of amylose and amylopectin tothe structural properties of starches from various sources. Starch Hizukuri, S. Relationship between the distribution of the chain length of amylopectin and the crystalline structure of starch granules. Carbohydr.Res. Hizukuri, S. Polymodal distribution of the chain lengthsof amylopectin and its significance. Carbohydr. Res. Kobayashi, S., Schwartz, S. J., and Lineback, D. R.Comparison of the structure of amylopectins from different wheat varieties. Cereal Chem. Mercier, C. The fine structure of corn starches various amylose percentage: Waxy, normal and amylomaize. Starch'25: Manners, D. J. Recent development in our understanding of amylopectin structure. Carbohydr. Polym. Manners, D. J. Structural analysis of starch components by debranching enzymes. In: Hill, R. D., and Munck, L., eds., New Approaches to Research on Cereal Carbohydrates.Elsevier Science, Amsterdam, pp. Hizukuri, S., and Abe, J. A new method of enzymic analysis of amylopectin structure. In: Meuser, F., Manners, D. J., and Seibel, W., eds., Plant Polymeric Carbohydrates. Royal Society of Chemistry, Cambridge, pp.

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Watanabe, T., Akiyama, Y., Takahashi, H., Adachi, T., Matsumoto, A., and Matsuda, K Structural features and properties of Naegeli amylodextrin from waxy-maize, sweet potato, and potato starches. Carbohydr. Res. Imberty, A., and Perez, S. Conformational analysis and molecular modelling of the branching point of amylopectin. Int. J. Biol. Mucromol. Manners,D. J., and Matheson, N. K The fine structure of amylopectin. Carbohydr. Res. Hizukuri, S., Tabata, S., and Nikuni, Z. Studies on starch phosphate. Part I. Estimation of glucosed-phosphate residues in starch and the presence of other bound phosphates. Starch Nielsen, T. H., Wischmann, B., Enevoldsen, K., and Moller, B. L. Starch phosphorylation in potato tubers proceeds concurrently with the de novo biosynthesis of starch. Plant Physiol. Bay-Smidt, A. M., Wischmann, B., Olsen, C. E., and Nielsen, T. H. Starchbound phosphate in potato as studied by a simple method for determination of organic phosphate and 31P-NMR.Starch Muhrbeck, P., and Tellier, C. Determination of the phosphorylation of starch from native varieties by 31P-NMR. Starch Muhrbeck, P., Svensson, E.,and Eliasson, A.-C. Effectof the degree of phosphorylation on the crystallinity of native potato starch. Starch Evans, I. D. An investigation of starchhurfactant interactions using viscometry and differential scanning calorimetry. Starch Eliasson, A.-C., and Ljunger, G. Interactions between amylopectin and lipid additives during retrogradation in a model system. J. Sci. Food Agric Callaghan, P. T., and Lelievre, J. The size and shape of amylopectin: A study using pulsed-field gradient nuclear magnetic resonance. Biopolymers Lelievre, J., Lewis, J. and Marsden, K The size and shape of amylopectin:A study using analytical ultracentrifugation. Carbohydr. Res

Banks, W., Geddes, R., Greenwood, C. T., and Jones, I. G. Physicochemical studies on starches: Part The molecular size and shape of amylopectin. Starch 24: Ring, S. G., Colonna, P., l’Anson, K. J., Kalichevsky, M. T., Miles, M.J., Morris, V. J., and Orford, P.D. The gelation and crystallization of amylopectin. Carbohydr. Res. 162: Cameron, R. E., Durrani, C. M., and Donald, A. M. Gelation of amylopectin without long-range order. Starch 46: Ring, S. G., Colonna, P., Miles, M. J., Morris, V. J., and Turner, R. Spherulitic crystallization of short chain amylose. Int. J. BioL MacromoL P Gallant, D., Derrien, Aumaitre, and Guilbot, In vitro degradation of starch. Studies by transmission and scanning electronmicroscopy. Starch Gallant, D., and Guilbot, A. Developments in starch ultrastructure knowledge. I. Wheat starch granules. Starch 25: Kassenbeck, P. Contribution to the knowledge on distribution of amylose and amylopectin in starch granules. Starch 30: Yamaguchi, M., Kainuma, K., and French, D. Electron microscopy observations of waxy maize starch. J. Ultrastruct. Res. 69: Gallant, D. J., and Bouchet, B. Ultrastructure of the maize starch granules. J. Fd. Microstruct. Gallant, D. J., Bouchet, B., Buleon, A., and Perez, S. Physical characteristics of starch granules and susceptibility to enzymatic degradation. Eur. J. Clin. 46(S~ppl. 2): Gallant, D. J., Bouchet, B., and Baldwin, P.M. Microscopy of starch Evidence of a new level of granule organization. Carbohydr. Polym. 32: Sterling, C. J. A low angle spacing in starch. Polymer Sci. 56: Oostergetel, G. T.,and van Bruggen, E. F. J. On the origin of a low angle spacing in starch. Starch 41: Jenkins, P. J., Cameron, R. E., and Donald, M. A universal feature in the structure of starch granules fromdifferentbotanicalsources. Starch Blanshard, J. M. V., Bates, D. R., Muhr, H.,Worcester, D. L., and Higgins, J. S. Small-angle neutron scattering studies of starch granule structure. Carbohydr. Polym Oostergetel, G. T., and van Bruggen,E. F. J. The crystalline domains in potato starch granules are arranged in a helical fashion. Carbohydr. Polym. 21: Cameron, R. E., and Donald, A. M. A small-angle X-ray scattering study of the annealing and gelatinization of starch. Polymer 33: Zobel, H. F. Starch crystal transformations and their industrial importance. Starch

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1: Leloup, V. M., Colonna, P., and Ring, S. G. Studies on probe diffusion and accessibility in amylose gels. Macromolecules 23: Biliaderis, C. G., and Zawistowski, J. Viscoelastic behavior of aging starch gels: Effects of concentration, temperature, and starch hydrolyzates on network properties. Cereal Chem. 67: Clark, H., and Ross-Murphy, S. B. The concentrationdependence of biopolymer gel modulus. Brit. Pol. J. 17: Kalichevsky, M. T., Orford, P.D., and Ring, S. G. The retrogradation and gelation of amylopectins from various botanical sources. Carbohydr. Res. Durrani, C. M., and Donald, A. M. Physical characterization of amylopectin gels. Polym. Gels Networks 3: Wu, J. Y., and Eads, T. M. Evolution of polymer mobility during aging of gelatinized waxy maize starch: magnetization transfer ‘H NMR study. Carbohydr. Polym. 20: Shi, Y. Ch., and Seib, P. The structure of four waxy starches related to gelatinization and retrogradation. Carbohydr. Res. 227: Bello-Perez, L. and Paredes-Lopez, 0. Effects of solutes on retrogradation of stored starches and amylopectins: calorimetric study. Starch

Starch Biliaderis, C. G., and Prokopowich, D. J. Effect of polyhydroxy compounds on structure formation in waxy maize starch gels: A calorimetric study. Carbohydz Potym. 23: Prokopowich, D. J., and Biliaderis, C. G. A comparative studyof the effect of sugars on the thermal and mechanical properties of concentrated waxy maize, wheat, potato and pea starch gels. Food Chem. I’Anson, K. J., Miles, M. J., Moms, V. J., Besford, L. S., Jarvis, D. and Marsh, R.A. The effects added sugars on the retrogradation of wheat starch gels. J. Cereal Sci. 11: Cairns, P., Miles, M. J., and Moms, V.J. Studies of the effect of the sugars ribose, xylose and fructose on theretrogradation of wheat starch gels by Xray diffraction. Carbohydz Polym. 16: Doublier, J.-L., and Llamas, G . A rheological description of amylose-amylopectin mixtures. In: Dickinson,E., and Walstra, P., eds., Food Colloids and Polymers: Stability and Mechanical Properties. Royal Soc. Chem., Cambridge, pp. Langton, M., andHermansson, A.”. Microstructuralchangesin wheat starch dispersions during heating and cooling. Food Microstruct. Autio, K.Rheological and microstructural changesof oat and barleystarches during heating and cooling. Food Structure Svegmark, K., and Hermansson, A.-M. Distribution amylose amylopectin in potato starch pastes: Effects of heating and shearing. Food Structure 1 0 Steeneken, P. A. M. Rheological propertiesof aqueous suspensionsof swollen starch granules. Carbohydz Polym. 11: Mestres, C., Colonna, P., and Buleon, A. Characteristics of starch networks within rice flour noodles and mungbean starch vermicelli. J. Cereal Sci. 7: Ring, S. G. Some studies on starch gelation. Starch 37: EIiasson, A.-C. Viscoelastic behavior during the gelatinization of starch. I. Comparison of wheat, maize, potato and waxy-barley starches. J. Tat. Stud. 17: Launay, B., Doublier,J.-L.,andCuvelier, G. Flow properties ofaqueous solutions and dispersions of polysaccharides. In: Mitchell, J. R., and Ledward, D. A., eds., Functional Properties of Food Macromolecules. Elsevier Applied Science, London, pp. Evans, I. D., and Haisman, D. R. Rheology of gelatinized starch suspensions. J. Tature Stud. 1 0 Wong, R. B. K., and Lelievre, J. Viscoelastic behavior ofwheat starch paste. Rheol. Acta 20: Svegmark, K., and Hermansson, A.-M. Shear-induced changes in the viscoelastic behavior of heat-treated potato starch dispersions. Carbohydz Polym. 13: Svegmark, K., and Hermansson, A.-M. Changes induced by shear and gel formation in the viscoelastic behavior potato, wheat and maize starch dispersions. Carbohydz Polym. 15:

Muhrbeck, P., and Eliasson, A.-C. Influence of pH and ionic strength on the viscoelastic properties of starch gels-A comparison of potato and cassava starches. Carbohydr. Polym. Kulp, K, and Ponte, J. G. Staling of white pan bread: Fundamental causes. Crit. Rev. Food Sci Nu& Krog, N.,Olesen, S. K, Toemaes, H., and Joensson, T. Retrogradation of the starch fraction in wheat bread. Cereal Foods World 34: Bulkin, B. J., Kwak, I., and Dea, I. C. M. Retrogradation kinetics of waxy corn and potato starches: A rapid, Raman-spectroscopic study. Carbohydr. Res. van Soest, J. J. G., de Wit,D., Tournois, H., and Vliegenthart, J. F. G. Retrogradation of potato starch as studied by Fourier transform infrared spectroscopy. Starch 46: Silverio, J., Svensson, E., Eliasson, A.-C. and Olofsson, G. Isothermal microcalorimetric studies on starch retrogradation. J. Therm. Anal. (in press) Orford, P.D., Ring, S. G., Carroll, V., Miles, M. J., and V. J. The effect of concentration and botanical source on the gelation and retrogradation of starch. J. Sci. Food Agric. 39: Colwell, K. H., Axford, D. W., Chamberlain, N., and Elton, G. A. H. Effect of storage temperature on the aging of concentrated wheat starch gels. J. Sci Food Agric. Longton, J., and G. A. Differential scanning calorimetry studies on the crystallinity of aging wheat starch gels. Starch 33: Eliasson, A.-C. Retrogradation of starch as measured by differential scanning calorimetry. In: Hill, R. D., and Munck, L., eds., New Approaches to Research on Cereal Carbohydrates. Elsevier Science, Amsterdam, The Netherlands, pp. Nakazawa, F., Noguchi, S., Takahashi, J., and Takada, M. Retrogradation of gelatinized potato starch studied by differential scanning calorimetry. Agric. Biol. Chem. 49: Jankowski, T., and Rha, C.K. Retrogradation of starch in cooked wheat. Starch 38: Wunderlich, B. Macromolecular Physics: Crystal Nucleation, Growth, Annealing. Academic Press, New York Fearn, T.,and Russell, P. L. A kinetic study of bread staling by differential scanning calorimetry. The effect of specific loaf volume. J. Sci Food Agric. 33: Zeleznak, K. J., and Hoseney, R. C. The role of water in the retrogradation of wheat starch gels and bread crumb. Cereal Chem. 63: Russell, P. L. The aging of gels from starches of different amylose/amylopectin content studied by differential scanning calorimetry. J. Cereal Sci. 6 Roulet, Ph., MacInnes, W.M., Wursch, P., Sanchez, R. M., and Raemy, A. A comparative study of the retrogradation kinetics of gelatinized wheat starch in gel and powder form using X-rays, differential scanning calorimetry and dynamic mechanical analysis. Food Hydrocoll.

Roulet, Ph., MacInnes, W.M., Gumy,D.,and Wursch, P. Retrogradation kinetics of eight starches. Starch 42: Marsh, R. D. L., and Blanshard, J. M. V. The applicationof polymer crystal growth theory to the kinetics of formation of the B-amylose polymorph in a wheat-starch gel. Carbohydr. Polym. 9: Wong, R. B. K., and Lelievre, J. Effects of storage on dynamic rheological properties of wheat starch pastes. Starch 34: Mita, T. Structure of potato starch pastes in the aging process by the measurement of their dynamic moduli. Carbohydr. Polym. 17: Kim, S. K, Ciacco, C.F., and D’Appolonia, B. L. A kinetic study of retrogradation of cassava starch gels. J. Food Sci. 41: Ciacco, C. F., and Fernades, J. L. A. Effect of various ions on the kinetics of retrogradationofconcentratedwheat starch gels. Starch 31: Germani, R., Ciacco, C.F., and Rodriguez-Amaya, D. B. Effect of sugars, lipids and type of starch on the mode and kinetics of retrogradation of concentrated corn starch gels. Starch 35: Matsukura, U.,Matsunaga, A., and Kainuma, K Structural studies on retrograded normal and waxy corn starches. J. Jpn. Soc. Starch Sci. 30: McIver, R., Axford, D.W.E., Colwell, K H., and Elton, G. A. H. Kinetic study of the retrogradation of gelatinized starch. J. Sci Fd. Agric. 19: Eliasson, A.-C. Differential scanning calorimetry studies on wheat starchgluten mixtures. 11. Effect of gluten and sodium stearoyl lactylate on starch crystallization during aging of wheat starch gels. J. Cereal Sci l : Russell, P. A kinetic study of bread staling by differential scanning calorimetry and compressibility measurements. The effect of different grists. J. Cereal Sci. l : Russell, P. A kinetic study of bread staling by differential scanning calorimetry and compressibility measurements.The effect of added monoglycerides. J. Cereal Sci. l : Russell, P. A kinetic study of bread staling by differential scanning calorimetry. The effect of painting loaves withethanol. Starch 35: Mandelkern, L.Crystallization OfPolymers. McGraw Hill, New York Goering, K. J., Jackson, L. L., Dehaas, B.W. Effectof some nonstarch components in corn and barley starch granules on the viscosity of heated starch-water suspensions. Cereal Chem. 52: Melvin, M. A. The effect of extractable lipid on the viscosity characteristics of corn and wheat starches. J. Sci. Food Agric. 30: Eliasson, A.-C., Carlsson, T. L., Larsson, K., and Miezis, Y. Some effects of starch lipids on the thermal and rheological properties of wheat starch gels. Starch 33: Takahashi, S., and Seib, P. A. Paste and gel properties of prime corn and wheat starches with and without native lipids. Cereal Chem 65:

Gray, V.M., and Schoch, T.J. Effects of surfactants and fatty adjuncts on the swelling and solubilization of granular starches. Starch 14: Eliasson, A.-C., Larsson, K, and Miezis, Y. On the possibility of modifying the gelatinization properties of starch by lipid surface coating. Starch 33: Eliasson, A.-C. Starch gelatinization in the presence of emulsifiers. Starch 37

Osman, E. M., and Dix, M. R. Effects of fats and nonionic surface-active agents on starch pastes. Cereal Chem. 3 7 Harbitz, Gel formation of potato starch in the presence of a surfactant. Starch 35: Gough, B. M., Greenwell, P.,and Russell, P. I. On the interaction of sodium dodecyl sulfate with starch granules. In: Hill, R. D., and Munck, L., eds., New Approaches to Research on Cereal Carbohydrates. Elsevier Science, Amsterdam, The Netherlands, pp. Krog, N. Influence of food emulsifiers on pasting temperature and viscosity of various starches. Starch 444. Nierle, W., and Baya, A. W. Lipids and rheological properties of starch. Part I. The effect fatty acids, monoglycerides and monoglyceride ethers on pastingtemperature andviscosity of wheat starch. Starch 42: Eliasson, A.-C. Viscoelastic behavior during the gelatinization of starch. 11. Effects of emulsifiers. J. Text S&. 1 7 Eliasson, A.-C, Finstad, H., and Ljunger, G. A study of starch-lipid interactions for some native and modified maize starches. Starch 4 0 Gudmundsson, M., and Eliasson A.-C. Retrogradation of amylopectin and the effects of amylose and added surfactantslemulsifiers. Carbohydr.Polym. 13:

Rao, P., Nussinovitch, A., and Chinachoti, P. Effects of selected surfactants on amylopectin recrystallization and on recoverability bread crumb during storage. Cereal Chem. 69 Miura, M., Nishimura, A., and Katsuta, K. Influence of addition polyols and food emulsifiers on the retrogradation rate of starch. Food Struct. 11: Conde-Petit, B., and Escher, F. Influence of starch-lipid complexation on the aging behavior high-concentration starch gels. Starch 46: Batres, L. V., and White, P.J. Interaction of amylopectin with monoglycerides in model systems. J. A m Oil Chem. Soc. 63: Biliaderis, C.G.,andVaughan,D. J. Electron spin resonance studies of starch-water-probe interactions. Carbohydr. Polym. 7 Svensson, E. Crystalline properties of starch. Ph.D. dissertation, Lund University, Sweden Maxwell, J. L., and Zobel, H. F. Model studies on cake staling. Cereal Foods World 23:

Wang, Y.-J., and Jane, J. Correlation between glass transition temperature and starch retrogradation in the presence of sugars and maltodextrins. Cereal Chem. 71: Kohyama, K., and Nishinari, K. Effect of soluble sugars on gelatinization and retrogradation of sweet potato starch. J. Agric. Food Chem. 39: Katsuta, K, Nishimura, and Miura, M. Effects of saccharides on the stabilities of rice starch gels. Mono- and disaccharides. Food Hydrocoll. Katsuta, K, Nishimura, A.,and Miura, M. Effects of saccharides on the stabilities of rice starch gels. Oligosaccharides. Food Hydrocoll. 6: Biliaderis, C. G., Prokopowich, D, J., Jacobson, M. R., and BeMiller, J. N. Effect of n-alkyl glucosides onwaxy maize and wheat starch retrogradation. Carbohydr.Res. 280: Rogers, D. E., Zeleznak, K. J., and Hoseney, R. C. Effect of native lipids, shortening, and bread moisture on bread firming. Cereal Chem 6 5 Biliaderis, C. G.,Izydorczyk, M. S., and Rattan, 0. Effect of arabinoxylans on breadmaking quality of wheat flours. Food Chem. 53: Alloncle, M., and Doublier, J. L. Viscoelastic properties of maize starch/ hydrocolloid pastes and gels. Food Hydrocoll. 5: Eidam,D.,Kulicke, W."., Kuhn, K., and Stute, R. Formation of maize starch gels selectively regulated by the addition of hydrocolloids. Starch Biliaderis, C. G., and Izydorczyk, M. S. Observations on retrogradation of starch polymers in the presence of wheat and rye arabinoxylans. In: G. 0. Phillips, D. J. Wedlock, and P. A. Williams, eds., Gums and Stabilizers for the Food Industry. IRL Press, Oxford, pp. Marzin, C., Doublier, J.-L., and Lefebvre, J. Effect of sodium caseinate on pasting and gelation properties of wheat starch. In: E. Dickinson and D. Lorient, eds., Food Macromolecules and Colloids. Royal Society of Chemistry, Cambridge, pp. Biliaderis, C. G. The structure and interactions of starch with food constituents. Can. J. Physiol. Phannacol. 69: Ponte, J. G., Payne, J. D,, and Ingelin, M. E. The shelf life of bakery foods. In: G. Charalambous, ed., Shelf Life Studies Foods and Beverages. Elsevier Science Publishers, Amsterdam, pp. Bulpin, P. V., Welsh, E. J., and Moms, E.R. Physical characterization of amylose-fatty acid complexes in starch granules and in solution. Starch Stute, R., and Konieczny-Janda, G. DSc-Untersuchungen an starken, Teil II. Untersuchungen an starke-lipid-complexen. Starch 35: Eliasson, A.-C. Starch-lipid interactions studied by differential scanning calorimetry. Thermochim Acta 95: Kowblansky, M. Calorimetric investigation of inclusion complexesof amylose with long-chain aliphatic compounds containing different functional groups. Macromolecules 18:

Eliasson, A.-C., and Krog, N. Physical properties of amylose-monoglyceride complexes. J. Cereal Sci. Raphaelides, S., and Karkalas, J. Thermal dissociation of amylose-fatty acid complexes. Carbohydr. Res. Eliasson, A.-C. On the thermal transitions of the amylose-cetyltrimethylammonium bromide complex. Carbohydr. Res. 172 Le Bail, P., Bizot, H., Pontoire, B., and Buleon, A. Polymorphic transitions of amylose-ethanol crystalline complexes induced by moisture exchanges. Starch Godet, M. C., Bizot, H., and Buleon, A. Crystallization of amylose-fatty acid complexes. Carbohydr. Polym. Dalvand, C. C. Investigations on starch and starch-emulsifier interactions in durum wheat pasta. Doctoral Dissertation, Swiss Federal Institute of Technology, Zurich, Switzerland Seneviratne, H. D., and Biliaderis, C. G. Action of a-amylases on amyloselipid complex superstructures. J. Cereal Sci. 13: Svensson, E., Autio, K., and Eliasson, A.-C. The effect of sodium dodecylsulphate on gelatinization and gelation properties of wheatand potato starches. Food Hydrocoll. (in press). Eliasson, A.-C. On the effects of surface active agents on the gelatinization of starch: a calorimetric investigation. Carbohydr. Polym. Kugimiya, M., and Donovan, J. W. Calorimetric determination of the amylose content of starches based on the formation and melting of the amyloselysolecithin complex. J. Food Sci. and Karkalas, J., and Raphaelides, S. Quantitative aspects of amylose-lipid interactions. Carbohydr. Res. Sievert, D.,and Holm, J. Determination of amylose by differential scanning calorimetry. Starch Mestres, C., Matencio, F., Pons, B., Yajid, M., and Fliedel, G. A rapid method for the determination of amylose content by using differential scanning calorimetry. Starch Riisom, T., Krog, N., and Eriksen, J. Amylose complexing capacities of cisand trans-unsaturated monoglycerides in relation to their functionality in bread. J. Cereal Sci. Ong, M. H., and Blanshard, J. M. V. Texture determinants of cooked, parboiled rice. II. Physicochemical properties and leaching behavior of rice. J. Cereal Sci. Ong, M. H., and Blanshard,J. M. V.The significance of starch polymorphism in commercially produced parboiled rice. Starch Bader, H., and Goritz, D. Investigations on high amylose corn starch films. Part Wide angle X-ray scattering (WAXS).Starch Bader, H., and Goritz, D. Investigations on high amylose corn starch films. Part Stress-strain behaviour. Starch

5 Microcrystalline Cellulose Technology Gregory S. Buliga, George W. Ayling, Gregory R Krawczyk, and E. J. McGinleyt

FMC Corporation Princeton, New Jersey

In the past several years there havebeenmany stabilizers, viscosifiers, gelling agents, fat mimetics, and textural agents introduced into the food market. Most of these are carbohydrate ingredients that are based on the old, one-dimensional starch or gum technology. The utilization of microcrystalline cellulose (MCC) technology to solve food product or processing problems is uniquely different from most hydrocolloids, and it possesses multidimensionalproperties to provide effective structure, texture, and physical stabilization in a vast number of food product systems [l].The multidimensional nature of these MCC-based ingredients is based on the ability to control their viscosity, gelling, surface area, thixotropy, or water binding through the interaction of microcrystallinecellulose with other hydrocolloids and processing parameters. The functional properties of these MCC-based ingredients include ice crystal control, foam stability, high temperature stabilization, emulsion stability, texture modifications, suspension of solids, and fat replacement +Deceased.

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TECHNOLOGYOFMICROCRYSTALLINE CELLULOSE Cellulose, the precursor of microcrystalline cellulose is a polysaccharide composed of hundreds of glucose units linked in a beta configuration (Fig. 1). Cellulose is the chief structural ingredient of the cell wall of pIants Wood contains about 50% cellulose, and cotton contains more than 90% cellulose. The cellulose polymer molecule is rodlike andhasmany hydroxyl groups available to participate in hydrogen bonding between molecules. The strong hydrogen bonding between chains results in many chains grouping together to form bundles, which are called fibrils. The fibrils are arranged in layers to form the cell wall of plant fibers. Within the bundles the cellulose molecules can occur in crystalline or paracrystalline (amorphous) structures or regions in the fibrils, depending on the arrangement and strength of interaction between the chains in the bundles. The raw material used to produce microcrystalline cellulose is a highly purified grade of plant cellulose, i.e., alpha cellulose, also referred to as special alpha or dissolving-grade pulp. The pulp used in the manufacture of MCC contains a high percentage of long-chain molecules and is relatively free of lignin, fats, waxes, noncellulosic carbohydrates, tannins, and resins. The same raw material is used to produce various cellulose derivatives,e.g.,sodiumcarboxymethylcellulose,which also is used in the food and pharmaceutical industries.

MANUFACTURING OF COPROCESSED MCC/HYDROCOLLOID GRADES To obtain crystalline cellulose, the plant cellulose, or pulp, is hydrolyzed to remove the amorphous regions, leaving only the crystalline bundles. The

first step requires treating the pulp with a dilute concentration of mineral acid in water. During hydrolysis, the acid penetrates the amorphous regions and converts them to soluble polysaccharides, short-chain oligosaccharides, and sugars, which are removed by a subsequent washing and filtration. The product that remains is the microcrystalline cellulose or simply the more crystalline or “organized’’ regions of the starting fibrous material. Only the physical form of the starting raw material has been altered in the course of the manufacturing; i.e., the cellulose in the fibrous formhas been converted to cellulose in a particulate form. In Fig. 2 the process to produce various grades of MCC is depicted in a simplified form.The purified MCC wetcake is mixed with deionized water, and the resulting slurry is neutralized and spray-dried to produce MCC-FD grades. These pure MCC grades are produced by controlling the atomization and drying conditions, thus resulting in porous MCC particles consisting of fragments of cellulose bound together by hydrogen bonding To develop the functional properties of hydrocolloid MCC grades, the hydrolyzed fiber is broken into smaller fragments and subjected to shear to liberate the cellulose microfibrils and crystallite aggregates. The functionality desired is based on the distribution of microfibrils and crystalline particles. This fracturing process of hydrocellulose fragments, which results in the liberation of microfibrils andcrystallite aggregates, is called [7]. Cellulose is an insoluble polymer. The intermolecular hydrogen bonds are low-energy bonds; but the considerable number and precise regularity of these bonds result in the formation of crystallite bundles of chains in native cellulose. When the attrited cellulose is dried, these same intermo-

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lecular bonds are formed between surfaces of adjacent microfibrils and/or crystallite aggregates. The dried product is not easily rehydrated. A dried microfibril-and-crystallitedispersion must be rehydrated and reattrited get to it back into a particulate form. To develop a dry, dispersible form of particulate MCC, a substance must be present during drying to act as a barrier to prevent formation of irreversible hydrogen bonds. If the microfibrils and crystallite aggregates are to disaggregate/dispersereadily, a material capable of acting as a dispersant must be present between particles. Manymaterials act as barriers if present in large enough quantities. Sugars, emulsifiers, fat, and polymers are examples. After the attrition step, the gums are combined with the MCC wetcake and the composition is dried. Again, the atomization and drying conditions will control the particle size andresidualwater content.

N. FUNCTIONAL PROPERTIES OF COPROCESSED MCC/HYDROCOLLOID PRODUCTS A small number of polymers has been found to act as both barriers and dispersants. These are typically anionic polymers like sodium carboxymethylcellulose (CMC), sodium alginate, or carrageenan. Functionality as a barrier-dispersant occurs at 5-15% of the composition of the coprocessed products. Other materials may function as barrier-dispersants but at higher levels. The current commercial hydrocolloid MCC grades contain CMC and/or alginate. These MCC products are referred to as cellulose gel, because of the gel network created between the microfibrils, crystallite aggregates, and gum upon complete dispersion in water. In Figs. 3a-c are micrographs of an MCC/CMC product. Examination of a dry particle (Fig. 3a) illustrates the

(b)

(c)

Figure 3 Micrographs of coprocessed MCCKMC product.

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173

porous nature of particles that are an aggregation of microfibrils and tallites. Studying the internal nature of the MCC/CMC particles (Fig. 3b) makes it clear that the CMC acts to coat the MCC particles, hence making it act as an effective barrier. Once the MCC/CMC particles are dispersed in water, the three-dimensional network can be identified (Fig. 3c). The ability to bind or entrap water by means of the three-dimensional network will control the texture or physical stability of a dispersion or emulsion. In the development of the MCC/CMC products, the ratio of gum to MCC as well as the molecular properties of the gum (MW, degree of substitution, polydispersity, uniformity of substitution) are important (Fig. For instance, a high ratio of CMC to MCC (i.e., more CMC) will ensure that an adequate amountof CMC is present to provide barrier propertiesfor the MCC particles,thusallowingcompletedispersion of theMCCparticulates.The molecular weight (low-high) of CMC utilized in the coprocessing with MCC also has an effect in controlling the dispersion and viscosity profiles of the end product. A high-Mw CMC will be much more effective bamer for the MCC particles than a low-MW CMC at a given MCC/CMC ratio. In addition to coating the MCC effectively, the CMC contributes to the setup viscosity of the dispersed MCC/CMC product. Shown in Fig. 5 are the initial and setup viscosities for dispersions prepared using two coprocessed MCC/CMC products: one with a low-viscosity CMC and one with a high-viscosity CMC. It is apparent that the dispersion viscosity parallels that of the CMC’s viscosity. The gel network formed by the MCC/CMC products can be characterized by dynamic rheological measurements. Figure6 illustrates the strain sweep curves(G’ and G” versus % strain) for an MCC/CMC (high-viscosity) product and an MCC/CMC (low-viscosity) product. Again, the strength of the gel parallels the viscosity(MW)of the CMC utilized during coprocessing

“High” Figure

“Low”

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Initial and setup viscosities for coprocessed MCC/CMC product.

with the MCC. Thus, it is possible to design a coprocessed MCChydrocolIoid product that can meet thespecific viscosity, gel strength,and dispersion specifications in a certain food system. Another tool that can be utilized to control functionalityis the drying methodology,Spray-dryingandbulk-dryingare two processesavailable.

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I l

1 Id

These differ mainIy in the solids concentration at which the MCC/hydrocolloid systems are dried. In these two processes the level of hydration and the sensible heat during drying are different. This has an effect on bonding dynamics between particles and particle/gum andin the overall residual water content. Coprocessed products that have been bulk-dried see higher temperatures during the drying process than products that have been spray-dried. The different drying methods make up part of the reason thatthe dried MCCI CMC products disperse in water at different rates (Fig. However, once the product has been dispersed in water, the viscosity (Fig. 8) and gel properties (Fig. 9) are virtually identical for either a spray-dried or a bulk-dried system. Most MCC/hydrocolloid systems are heat stabIe. Temperaturechanges have little or no effect on the functionality and viscosity of MCC/CMC dispersions, as seen in Figs. loa-b. The viscosity of a dispersion of MCC/CMC is independent of temperature. It is not until temperatures above 80°C that a slight decrease in viscosity is found. This property is extremely important in the preparation of heat-stable food products, especially when acids are present. The MCC/hydrocolloid products will hold up under extremely high temperatures, including those used during baking, retorting, UHT processing, and microwave heating, with minimal loss in viscosity, consistency, or color. The viscosity versus concentration curves for MCC/

of Shearing

(MCC/CMC)

High Visc. SD Process

High

,

BD Process

Low MSC.SD 'rocess 0

50

150

200

l

Viscosity vs. shearing time profiles fo? several MCC/CMC products.

A,B (initial visc.) A,B' (se'up visc.) A,A' SD 'rocess

j

3rocess

1.5

0.5

2.0

Percent Solids

Wscosity vs. concentration curves for a spray-dried and bulk-dried MCC/ CMC product.

TIS

-.. -

"

-..

G' = i

-

-

G' =BD

I

G' = SD

--

=

"

1oa

I

I I 1

I I IIIII 10.

I

I

I 10'

% Straln

Strain sweepcurvesforaspray-driedandbulk-driedMCC/CMC product.

I I 10'

5

(U

2

(U

B

B

W

-1 (U

(v

0

0

g!

0

h:

%

0 0-

m

G

al. CMC dispersions, before and after retorting (250’F for minutes), indicate only a slight loss in viscosity after retorting (Fig. lob). Nonionic galactomannan such as guar, locust bean gum (LBG), and tara, rather than provide a barrier-dispersant effect, tend to act as binders and hold the cellulose microcrystals in preformed shapes, as in the case of The ratio of guar to MCC plays a the spherical forms of MCC/guar major role in determining the dispersibility, particle shape, and rheological properties of final MCC/guar product (Fig. 11). In the case of MCC/10% guar, the interaction between guar and MCC acts to form “stronger” (less shear-sensitive) spherical particles (Fig. 12a), which have limited hydration and remain as intact particles after dispersion. In the case of MCC/15% guar, there is excess guar beyond what is needed to coat the MCC. With added shear, these products will break open to open up further the overall MCC/guar structure. This leads to a more spongelike (jagged) spherical shape (Fig. 12b) and gives rise to increased hydration and a higher dispersion viscosity. The effect of product composition on the dispersion profile (viscosity versus shear time) is represented in Fig. 13. The higher percentage of guar provides a higher viscosity, less resistance to shear, and a greater structural deformation, as shown. These types of structures may also be created the interaction of MCC with a glucomannan gum such as konjac. The range of properties in this case will be similar to those already described.

MCC/lO% Guar Spherlcal Aggregates Llmlted Hydratlon Intact form Heatlshear stable

Agglomerate of MCClGuar Aggregates +H,O

Mild Shear

Increased Shear

MCC/iSOAGuar Spherlcal Aggregates

Increased H,O absorptlon Spherlcal sponge-llke More vlscous

MCC/guar technology: the effect of the guar-to-MCC ratio.

Technology Microcrystalline Cellulose

179

”

(W

(a)

Figure

V.

Micrographs of two MCC/guar products.

APPLICATION OF COPROCESSED MCC/HYDROCOLLOID TECHNOLOGY TO FOOD SYSTEMS

As stated earlier, the physical properties of “cellulose gel” dispersions are quite different from the properties of gum solutions, starch gels, and other water-soluble materials commonly used in food technology. When MCC/ hydrocolloid grades are properly dispersed, the cellulose particulates and gum set up a network.It is the formation of this insolublecellulose structural network that provides the functionality. The formed gel possesses certain solidlike properties of elasticity, exhibiting relatively high yield stresses and atime-dependent type offlowbehavior. Thixotropic properties imparta variety of desirable characteristics suitable for oil-and-water emulsions or dispersion-type products.

A.

Ice Crystal Control and Texture Modifications

Body and texture are two of the most desirable characteristics that frozen dessert manufacturers try to impart to and maintainin their products. Effective levels of microcrystalline cellulose will produce frozen dessert dairy products with improved body and texture qualities, improved extrusion qualities, and good heat shock resistance. Heat shock, an industry term for the

/" 15% Guar

10

20

30 Time (mlns)

Dispersion profile curves for

40

l

MCC/guar products.

cyclical temperature conditions to which ice cream is exposed, causes partial thawing and refreezing of product. This slow, quiescent process favors development of large ice crystals. It is believed that the free water generated by the melting crystals is not reabsorbed immediately or completely by the other solids in the mix, probablydue to the alteration of the solubility properties of these materials. Additionally, when the temperature decreases, the unabsorbed free water refreezes, preferentially on the larger crystals, contributing to the progressive textural deterioration of the product through an increase in the size of the ice crystals. MCC, with its unique water absorption capability,will compensate for the inability or slowness of the other solids in the mix to reabsorb free water upon partial melting of ice crystals during heat shock. It is believed that particulate MCC, with its tremendous surface area and wicking action, has the unique ability to manage the free water produced in the frozen product MCC also helps to minimize the agglomeration during heat shock (Fig. of these dairy solids, ensuring product homogeneity. As little as MCC inan ice cream mix can presewe the original texture of frozen desserts through numerous freezehhaw cycles by maintaining the three-phase system of water/fat/air. In additionto conventional butterfat-basedice cream, MCC, when utilized in conjunction with stabilizers, supplements the body of and controls the texture of frozen dessert novelties, vegetable-fat-based products, as well as low-solids and/or artificially sweetenedformulations (see Appendix A).

The effect of MCC/CMC on ice crystal

in frozen desserts.

B. EmulsionStabilization Effective stabilization against the coalescence of oil globules in an emulsion system can be obtained utilizing the “cellulose gel” network. MCC functions as an emulsion stabilizer and thicker because of the strong affinity cellulose has for both oil and water. This results in some orientation of the solid particulates at the oil-in-water interface. In addition, the viscosity developed by the MCC/hydrocolloid products acts to thicken the water phase between the oil globules, preventing their close approach and subsequent coalescence. This combined functional effect canbeutilized to stabilize emulsions and dispersions in food products under adverse conditions of both processing and long-term shelf stability (see Appendix B).

C.

Foam Stability and Syneresis Control

In aerated food systems, foam stability depends primarily on the types of additives present andtheir ability to produce the necessary structural strength in preventing the coalescence and subsequence collapse of the air bubbles. MCC is not a whipping aid or film-forming material, but it does provide effective foam stabilization in a variety of whippedand/oraerated food systems. MCC dispersions act to thicken the water phase between air cells and provides added structural integrity to the protein film surrounding the air cells. The cellulose gel network created improves the body and texture, foam stiffness, and stability of both dairy and nondairy whipped topping

al.

products (see Appendix C). In addition, MCC is effectively used to stabilize mousse products, marshmallow toppings, and confectionery products and controls overrun in frozen desserts.

D. Fat Replacement The inherent rheological properties of cellulose gel dispersions (asmentioned earlier) allows the MCCfiydrocolloid products to be utilized in many reduced-fat or nonfat food applications The consistency of oil-in-water emulsions can vary from a thin fluid material at low oil levels to a thick viscous paste atveryhigh oil levels. The water-holdingcapacity of the cellulose gel network and the thixotropic nature of the gels allow the maof oil/fat to be removed while maintaining the required rheological and textural properties found in the full-fat counterpart of these same food products. There have been a number of reduced-fat or nonfat salad dressings and frozen desserts introduced into the food market over the past 7 years in which MCC products plays a key role in controlling texture, stability, and flavor release.

E. Suspenslon of Solids In cocoa or dairy beverages, many soluble hydrocolloids have been blended and coprocessed with the cocoa or other ingredients, but they do not successfully provide suspension functionality under conditions of minimum agitation. These hydrocolloids swell, rather than dissolve, with minimum agitation. With proper dispersion, the coprocessed MCCfiydrocolloids form their unique "cellulose gel" network. The network imparts the functional properties necessary to suspend solids effectively in the food systems without a significant increase in the viscosity of the product (see Appendix D).

Among the more recent developments in MCC technology is the entry of a new product based on the coprocessing of MCC with alginate, Avicelm AC, and a new MCC product for bakery applications, Indulge", plus the new applications of Novagel"' as a fat replacer.

AvicelQDAC is a new coprocessed product composed of MCC and of alginate [lo]. This product provides a three-dimensional MCC colloidal network similar to CMC products, but instead it makes use of the barrierdispersant properties of calcium alginate and sodium alginate. It is the second "all-natural" colloidal MCC product developed. The AviceP RCN-30 product using the banier-dispersant of maltodextrin and xanthan gum was the first such product offered.AvicelQpAC extends product functionality into new areas of usage for colloidal MCC. The product can be dispersed under high-shear conditions provided by a colloid mill or homogenizer in either milk or water. The product may also be dry-blended with other ingredients and dispersed under low-shear conditions of a Lightnin' Mixer.Inthese cases a sequestrant, such as tetrasodium pyrophosphate, may be needed to activate the colloidal network fully. This product offers a number of new benefits. The product may be used at a lower use level in a dry-mix food system, such as a hollandaise sauce, to providehigher a MCC colloidal content over the current MicroQuickQDproduct, a composite of MCC, CMC, and sweet dairy whey. The product offers low-pH stability without the need of an added protective colloid down to pH 3.2. It further provides ease of dispersion into a milkbased system, with the added benefit of providing milk-gelling properties. These two attributes now extend usage into cultured dairy products such as low-fat sour cream to provide added stability with improved texture and a creamy mouthfeel. This product also offers greater ice crystal control in frozen desserts at a use level lower than with previous RC/CL MCC products. Finally, the colloidal MCC network works to strengthen the alginate gel and provide heat stability to control boil-out in bakery fillings (see Appendix E).

The development of reduced-fat bakery andconfectionery applications provided new opportunities for MCC ingredients. In the case of low-moisture foods, one need was for an MCC product to be used as a functional bulk filler with reducedwater and oil absorptivity. The Indulge" LM-310 product is one such ingredient. It is MCC but processedto modify the physical properties to provide a more dense MCC with a reduced surface area. This product allows for a substantial reduction of fat and calories in confectionery fillings while providing a short, creamy texture to the filling. This MCC product has also been used in the cookie base cake to produce a lowercalorie/lower-fat cookie with a good, crisp/crunchy texture (see Appendix

et al.

1 WaterAbsorption

VariousFiber

Sources source Fiber Oat fiber Sugar beet 590 Solka Corn bran LM-3

% water absomtivitv

310

F). Because the LM-310is less absorptive than often-used fiber sources, such as oat fiber, corn bran, or a cellulose floc (see Table l), the cookie dough is less sticky, has good machinability,uses less water in the mix, and reduces the bake time. C. Novagel'" as a Fat Mimetic In Cheese The use of the Novagel" products as fat replacers has been discussed earlier. There have been several applications developed from this technology. The first is in low-fat and nonfat natural and process cheese [11,12]. In full-fat cheese, the protein matrix that forms the structure is most often modified by the inclusion of fat globules. As the fat level is reduced, the structure becomes very firm and cohesive; this results in an undesirable "rubbery" texture. The addition of the Novagel" particulates with added water to the formula of a reduced-fat cheese will interrupt the protein structure. This in turn will modify and soften the texture to restore a creamy mouthfeel. An additional benefit is an improved appearance; the cheese becomes more opaque, with a richer overall color. Finally, for process cheese there is an improvement in processing, especially in sliceability and handling, without the usual stickiness found in these higher-moisture process cheese products (see Appendix G). In natural cheese the effect is quite similar. Using scanning electron microscopy, it can be observed thatthe casein microstructurein a reduced-fat cheddar cheese would be a very dense, continuous matrix. With the added Novagel" particulates, the matrix resembles the porous cellular structure of the full-fat control. The texture differences have been measured by instrumentation, such as a Kramer Shear Press, and the eating quality is much improved. The application has' now been extended to other natural cheese products with the introduction of two new products: Novagel"" NC-200, a

Technology Cellulose Microcrystalline

185

composite of MCC/guar with carrageenan, and Novageln NC-250, a composite MCC/guar with alginate.

D. Novagel'" as a Fat Mimetlc ln Tablespreads Research has demonstrated the ability to influence and modify the texture of low-fat table spreads. Table spreads are water-in-oil emulsions whose structure and texture is determined by a certain amount of solid fat crystals within the fat phase. In reduced-fat or low-fat spreads, the oil continuous phase is still a major factor. Here the arrangement of fat crystals determines the distribution of melt-point types and provides some of the form and texture of the finished spread. However, as the percentage of the fat phase is lowered and the percentage of the aqueous phase is increased, two considerations take on more importance. The first is the formation and stability of the emulsion, which requires the help of a surface active agent, known as a surfactant or an emulsifier;the second is the structure of the aqueous phase. In the aqueous phase, a combination of several components can be used to provide structure. Keeping in mind the importance of the lipid phase, it is possible to structure better the aqueous phase of a table spread by the use of two different AviceP cellulose gel products [13,14]. A colloidal MCC/CMC ingredient, RC-591F, is used to provide structure and stability to the aqueous phase by the development of a weak, soft, cuttable gel. This gel is thixotropic in nature. This behavior allows the aquephase to be fluid under shear and permits formation of a fine water-inoil emulsion. Once formed, however, the droplets and domains of the aqueous phase are then stabilized by a gel reinforced with microcrystals of MCC, which appear to orient at the water/oil interface. In addition to the AviceP, there is Novagel" RCN-15 cellulose gel. This composite of MCC and guar behaves as spherical, globule arrangements of flocculated microparticles. These microparticles act to provide a fatlike mouthfeel whichis especially important on melting and spreading. The result is to enhance the stability, the mouthfeel, the melt properties, and thespreadability of the finished product (see Appendix H).

E. Novagel" as a Fat Mimetic In Bakery/Confectionery Coatings Another application of Novagel" as a fat replacer is in bakery and confectionery coatings and icings [15]. To replace fat in low-fat chocolate coatings and icings, the dispersion of sugar, cocoa, and/or milksolids in a continuous fat phase now becomes a dispersion of sugar, cocoa, and Novagel" particulates in a continuous aqueous phase of sugar syrup. Novagel" BC-300,

et al.

developed for these applications, provides several functional benefits. The texture is improved to mimic the rheology of a chocolate coating, which provides an appealing mouthfeel and flavor release. In processing, the coating may be used for enrobing, dipping, or extruding. It may also be cooled, stored, and remeltedfor use later. The set time of such a coatingis decreased and, importantly, such a formulation provides for control of sugar bloom (see Appendix I).

Microcrystalline cellulose (cellulose gel) is produced from purified alpha cellulose and coprocessed with various hydrocolloids, which act as barrierdispersants that contribute to the unique textural and functional properties in aqueous food systems. To develop the functional properties of cellulose gel,thehydrolyzed cellulose fibers are broken into small fragments and subjected to high shear during processing to liberate the microcrystals and crystallite aggregates. Barrier-dispersants enhance the functional properties of the colloidal particles by preventing themfrom reaggregating during processing and dispersing them during hydration to form a three-dimensional gel network. The three-dimensional gel network set up by the dispersed particles of cellulose products coprocessed with sodium carboxymethylcellulose (NaCMC), sodium alginate, maltodextrin, and/or xanthan gum can greatly contribute to the functional stability and textural properties in various food systems. The gel formed from these functional stabilizers possesses certain properties of elasticity that exhibits relatively high yield stress. Temperature has little effect on the functionality and viscosity of a colloidal dispersion. It has provided functional stability in food systems processed through UHT, aseptic, retort, and microwave.Cellulose gel coprocessed with various soluble polymers acts to simulate the mouth feel and rheological properties of fat in food systems. Guar gum physically interacts with dispersed crystalline cellulose during processing andonhydration to form spherical flocculates of cellulose gel and guar. These hydrated aggregates behave like a continuous fluid, creating the smoothness most closely associated with fat dispersed in water.

APPENDIX

ICE CREAM PRODUCTS

Benefits of AviceP Cellulose Gel and Novagel" Cellulose Gel Impart smooth texture and creamy mouthfeel Provide heat shock stability by controlling ice crystal growth Improve extrudability and meltdown properties

Full-Fat and Low-Fat Ice Creams

Full-fat Ingredient Butterfat MSNF Sugar Corn syrup solids Avicel" RC-581 Aqualonm CMC Carrageenan Emulsifier Total solids Serum solids Total fat

Low-fat ice cream

10.00% 10.00 12.00 6.00 0.40 0.10 0.02 0.25 38.77% 10.00 10.00

ice cream

4.00% 13.00 11.00 5.00 0.40 0.10 0.02 0.30 33.82% 13.00 4.00

1. Assemble all liquid ingredients in a vat. Start heat and agitation. 2. Dry-blend powder ingredients. Add slowly tothe liquid ingredients under good agitation. Mix for 30 minutes to allow for dispersion and hydration of the powder ingredients. 3. Pasteurize mix. 4. Homogenize at 2000 psi (first stage) and 500 psi (second stage). Cool mix rapidly to 40°F Age mix overnight (if desired). Freeze to appropriate draw temperature, pack into containers, and place into hardening room.

et al.

European Ice Cream Using AviceP AC-815

Ingredient Skim Heavy cream Avicel" AC-815 Myvatex mighty MSNF Sugar Corn SYNP solids Procol@ G2 guar Dangelm 15 Total solids Serum solids Butterfat

45.83% 31.5 0.4 0.2 4.67 12 5.25 0.13 0.02 34.99% 11.12 5.9

Dry-Mix Soft-Serve Ice Cream

Ingredient Whole milk powder (28% fat) MSNF Sugar syrup Corn solids (36DE) (1ODE) Maltodextrin AviceF CL611 cellulose gel Aqualona CMC Lactarin" MV 306, Carrageenan Water Total solids Serum solids Butterfat

12.42% 1.89 12.00 1.00 0.10 0.02 67.07 32.93% 10.83 3.48

1. Assemble all liquid ingredients in a vat. Start heat and agitation. 2. Dry-blend powder ingredients. Addslowly to the liquid ingredients under good agitation. Mix for 30 minutes to allow for dispersion and hydration of the powder ingredients.

echnology Cellulose Microcrystalline

189

Pasteurize mix. Homogenize at 2000 psi (first stage) and 500 psi (second stage). 5. Cool mix rapidly to (5°C). Age mix overnight (if desired). Freeze to appropriate draw temperature, pack into containers, and place into hardening room.

APPENDIX B: "LITE" RANCH SALAD DRESSING Benefits of AvIceP Cellulose Gel and Novagel'" Cellulose Gel Impart short texture and creamy mouthfeel Contribute the body and mouthfeel of high-fat emulsions Add rheological properties of fat by structuring water Add opacity and improve cling AviceP cellulose gel forms a colloidal dispersion in water that can be used to replace some of the oil in reduced-fat salad dressings. Avicel cellulose gel produces a short, creamy-textured reduced-fatsalad dressing with good opacity and cling. Novagel" cellulose gel is recommended for additional rich, creamy body and mouth coating. Both formulations represent a 70% reduction in fat.

Formulation

Ingredient Water Soybean oil Vinegar, white distilled 120-grain Sugar Cultured buttemilk powder" Novagel" RCN-15 cellulose gelb Salt AvicePJ CL611 cellulose gelb Powdered egg yolks" Natural buttermilk flavord Keltrol" xanthan gum" MSG Garlic powder Onion powder Potassium sorbate

Avicel" CL611

Avicel" CL-611 and Novagel" RCN-15

71.80% 10.00 5.00

69.30% 10.00 5.00

3.50

3.50 2.50 2.00 1.50 0.50

-

2.00 1.50 0.50

0.30 0.18 0.18 0.10

.

0.30 0.18 0.18 0.10 Table continues

Bullga et al.

Ingredient

Avicela CL611

Mustard flouf Ground black pepper Parsley Calcium disodium EDTA Ribotide

0.02 0.01 0.01

Avicela CL611 and Novagel" RCN-15

0.02 0.01 0.01 100.00%

'Buttermilk powder m1741 Corp., Food Ingredients Div. Wenningsen" Q p e Y-l dNatural buttermilk flavor Tastemakef 'Kelco, a Unit Monsanto 'French's, Durkee French" %C

Procedure: ColloidMill 1. Disperse Avicel@CL611 cellulose gel in the water for 5 minutes using a Silverson@Mixer or LightninQP Mixer (or equivalent).Using polarized light under lOOx magnification, check dispersion microscopically to determine if fully dispersed. 2. Disperse Novagel" RCN-15 cellulose gel (when added) into the Avicel mixture for 5 minutes using a Silverson Mixer or Lightnin' Mixer (or equivalent). Again, check microscopically using polarized light to determine if fully activated into the spherical form. Add dry blend of xanthan gum and some of the sugar (1:lO ratio) to the Aviceldispersion.Mix for minutes using a Silverson Mixer or Lightnin' Mixer (or equivalent). Transfer this mixture to a Dixie Mixer (or equivalent). Slowly add remaining sugar, buttermilk powder, calcium disodium EDTA, ribotide, potassium sorbate, and egg yolk powder. Mix minutes. Add soybean oil and buttermilk flavor. Mix 5 minutes. 6. Add vinegar, MSG, mustard, garlic, onion, black pepper and parsley. Mix 5 minutes. 7. Mill with Fryma@colloid mill (or equivalent) at mm. 8. Deaerate and fill into containers.

Nutrition Information [Serving Size = 2 T (30 g)] “Lite” ranch dressing Calories Calories from fat Total fat Cholesterol 1Carbohydrate g Sodium

mg

Full-fat ranch dressing 140 126 14 7 mg

4g g mg 4g 140 mg

APPENDIX C: WHIPPED TOPPINGS Benefits of Gelcarin@ Carrageenan, and Avicei@ Cellulose Gel Controls syneresis Stabilizes the structural integrity of the foam Provides a smooth, creamy texture

Comments Gelcarin@GP carrageenan provides body, controls syneresis, and eliminates separation of the fat phase. The iota carrageenan maintains overrun by increasing the product’sviscosityand forming a lattice network with casein micelles.The lattice network stabilizes the fat, air, and protein matrix, resulting in a stabilized whipped cream that will retain stiffness and body. The AviceP CL611 cellulose gel promotes foam stability and stand-up and stabilizes the topping under adverse storage conditions.

Formulation Ingredient Water Milk fat

Hydrogenated vegetable fat Sucrose Corn syrup Nonfat milk solids Avicel” CL611 cellulose gel”

60.00%

-

-

24.00

6.00

5.00

5.00

-

0.40-0.60

0.40-0.60 Table continues

et al.

Ingredient GP Gelcarin@ 359 carrageenan" Sodium "heen@ 60b MyveroP 18-35'

0.30

0.01-0.05 2.50 0.30

0.15

0.12

-

TMC Food Ingredients Div. ween" ICI Americas 'Myverol" 18 emulsifier, Quest (Eashan)

Procedure 1. Disperse the AviceP CL611 cellulose gel in the water with rapid agitation. 2. Dry-blend the Gelcarin@GP 359 carrageenan with sucrose and disperse into the water slurry with continuous agitation. Add milk powder and/or sodium caseinate to the slurry. 3. then add the corn syrup solids. Heat the system to 63°C (145°F); 4. 5. Separately heat the hydrogenated vegetable fat and emulsifiersto 60°C (140°F). 6. Add the melted fat andemulsifier to the aqueousphasewith constant agitation. 7. If dairy based, add the fresh cream to the mixing vat at this time and heat. for 30 minutes. 8. Pasteurize at 71"-77°C (160"-170"F) 9. Homogenize at 2000 psi first stage and 500 psi second stage. 10. Cool mix to 2"-5°C (35O-40"F) and age for 24 hours. 11. Whip in planetary mixer with wire whip at speed #3 for minutes.

APPENDIX D: CALCIUM-FORTIFIED MILK SYSTEMS Benefits of AviceP Cellulose Gel Suspends calcium salts better than commercial products with carrageenadguar gum. Imparts creaminess to low-fat/skim milks, giving richer mouth feel. Combinations of AviceP cellulose gel and SeaKemm carrageenan give improved results, both in terms of salt suspension and organoleptic properties, over use of either product alone.

Microcrystalline Cellulose Technology

Formulation Low-fat milk fortified with: tricalcium phosphate (formulation A) calcium carbonate (formulation B) Skim milk fortified with: tricalcium phosphate (formulation C) calcium carbonate (formulation D)

Technical Discussion AvicelQPcellulose gel, SeaKemQPcarrageenan, and combinations of the two were evaluated in the laboratory for use in suspension of calcium salts in low-fat and skim milk systems. The results show that Avicel" RC-581 cellulose gel, in conjunction withcarrageenan, is useful in producing and maintaining stable calcium suspensions in milk systems.Inaddition, this combination modifies the rheological (mouthfeeVtexture) properties of lowfat milk to resemble closely those of a full-fat (3.5-4%) milk product. Details of the results follow.

Single-Component Stabilizer in Skim Milk The initial experimental work evaluated performance of several stabilizers individually. Results withAvicelQPRC-581 cellulose gel alone, SeaKem" CM 614 carrageenan alone, and SeaKeme CM 611 carrageenan alone indicated that, at levels needed to obtain good suspension of tricalcium phosphate (TTCP), gelation occurred after a short time in storage (overnight, 40°F). Morespecifically,when SeaKeme CM 611 carrageenan was used alone at 0.015%, the consistency of the milk was acceptable (viscosity = 125 buta trace sediment formed uponstorage. At the rate necessary to maintain a stable TCP suspension, a rigid gel structure formed during overnight storage.

Two-Component Stabilizers in Skim Milk With a combination of stabilizers, it was possible to overcome the stability problem and achieve excellent suspension of calcium salts. AviceP RC-581 cellulose gel at 0.3% and SeaKemQP CM 611 carrageenan at 0.015% gave the best results in terms of good stability with improved texture and creamier mouthfeel. The effective rates were similar in trials run with both TCP and calcium carbonate (CaCO,).

hosphate te

Bullga et al.

Two-Component Stabilizers in Low-Fat (1%) Milk In the case of low-fat milk, SeaKemQD CM614 carrageenan was preferred over SeaKemQD CM611 carrageenan because it exhibited less of a tendency to develop excessive viscosity buildup and gelation on cooling and storage (40°F). AvicelQDRC-581 cellulose gel at 0.3% and SeaKemQD CM 614 carrageenan at 0.01% gave the best results, both withTCP and with CaCO,. This stabilizercombinationalsomodifiestherheological(mouthfeel/texture) properties of low-fat milk to resemble closely those of a full-fat (3.5-4%) milk product. Moreover, this system gave more stable calciumsuspensionthana well-known commercial calciumPCP)-fortified milk stabilized with a combination of carrageenan and guar gum.

Formulation Low-fat milk fortifiedto provide 50%U.S. RDA for calcium per8-02. glass.

Ingredient Low-fat m w 1% Tricalcium Calcium Avicelm RC-581 cellulose gelb SeaKemm CM 614 carrageenanb

(%

A weight) 99.537% 0.153

0.300 0.010 100.000%

B (% of weight)

99.537%

-

0.153 0.300 0.010 100.000%

‘Low-fat protein-fortified milk contains approximately 352 my! calcium per Iglass (“Nutritive Values of the Edible Part of Foods,” The-& w e of Nutrition by M. k l i n , 2nd f :d.1 bFMC Corp., Food Ingredients Division.

Procedure 1. Dry-blendcalciumsalt, , rricelQDRC-581 cellulose gc:l, and carrageenan. Add slowly to milk with good agitation. minutes. 2. Pasteurize mix at 160°F for 30 minutes. 3. Homogenize at 2000 psi (1st stage) and 500 psi (2nd stage). 4. Cool mix to 40% 5. Store inrefrigeratorat 40°F.

Mlcrocrystalllne Cellulose Technology

Formulation Skim milk fortified to provide 50% U.S. RDA for calcium per 8-oz. glass.

C Ingredient

(% of weight)

D (% of weight)

-

0.210

Skim milk’

Tricalcium phosphate Calcium carbonate Avicelw RC-581 cellulose gelb SeaKemmCM carrageenanb

-.

0.300

‘Skim milk contains approximately296 mgcalcium per 8-02.glass. (“Nutritive Values Part Foods,” The Science ofNufn’tion,2nd ed.) hFMC Corporation, Food Ingredients Division.

the Edible

Procedure 1. Dry-blend calcium salt, AviceP RC-581 cellulose gel, and carrageenan. Add slowly to milk with good agitation. Mix 5 minutes. 2. Pasteurize mix at 160°F for minutes. Homogenize at 2000 psi (1st stage) and 500 psi (2nd stage). Cool mix to 40°F 5. Store in refrigerator at

APPENDIX E: PINEAPPLE BAKERY FILLING Benefits of Aviceim AC-815 Cellulose Gel Heat stability, no “boil-out” Imparts opacity and pulpy appearance without addition of fruit solids Easy to use, one-kettle processing

Comments AviceP AC-815 cellulose gel imparts heat stability, opacity, and pulplike appearance to pineapple bakery filling. The filling can’be deposited on the pastry and then baked to produce an appealing pastry-with no boil-out. The formulation is high in water activity and is designed for short-shelf-life pastries.

et al.

Formulation Ingredient

weight

Corn syrup, 63 DE Water Sucrose Water Starch" Citric acid solution Avicel" AC-815cellulose gelb Pectin" Flavord Sodium citrate Sodium benzoate Color (FDBrCyellow

% by

.oo 0.75 0.08 0.05 suit)

'National Flo@ 67 starch,NationalStarch & Chemical Company %C Corporation, Food Ingredients Division 'BB Rapid-set pectin, Hercules Inc. dArtificial pineapple flavor, #503.093C, Firmenich Inc.

Procedure 1. Dry-blend AviceP AC-815 cellulose gel with about 10% of crose; add to water and at highest speed for 3 minutes in Groen kettle. 2. Add sodium citrate and mix at the highest speed for 10 minutes. Blend pectin with about 10% sucrose, add to Groen kettle, and mix for minutes. Add remaining sucrose,color,andstarchslurry,andmix minutes. 5. Heat to 180°F while stirring constantly. Hold at 180"-190°F for 10 minutes. 7. Add corn syrup, return temperature at 185"F, add water to adjust to 63% solids. 8. Add citric acid, sodium benzoate, and flavor and deposit at not less than 180°F pectin does not gel prematurely.

APPENDIX F SANDWICH CREME COOKIE Benefits of Indulge" Cellulose Gel Allows substantial fat and calorie reduction Provides a short, creamy texture to the filling throughout the shelf life of the cookie Filling can be readily pumped and deposited onto base cake Produces soft, nonsticky cookie dough to facilitate efficient processing Reduces baking time of the cookie due to less water in dough

Comments Rotary-molded creme-filled sandwich cookies with 50% less fat and 25% fewer calories can be prepared using a combination of Indulge" cellulose gel and Raftilosea oligofructose. Indulge" LM-310is a specially processed cellulose gel designed to function in low-moisture systems. It allows these reduced-fatheduced-caloriecookies to be formulated with standard leavening systems and baking ingredients and to be processed on standard equipment. The resulting cookie is similar in mouth feel and texture to its fullfat counterpart. A , = (filling)

Formulation of Base Cake Ingredient

weight

Flour Sucrose 6X IndulgeN LM-310cellulose gel" Raftilosea (oligofructose)b Water Shortening High-fructose corn syrup" Cocoa powder (10-12% fat) Ammonium bicarbonate Sodium bicarbonated Salt Datem emulsifier" Sodium stearoyl lactylate Lecithin'

% by

38.32% 16.42 10.28 7.83 6.88 6.86 6.21 4.89 0.49 0.49 0.37 0.32 0.32 0.32

lorn% %*MC Corp., Food Ingredients Div. bRaftilose@, Rhone Poulenc ?sosweet@ 100, A.E. Staley Manufacturing Co. dFMC Corp., Alkali Chemicals Div. 'Datem 1118 emulsifier, Quest International %entrolex@F, Central Soya

al.

Procedure 1. Scale shortening and emulsifiers (datem, SSL, and lecithin) and put into a Hobart@ mixing bowl. Cream well at medium speed for minutes. 2. Add Indulge" LM-310 cellulose gel to the creamed mixture and continue mixing at #l speed for 2-3 minutes. 3. Add Raftilosem and sugar and mix for 2 minutes at #l speed. 4. Incorporate cocoa powder mix at #l speed for 1 minute. Mix in high-fructose corn syrup. Dissolve ammonium bicarbonate in water and then add to above and mix well. Add the remaining water (part of this was used for ammonium bicarbonate solution) and mix at #l speed for 3-4 minutes. Add flour, salt, and sodiumbicarbonate and mix for 3-4 minutes at #l speed. Make cookies by using rotary molder. 10. Bake at 400°F for minutes (final moisture 3.5-4%).

Formulation for Cookie Filling Ingredient

Sucrose 1Ox syrupa High-fructose corn Glycerin emulsifiedb Shortening, d Litesse" Indulge'" LM-310 cellulose gel' Litessem solutiond Vanilla flavor

% by weight 61.9%

15.0

3.0 3.0 0.1

SsosweeP 100, Staley Manufacturing bBetricing,Van Den Bergh Foods 'FMC Corp., Food Ingredients Div. dPfizer, Inc.

Procedure 1. Scale high-fructose corn syrup, glycerin, and Litesse@ (70%) and vanilla flavor into Hobart@ mixing bowl. Mix at#l speed for 23 minutes using mixing paddle.

echnology Cellulose Microcrystalline

2. Add Litesse powder and Indulge" LM-310 cellulose gel. Mix well at #l speed for 1-2 minutes. 3. Add powdered sugar and mix for 2-3 minutes at #l speed. 4. Incorporate emulsified shortening and mix to a smooth consistency (#l speed for 3-4 minutes). 5. Creme is ready for use.

Nutritional Information for Sandwich Cookie Reduced-fat and reduced-calorie cookie

Full-fat and full-calorie cookie

21% fat

7% fat 3.5 cal/g reduction] caloric [28% 0.07 g fatlg reduction] fat

4.8 CaVg g faVg

J Cake

Base 8% fat 3.45 cavg 0.08 g fatlg

\ Filling 5% fat 3.78 CaVg g fatlg

APPENDIX G:NONFAT PROCESS CHEESE Novagel" RCN-15 cellulose gel and Marine Colloids" carrageenan can be used in nonfat process cheese to impart body and textural characteristics more typical of full-fat process cheese. The following formulation can be used to produce process cheese loaves or slices by working within the recommended ingredient ranges.

Benefits of Using Novagel" RCN-15 Cellulose Gel and Marine Colloids'" Carrageenan Reduces stickiness to touch and tastebite Provides creamy mouth feel without gumminess Provides smooth consistency Reduces rubbery texture Improves slicing properties Adds opacity

et al.

Formulation Ingredient

% by weight

Standard skim milk cheese Nonfat dry milk solids" Sweet dairy whey powderb Buttermilk solidsb Lactic acid (10% solution) Sodium chloride Disodium phosphate dihydrate' Novagel" RCN-15 cellulose geld Sodium citrate Gelcarin@GP 911 carrageenand SeaKem@ GP418 carrageenand Sorbic acid Apo carotenal solution #73' Annatto extract' Water

54.0-56.0% 3.80 3.80 3.00 3.00 2.10 1.40 1.00 0.80 0.60 0.30 0.10 0.02 0.02 sufficient achieve approx. 58% in finished product

'Maryland and Virginia Milk Producers b n d O'Lakes, Inc. 'FMC Corp., Food Phosphate Marketing dFMC Corp., Food Ingredients Div. Xoffmann-LaRoche, Inc. 'Haarmann & Reimer Corp.

Inc.

Procedure 1. Prepare a blend of all dry ingredients to be added to the product. 2. Grind skim milk cheese to fine particles. Add cheese to cooker and begin heating. Add water and then add the dry blend, lactic acid, flavor, and color to the mixture. 5. Heat to approximately 165°F for about minutes or to set standard in customer operation.

Notes a. Adjust lactic acid level to desired pH range of finished product. Approximately 5.7 pH. b. A product for shredding may require firmer structure. If so, either increase the level of Gelcarin@GP 911 carrageenan or decrease the moisture level in the finished product.

Microcrystalline Cellulose Technology

Stabilize water-in-oil emulsion at low fat level Provide fatlike rheologyandspreadability characteristic offull-fat tablespreads Impart full-fat body and mouthfeel

Ingredient Fat Phase Soybean oil 16 10.4 20.00% Hydrg. oil 9.312 11.64 Palm 7.90 6.32 Dimodan ok 0.35 0.28 Triodan R90 0 0.00 Butter flavor 0.04 0.05 Beta-carotene 30% 0.00163 0.00 Antioxidant 0.06 0.0464 22.4 Total 32.0 Aqueous Phase Water 46.736 Maltodxtrn lODe 0 Salt 0.4 A v i d @RC-591 0.64 Novagel" RCN-15 0 Xanthan gum 0.064 K sorbate 0.20 0.16 Total 60 48

6.408 5.04 0.28 0.2 0.04 0.00163 0.0304

13.00% 8.01 6.30 0.35 0.25 0.05 0.00 0.04

9.208 11.51% 7.15 5.72 4.52 5.65 0.28 0.35 0.2 0.25 0.05 0.04 0.00163 0.00 0.04 0.0304 20.0

58.42% 49.88 62.35% 51.528 0.00 4 5.00 4 0.50 1.44 1.44 1.80 0.80 1.2 1.52 1.50 0.00 0.8 1.21.00 0.12 0.08 0.152 0.15 0.10.20 6 0.16 0.20 57.6

64.41% 5.00 1.80 1.90 1.50 0.19

" "

Grand total

80.0

100.00% 80.0

100.00% 80.0

100.00%

Ingredients: RBD soybean Karlshamms Capital Soya Part. hydrog soy, Karlshamns Akoleno "S"; Van den Bergh Cirol IC Palm oil, Fuji No. 2152 Dimodan O K distlld. mono-, diglycerides hydg. soy; Triodan R 9 0 polyglycerol poyricinoleate, Danisco Maltodextrin, lODE grain processing maltrin 100 Salt, fine flake, Cargill Avicel" RC-591, Novagel"' RCN-15, FMC Xanthan gum,Ketrol" F or RD, Kelco Potassium sorbate, Monitor K, pwd.; Tenox R antioxidant, Eastman" Beta-carotene, code #65646, Hoffman La Roche Nat. and art. butter flavor Danisco

et al.

Procedure Portion 1. Disperse Avicela RC-591 in available water. 2. For the 25% (and 28%) fat formula, add the Novagel" RCN-15 and mix in a high-shear mixer. 3. Add xanthan gum andallow for complete incorporation (5 minutes). 4. Incorporate the remaining aqueous portioningredientsandmix thoroughly minutes). 5. Heat aqueous portion to 45"-50°C(113"-122"F).

Lipid Portion 1. Heat the combined fats to 60°C (140°F) and hold at this temperature for 15 minutes. 2. In a portion of the heated fats, melt the emulsifiers (8OoC/176"F); add back to the main portion of the fats. Add fat-soluble flavor and color. 3. Cool fat phase to 45"-50"C(113-122°F).

Emulsification and Crystallization 1. Add the aqueous portion to the lipid portion very slowly under controlled mixing to form water-in-oil emulsion. Maintain minimum temperature of 40"C/104°F. 2. Pass through scraped surface chilling unit with an exit temperature of -15"C/59"F.

APPENDIX I: REDUCED-FAT AND FAT-FREE COATINGS FOR GRANOLA BARS Benefits of Novagel Cellulose Gel Allows enrobing with 0-3% fat coatings Facilitates rapid set of the coatings for greater throughput Good flavor release

Comments Fat levels can be reduced from 35% to 3% fat to prepare reduced-fat yogurt coatings and chocolate-flavored coatings with Novagel" BC-300 cellulose gel. Fat-free chocolate-flavored coatings can also be prepared, without sac-

echnology Cellulose Microcrystalline

se" b yogurt

203

rificing texture or mouthfeel. The coatings should be applied after the granola bars have been prepared. It is also important to try and balance the water activity between the coating and the medium coated, in order to minimize water migration. of granola bar = 0.52; of coating =

Formulation for3% Fat Yogurt-Coating ~~~

Ingredients Powdered sugar-lox Water Glycerin Crystalline Nonfat Partially hydrogenated soybean oil' Novagel" BC-300 cellulose Salt Polysorbate 60 Titanium dioxide Vanilla

weight

geld

~~

% by

68.60% 13.00 8.00 1.00 4.00 2.50 2.00 0.40 0.20 0.20 0.10 100.00%

'A.E. Staley Manufacturing %eatreme 3315, Kerry Food Ingredients 'Shurset K, Anderson ClaytodHumko Products d F h K Corp., Food Ingredients Div. 'N and A vanilla flavor Tastemaker Inc.

Procedure 1. Disperse Novagel" cellulose gel into room-temperaturewater with high-shear mixer, i.e., Silverson@,until a sour cream-like consistency is achieved. 2. Using a propeller mixer on high speed (140 rpm), rapidly incorporate Novagelm cellulose gel into glycerin to produce a smooth mixture; heat to 140% 3. Melt soybean oil and polysorbate together and add to beaker. 4. Add fructose to the dispersion; let it mix for 5 minutes. 5. Blend sugar, salt; add rapidly, and let it mix for minutes, covered, at 140°F. Blend yogurt powder and flavor; then add to mixture. 7. Enrobe at 140°F.

al.

Formulation for3% Fat and Fat-Free Chocolate-Flavored Coating

Ingredient 68.4% Powdered sugar- lox 14.2 Water Glycerin Chocolate liquor Cocoa, defatted Crystalline2.5 fructose Novagel" 2.0 BC-300 cellulose gel" Salt 0.4 100.0% 'FMC

3% fat

Fat-free

(% by weight)

(% by weight)

67.1% 14.0 8.0 6.0

8.5

-

2.5 2.0 0.4 100.0%

Corp., Fwd Ingredients Div.

Procedure 1. Disperse Novagel"BC-300 cellulose gel into room-temperature water with high-shear mixer, i.e., Silverson@,until a sour creamlike consistency is achieved. 2. Using alow-shearpropellermixeronhigh (1300 rpm),rapidly incorporate Novagel" dispersion in to glycerin, the final mixture should be smooth. Heat the mixture to 140°F. 3. Add fructose to the dispersion; let is mix for minutes. 4.Blend sugar andsalt;addrapidly. Let it mix for 30 minutesat 140°F. Keep covered to reduce moisture loss. 5. Add melted chocolate liquor; for fat-free formula, add cocoa powder. Mix for 10 minutes; keep covered. Enrobe or dipat 140°F.

REFERENCES 1. Thomas, W. R. Microcrystalline cellulose. In:Glicksman, M.,ed., Food Hydrocolloids, Vol. 111, CRC Press, Boca Raton, Fla., pp. 9-42 (1982). 2. McGinley, E. J., Thomas, W. R., Champion, S. A., Phillips,G. O., and Williams, P. The Use of Microcrystalline Cellulose inOil and Water Emulsions, Gums and Stabilizers for the Food Industry, Vol. 2, pp. 241-249 (1984). McGinley, E. J., andTuason, Jr., D. C. Application of Microcrystalline Cellulose, Gums and Stabilizers the Food Industry,Vol. 5, pp. 405-414 (1989)

4. Ward, K. Occurrence of cellulose. In: Cellulose and Cellulose Derivatives, Part I, Vol. 5, pp. 9-27. 5. Ott, E., and Tennent, H. G. Introduction. In: Cellulose and Cellulose Derivatives, Part I, Vol. 5, pp. 1-8 (1954). E. Dispersing and Stabilizing 6. Durand, H.W., Fleck, E. G.,andRaynor,G. Agent Comprising beta-1,4 Glucan and CMC and Method for Its Preparation. U.S. Patent 3,539,365 (1970). 7. Harrop, R., Phillips, G. O., Robb, I. D., and Williams, P. A. Dispersion stability of microcrystalline cellulose by polyelectrolytes. Prog. Fd. Nutr. Sci. 6:331340. 8. Tuason, D. C., McGinley, E. J., and Selinger,E. The development of Novagel" RCN. Food Technology International Europe. London, Sterling Publications (1995). 9. McGinley, E. J., and Tuason, Jr., D. C. Fat-like Bulking Agent for Aqueous Foods Comprising Microcrystalline Cellulose and a Galactomannan Gum.U.S. Patent 5,192,569 (1993). and McGinley, E. J. Colloidal Microcrystalline Cellulose 10. Tuason, Jr.,D.C., and Barrier Dispersant Composition and Method for Manufacturing. U.S. Patent 5,366,742 (1994). 11. Bullens, C. W., Krawczyk, G. R., and Geithman, L. M. Development of Reduced Fat Cheese Products Using Carrageenan and Microcrystalline Cellulose. Food Technology @(l): 79-81 (1993). 12. Krawczyk, G . R. Application of Avicel Cellulose Gel in Lowfat Pasteurized Process Cheese. Presented at 4th IBC Conference on Fat and Cholesterol Reduced Foods (1993). 13. Krawczyk, G. R., Buliga, G. S., Bertrand, D. T., and Humphreys,W. M. Lowfat Spread Technology, Inform 7(6) (June 1996). 14. Humphreys, W. M. Low Fat Spread and Process for Making Same. U.S. Patent 5,338,562 (1994). 15. Izzo, M., Stahl, C., and Tuazon, M. Using Cellulose Gel and Carrageenan to Lower Fat and Calories in Confections. Food Technology 45-49.

This Page Intentionally Left Blank

Cyclodextrins Z. Helena Qi and Matthew L. Romberger Cerestar USA, Inc. Hammoncl, Indiana

Cyclodextrins comprise a series of cyclic oligosaccharides produced from starch by the action of cyclodextrin glucosyltransferase (CGTase). Cyclodextrins were first discovered byWliers in 1891 [l]as degradation products of potato starch. The foundations of cyclodextrin chemistry were laid down, however,between 1903 and 1911 bySchardinger, during which time he elucidated the definitive structure of cyclodextrins [2-41. For this reason cyclodextrins were referred to as Schardinger dextrins in some of the earlier literature. They are also known as cycloamyloses or cycloglucans. Cyclodextrins are composed of D-glucopyranose residues bonded by a-(1-4) linkages. They are each designated by a Greek letter to denote the number of glucopyranose units. Three well-characterized and commercially available members of this family are a-, and y-cyclodextrins, made up of 6, 7, and 8 glucopyranose residues, respectively. Purification and characterization of a-, t-, q-, &cyclodextrins, with '9, 10, 11, 12, and 13 glucopyranose units, respectively, have been accomplished more recently [5-71. Cyclodextrins with fewer than 6 glucopyranose units are not known to exist naturally. Within a cyclodextrin molecule, each D-glucopyranose residue assumes the C l (chair) conformation. The molecular geometry of a cyclodextrin can be described as a truncated cone or as a doughnut with a hydrophilic outer surface and hydrophobic inner cavity. All secondary hydroxyl groups (C2OH and C3-OH) are situated on the wider end of the cavity, whereas all

primary hydroxyls (C6-OH) are situated on the narrower end. The unique structure enables cyclodextrins to form molecular inclusion complexes with various organic and inorganic “guest” compounds [8].

II.

PHYSICOCHEMICALPROPERTIESOF CYCLODEXTRINS

A.PhysicalProperties Listed in Table 1 are several important dimensional and physical parameters of four cyclodextrins. Cyclodextrins are fairly water soluble. However, cyclodextrin exhibits a remarkably lower solubility than does a- or y-cyclodextrin. This unusual phenomenon has been attributed to a rigid structure owing to a complete secondary hydrogen-bond belt found in the P-cyclodextrin molecule. The hydrogen-bond belt in the a-cyclodextrin molecule is incomplete due to one glucopyranose residue being in a distorted position As a consequence, only four of the six possible hydrogen bonds can be established. The y-cyclodextrin molecule is a noncoplanar, moreflexible structure, and is therefore the most soluble of the three. Although more soluble than p-cyclodextrin, 6-cyclodextrin also has a low solubility compared with thatof a-or y-cyclodextrin, which has been ascribedto the same mechanism as p-cyclodextrin and an odd number of glucose units At elevated temperatures the aqueous solubility of all cyclodextrins increases. The temperature-solubility correlation of cyclodextrins, especially that of P-cyclodextrin, has beenutilized in the preparation of inclusion complexes. Beta-cyclodextrin dissolves well in DMSO and DMF, but it is practically insoluble in common organic solvents such as methanol, ethanol, isopropanol, acetonitrile, and tetrahydrofuran. Although insoluble in many pure organic solvents, p-cyclodextrin becomes more soluble in some solvent/

Important Properties

Glucose residues Molecular weight Solubility (g100 mL, 25°C) Specific rotation Cavity diameter (A)

Crystal water

a-,

6 973 14.5 150.5 4.7-5.3 10.2

and S-Cyclodextrins

7 1135 1.85 162.5 6.0-6.5 13.2-14.5

8

1297 23.2 177.4 7.5-8.3 8.1-17.7

9 1459 8.19 187.5 10.3-11.2 13.75

Cyclodextrins

water mixtures. Whereasthe solubility of P-cyclodextrin generallydecreases as the concentration of a solvent increases, maximums in solubility have been shown in the aqueous mixtures of ethanol, isopropanol, acetronitrile, and 20%, respecand tetrahydrofuran at approximately 30%, tively [ll]. Departure from these solvent/water ratios reduces the solubility of P-cyclodextrin in corresponding solutions. Certain additives, including bases, acids, and salts, have been found to enhance the aqueous solubility of P-cyclodextrin[12]. The most concentrated P-cyclodextrin solutions can be made with strong bases, such as dium hydroxide. Ureaalso has significant effect [13]. For example, the aquesolubility of P-cyclodextrin increases more than an order of magnitude when transferred from water to 8 M urea. Although inorganic acids do not normally change the P-cyclodextrin solubility, some organic acids, such as pura-toluene sulfonic acid, have shown a remarkable solubilizing capacity, probably due to the hydrogen-bonding interactions.Effects of salts are mixed [14]. Perchlorate exhibits significant solubility enhancement, while sulfates have an opposite effect. The alkali chlorides slightly improve the P-cyclodextrin solubility. Cyclodextrins are hygroscopic. The amount of water picked up and the rate of absorption vary with the molecular surface area or molecular weight. In one experiment, dehydrated cyclodextrins were exposed to an atmosphere of 85% relative humidity at 30°C; a- and P-cyclodextrins absorbed moisture to their equilibrium levels within 24 hours, while y-cyclohours to reach its equilibrium. The equilibrium moisdextrin took about ture levels for a-,P-, and y-cyclodextrins are 12%, 13.5%, and 17% by weight, respectively. It is striking that even when at their equilibrium moisture levels, cyclodextrins remain free-flowing powders with a dryappearance and feel.

Chemical Properties Cyclodextrins are nonreducing oligosaccharides. Periodate oxidation of cyclodextrins will open the glucopyranose ring, butno formaldehyde or formic acid is formed, consistent with the fact that they do not contain free end groups. Hypochlorite, a strong oxidizing agent, at 50°C and at a laundry bleach concentration, can rapidlyoxidize P-cyclodextrin. Hydrogen peroxide oxidizes P-cyclodextrin at a much slower rate. Cyclodextrins are stable toward alkali, even at elevated temperatures. Exposure of cyclodextrins to 0.35 M sodium hydroxide at 70°C results in no detectable degradation. The stability of cyclodextrins in alkaline solution is similar to that of cellulose.

and Romberger

Although cyclodextrins are more resistant to acid hydrolysis than is starch, strong acids such as hydrochloric acid will hydrolyze the cyclodextrins to yield a mixture of oligosaccharides ranging from the opened ring down to glucose [15]. The rate-limiting step is the ring opening reaction, and the rate of acid hydrolysis increases as functions of both increased temperature and the acid concentration. The stability of the intact ring against acid hydrolysis is about two to three times higher than that of the acyclic dextrins. In the presence of weak acids such as organic acids, hydrolysis of cyclodextrins is minimal [16]. The thermal stability of cyclodextrins is far greater than that of common starch. differential,scanning calorimetry. studyshows that melting of cyclodextrin crystals and the thermal decomposition of cyclodextrins occur simultaneously at around 300°C. However, no defined melting point can be reported, because the same cyclodextrin can contain different crystal forms. The observed thermoanalytical data depend uponmultiple factors, including the moisture content, crystal structure, as well as instrumental parameters.

Numerous methods have been developed to analyze cyclodextrins. These include direct detection by gel electrophoresis, capillary zone electrophoresis, cyclic voltammetry, nuclear magnetic resonance (NMR) spectroscopy, polarography, and mass spectroscopy, as well as indirect analysis by fluorimetry and spectrophometry. few examples of thin-layer chromatographic (TLC) and liquid chromatographic (LC and HPLC) systems as well as relevant literature references are compiled in Table 2. For detection on TLC plates, iodine vapor, 1% alcoholic iodine solution, or 5% concentrated sulfuric acid in methanol are commonly used.

Production of cyclodextrins from starch generally consists of the following steps:

1. Liquefaction, or prehydrolysis, of starch to maltodextrins with either alpha-amylases, CGTases, or with acids 2. Enzymatic conversion of the maltodextrins to a mixture of cyclic and acyclic dextrins (with CGTases) Separation and purification of a cyclodextrin product from the resultant mixture

Cyclodextrins Table

Examples of Chromatographic Systems for the Separation

and Determination of Cyclodextrins

m e TLC

Stationary Reference and phase mobile Kieselgal G:

Butanol/aceticacidhater (63:l)

17

Microcrystalline cellulose:

Butanollethanolhater(4:3:3) Butanollethanolhater(4:3:5) LC

18

19

Cellulose column:

Butanollethanollwater 20 (42:29:29) Activated carbon column:

21

Butanollwaterlgradient Mokelect G15lG2.5 column:

Water HPLC

22

pL3ondapaklcarbohydrate:

Acetonitrilehater pBondapaklpheny1: Water Econosillamino:

24

Acetonitrile/water

Many papers and patents have been dedicated to each phase of cyclodextrin production, and great efforts are still being made for process improvement. CGTases isolated fromvarious microbes thus far produce a mixture of a-, and y-cyclodextrins.Rvo different industrial approaches are currently used to produce pure cyclodextrins,the nonsolvent (or uncontrolled)process and the solvent (or controlled) process.

A. The Nonsolvent Process Differential solubility of cyclodextrins in water made it possible to isolate p-cyclodextrin through selective crystallization without using any added organic solvents. A typical nonsolventprocess includes inactivation of CGTase after the cyclization reaction, hydrolysis of residual starch with glucoamylase, and crystallization of p-cyclodextrin. The products thus obtained are pure p-cyclodextrin and the mother liquor,containing all three cyclodextrins as well as glucose, maltose, and other oligosaccharides. Isolation of a- and y-cyclodextrins from a conversion mixture by selective precipitation or by chromatography is too expensive for industrial production.

and Romberger

B. The Solvent Process By addition of water-immiscible solvents known as complexants to a cyclization reaction, the equilibrium of enzymatic conversion can be shifted in favor of cyclodextrin production. These complexants form much less soluble inclusion complexes with particular cyclodextrins, thus allowing their separation through precipitation. Reasonably high yields of the specific cyclodextrins can be obtained using this method. Some well-known complexants are l-decanol (for a-cyclodextrin), toluene and cyclohexane for (p-cyclodextrin) [25], cyclotetradec-7-en-l-one or cyclohexadec-8-en-one [26], and cyclododecanone[27a]with or without[27b]acosolvent (for y-cyclodextrin). The two approaches just described have certain disadvantages. There is a great demand for commercial processes that could produce pure cyclodextrins more economically. Active research is now directed toward modifying CGTases by protein engineering to develop mutants specific for one type of cyclodextrin.

IV. CHEMICAL MODIFICATIONOF CYCLODEXTRINS Cyclodextrins are modified in order to alter their solubility behavior and complexation properties and to introduce groups with certain specific functions. Chemical modification of cyclodextrins has been the subject of numerous studies and publications. Interested readers may refer to the comprehensive reviews by Croft and Bartsh [28] and Wenz [29] and relevant book chapters [8,30]. Only a brief discussion will be given for terms used to describe the chemically modified forms, as well as reactions currently used in commercial production of cyclodextrin derivatives.

A.Degree

of Substitution

Degree of substitution (D.S.) is an important and somewhat unique parameter used for commercial cyclodextrin derivatives. It refers to the average number of hydroxyl groups of a cyclodextrin molecule that have been converted upon during chemical modification. An alternative to D.S. is molar substitution (M.S.), which describes the number of hydroxyl groups converted per mole of glucose. Therefore, the maximum values of D.S. are 18, 21, and 24 for a-, and y-cyclodextrins, respectively, whereas 3 is the maximum M.S. value of each. Complication may arise when reagents such

3

as epoxides used to modify cyclodextrins undergo simultaneous self-polymerization, when the values of D.S. or M.S. do not truly reflect the actual moles of reagents consumed by each mole of a cyclodextrin. In any case, the degree of substitution per se cannot characterize a cyclodextrinderivative unambiguously, since it is an averaged value. For example, a product with D.S. of 7 may contain a distribution of compounds having degrees of substitution ranging from to 10, to 9, or 6 to 8, depending on reaction and process conditions. Moreover, the value of D.S. does not provide any information about substitution positions. Thus,even a monosubstituted product (D.S.of 1) could contain three compounds corresponding to substitutions on C 2 , 6 , andC6. Despite the lack of accuracy, the D.S.parameter is widely used in characterizing a chemically modified cyclodextrin. The control over D.S. can provide assurance that a productof similar characteristics is manufactured consistently.

B. HydrophilicCyclodextrinDerivatives Derivatives that have greater aqueous solubility than their parental cyclodextrins are defined here as hydrophilic derivatives. Increased solubility of cyclodextrins, particularly P-cyclodextrin, is desired in many applications. Substitution of hydroxyl groups disrupts the regular hydrogen-bonding network within a cyclodextrin molecule, and reduces the tendency of crystal formation, thus resulting in an increase in aqueous solubility. Ether or ester linkages are most common as a result of derivatization. For example, hydroxyethyl, hydroxypropyl, and methyl ethers of P-cyclodextrin can be soluble in water at up to 60 dl00 g. At a D.S. range of 3-8, acetylated cyclodextins are highly soluble as well. Ionic functional groups such as carboxyalkyl, alkylamine, alkylsulphone,sulfate, and phosphate can provide additional hydration potential for high water solubility as well as electrostatic interactions with guest compounds bearing oppositely charged groups. crosslinked cycloAt a low degree of polymerization (no more than dextrins also exhibit increased aqueous solubility. This type of derivative is frequently used to solubilize guest compounds that have low or very low solubility in water.

C. HydrophobicCyclodextrlnDerivatives Cyclodextrins can be chemically modified to produce derivatives that are much less water soluble than their parents. A reduced aqueous solubility is necessary, for example, in applications where a low residue of cyclodextrin is required. Extensive crosslinking, with epichlorohydrin or similar crosslinking reagents,canresult in cyclodextrin polymers as water-insoluble

beads. Binding of a guest compoundto a cyclodextrin polymeris dependent upon the ability of the guest both to diffuse into the beads and to bind with the cavity of the cyclodextrin. Cyclodextrin polymers are typically used to remove materials from aqueous or to release materials over a period of time. Large volumes of aqueous solutions may be treated. The absorbed components can be recovered from the polymer, and the polymer can be regenerated for repeated uses. In addition to polymerization, low aqueous solubility of monomeric .cyclodextrins can also be achieved by extensively substituting the hydroxyls of a cyclodextrin with hydrophobic functional groups. For example, when exhaustively acetylated, cyclodextrins become practically insoluble in water.

The most important characteristic of cyclodextrins is their ability to form inclusion complexes with a wide variety of guest compounds without formation of covalent bonds. The terminology of Einschllrssverbindungen (inclusion complex)was first introducedin 1951 by Schlenk [31]. Several other terms have also been used to describe the host-guest association, such as adduct, clathrate, cryptate, supramolecular compound, and complex. The formation of a cyclodextrin-guest inclusion complex is truly a molecular phenomenon. Most often, complexation occurs with 1:l (cyclodextrin to guest) stoichiometry, although exceptions, including 2:1, 1:2, and 2 2 complexes, are well known. In contrast, other encapsulation vehicles, such as liposomes, entrap many molecules within a wall or sphere.

Various theories have been developed as thermodynamic driving forces of forming cyclodextrin inclusion complexes.One explanation involves the release of “high-energy water.” The cavity of “empty’ cyclodextrins contains water in aqueous solutions, which is incompatible with the apolar interior of the cavity. If a guest molecule with a nonpolar portion of suitable size is presented to the cavity, it can displace the water in the cavity. Releasing the water in the cavity to the bulk solvent water then lowers the free energy of the system and is therefore thermodynamically favored. Furthermore it is also thermodynamically morefavorable for the apolar guest to associatewith the apolar cavity, a driving force of complexation known as hydrophobic interaction.X-ray crystallographic studies haverevealedachange in the conformation of the ring of the cyclodextrin molecule upon complexation. As a result, release of conformational strain occurs and is described as a

driving force in favor of complexation. In addition, a variety of noncovalent interactions, such as van der Waals forces, hydrogen bonding, dipole-dipole interaction, and London dispersion forces, are also considered to be responsible for the formation of a stable complex. In most cases, several forces seem to contribute collectively to the complexation process.

B. Suitable Guest Molecules Since the cavity diameter of a cyclodextrin varies with the number of glucose units in the ring, selecting an appropriately sized cyclodextrin to fit the geometric parameters of a guest compoundis of primaryimportance in forming an inclusion complex. For instance, propionic acid is compatible only with a-cyclodextrin and not with the larger cavity volumes of or cyclodextrin. Conversely, naphthalene is too large for a-cyclodextrin, but fits "snugly" within the cavity of p-cyclodextrin. Additionally, complex formation does not always require inclusion of the entire guest molecule into a cyclodextrin cavity. Even very large molecules can be complexed as long as their pendant groups or side chains fit well with the cyclodextrin cavity. Polarity is another factor. Water-insoluble or sparingly water-soluble organic molecules are generally good candidates because of the favorable hydrophobic interaction. Owing to the apolar nature of the cavity, ionic organic moieties generally do not bind well with cyclodextrins. Amphoteric materials may be exceptions. For example, benzoic acid is very well complexed by P-cyclodextrin, with its benzene ring inside the cavity and the carboxyl group forming a hydrogen bond with one of the secondary hydroxyl groups of the cyclodextrin. When cyclodextrins are modified with ionizable side chains, electrostatic interaction between the ionic groups of the hostand the guestcan be asignificant factor contributing to the complexation.

C. Factors InfluencingComplexation It is important to keep in mind that the complexation reaction between a cyclodextrin and a guest molecule is a rapid and reversible process in lution. Any factors shifting the dynamic equilibrium may affect the stability of the resultant complex. High temperature increases molecular motion and therefore generally favors the dissociation of a cyclodextrin complex. For instance, the stability constant of the a-cyclodextrin-benzoic acid complex decreased from 1397 M" to M" as temperature was raised from 15°C to [32]. the other hand, heat can increase the solubility of a cyclodextrin and the guest

molecule, allowing necessary molecular contact that a complex can form within reasonable time frame. Balancing these effects is of practical importance in preparing complexes, especially when P-cyclodextrin is used. Testing is usually needed to determine the optimal use of heat in each complexation reaction. Theoretically, cyclodextrin complexes can be formed without the presence of any solvent. It is not practical, however, because mostcomplexation reactions are too slow under anhydrous conditions. In fact, complexation reactions usually occur in the presence of a solvent, and generally that solvent is water.Water favors the formation of acomplex on the basis of hydrophobic interaction. The main disadvantage using water is its subsequent removal in the large-scale production of a dry complex. Various methods of preparing cyclodextrin complexes are employed, which differ mainly in the amount of water used. Sometimes, organic solvents have to be used to dissolve highly insoluble guest compounds in order to prepare a complex within a reasonable time period. Choice of solvents is restricted due to their possible competition with the guest for the cyclodextrin cavity, or the formation of a ternary complex. Small and polar molecules, such as methanol, ethylene glycol, and 2-methoxyethanol, are preferable. Usually, a minimum amount of solvent usage is recommended. With an ionizable guest, pH plays an important role in complexation. For example, sodium benzoate, which is very water soluble in water, forms a soluble complex with cyclodextrin. As pH decreases, the benzoate anion is protonated to form benzoic acid. During this change, a more stable but much less soluble complex precipitates out of the solution. Also, when the solution pH is high enough, the hydroxyl groups of cyclodextrin will deprotonate. This interferes with the host-guest binding in a similar way. Thus, heating in alkaline solution is typically used as means to dissociate a cyclodextrin complex. The stability and solubility a cyclodextrin inclusion complex may be influenced by inorganic salts. Interaction of salts with the hydroxyls increases the solubility of cyclodextrins, while their dehydrating effect decreases it. Cyclodextrins have also been found to form coordination complexes with transition metal ions and metallocenes through their hydroxyl groups.

Molecular inclusion complexation is the basis increasingly widespread applications with cyclodextrins. The breadth of industries that can benefit from cyclodextrin technology range from the agricultural, chemical, phar-

maceutical, analytical, and diagnostics industries to the food, cosmetics, toiletry, and coatings industries. Essentially any industry that involves organic chemistry can use cyclodextrins to improve certain current practices. Some of these applications are briefly summarized next, and commercialexamples are cited in Table 3. Frequently observed benefits associated with cyclodextrin complexation include: Modification of aqueous solubility Alteration of chemical and physical stability Selective binding Conversion of liquid or gas to solid Reduction of malodor or off-taste

Increased aqueous solubility has been obtained on a large number of guest compounds by the use of cyclodextrins. Among hydrophilic derivatives, hydroxyalkyl and methyl cyclodextrins are frequently used as effective solubilkers. Bioavailability enhancement of certain drugs is also partially attributedto the solubilizing effect of cyclodextrins. While enhancedwater solubility of various organic compounds is desirable in many areas,the effect seems to have most widespread and profound impact in the pharmaceutical industry. Many pharmacologically active substances are not easily formulated into suitable forms, due to insufficient aqueous solubility. Dramatic solubility enhancement has been achieved on a large number of these hydrophobic drugs through complexation with soluble derivatives of cyclodextrins. Pitha [33] has demonstrated thatcrystalline drugs with substantially low solubility could be made water soluble via complexation with amorphous, hydrophilic cyclodextrins. According to Pitha, the rationale for using randomly substituted or amorphous material is the improbability of crystal formation. The maximum number of individual isomers is achieved when half of the available hydroxyls are substituted. In the case of a modified pcyclodextrin, 50,388 different isomers [34] are possible at D.S. = 10 or 11. On the other hand, decrease in water solubility of some hydrophilic molecules upon complexation with p-cyclodextrin or certain hydrophobic derivatives of cyclodextrin has been utilizedas a means of sustained release.

The cavity of cyclodextrinsprovides an isolated microenvironmentfor many labile substances that significant stability can be gained against degra-

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222

dation due to heat, light, oxidation, hydrolysis, etc. Much prolonged shelf life of vitamins in their cyclodextrin complexes are well-born examples. Another example is the one-partheat-curable organopolysiloxane system developed by Lewis and Sumpter [35]. The cyclooctadiene platinum catalyst is greatly stabilized by forming a P-cyclodextrin complex,which has a shelf stability of more than 7 months at 25°C compared with that of 12 hours shown by the uncomplexed catalyst. Once the hydrosilation polymer was heated to 150°C,the catalyst was quickly released’to cure the silicone.

Physical Active ingredients such as flavors, fragrances, certain pharmaceuticals, and insecticides lose strength over time due to evaporation or sublimation. The losses may be eliminated or reduced upon complexation with cyclodextrins. L-Menthol sublimes readily at room temperature, while its cyclodextrin complexes exhibit great stability. Thermal analysis shows that inclusion with cyclodextrin has substantially increased both the melting and boiling temperatures of menthol [36]. The irritating effect of mustard oil to eyes and respiratory tract is suppressed uponits complex formation with cyclodextrin.

The selectivity and affinity of cyclodextrinsfor various guest molecules arise from the well-defined dimensions of the cyclodextrin cavities. For instance, 80%-90% of cholesterol can be removed from whole eggs, lard, tallow, or cheese with cyclodextrin [37]. Through cyclodextrin technology,the cholesterol-cyclodextrin complex can be formed and precipitated from solution. The complex can then be removed by physical separation means such as centrifugation. In this process, quantities of undesirable materials can be greatly reduced or eliminated from multicomponent mixtures with simultaneous recovery of cyclodextrins for repeated processing. Cyclodextrin derivatives with decreased aqueous solubility are particularly useful for separating components through selective complexation. Usually, no or a very low level of residual cyclodextrin is left in the processed materials. Therefore, they are often used to isolate or enrich specific components from a mixture or from natural sources. Some examples are removal of bitter-tasting compounds from citrus juice, removal of phenylalanine from protein hydrolysates, and extraction of flavors and medicinal actives from plants or fermentation mixtures. Cyclodextrins are chiral molecules. They are known to bind preferentially with certain chiral isomers. Therefore,cyclodextrins are widely used

for chromatographic separations, either as a stationary-phase or as a mobilephase additive.

Conversion of a volatile substance from its gaseous or liquid state to a stable, solid powder byforming a cyclodextrincomplex has great practical benefits, for instance, precise dosing, uniform distribution, and facilitating package, storage, and transport. Thus, the C02-a-CD complex [38] can be utilized as an emulsifying or foaming baking powder for sponge cakes,andthe ethanol-P-CD complex [39] can be used for food preservation.

Beta cyclodextrin itself has a sweet taste. But its threshold value is lower than that of sucrose [40]. Because of the relatively small amount being used, the taste of P-cyclodextrin in the complex does not have anoticeable impact on that of the final product. As additives in food and oral pharmaceutical preparations, cyclodextrins have been found to be effective in masking certain undesirable tastes or odors. For example, the bitterness of naringin present in grapefruit juice is greatly reduced by the addition of 0.3% P-cyclodextrin prior to a heat treatment [41]. Soy bean products are freed from the characteristic grassy smell and stringent taste by mixing with cyclodextrin 1421.

Although it has been over 100 years since the discovery of cyclodextrins, their commercial potential has yet to be fully tapped. Their unique structure and their ability to form molecular inclusion complexes and to alter the behavior of their included materials have allowed them to find applications in more ad more disciplines that use organic chemicals. As complexation techniques are improved, new derivatives are being manufactured at lower costs;and as more regulatoryhurdles are crossed,newapplications will certainly be developed at an accelerating speed. Cyclodextrins will truly be “great solutions seeking problems.’’ I

1. Villiers, A. Compt. Rend. Acad Sci. Paris 112: 536 (1891). Schardinger, F.Z. Unters. Nahrungs-, Genussmitlel, Gebrauchsgegenstande6 (1903).

3. Schardinger, F. Wen, Klin. Wochenschr. 17: 207 (1904). 4. Schardinger, F. Zenk Bakteriol. Parasitenk 11 2 9 188 (1911). 5. Endo, T., Ueda, H., Kobayashi, S., and Nagai, T. Carbohydr. Res. 269: 369373 (1995). 6. Miyazawa, I., Ueda, H., Nagase, H., Endo, T., Kobayashi, S., and Nagai, T. European J. Pharm. Sci. 3: 153-162 (1995). 7. Endo, T., Nagase, H., Kobayashi, S., and Nagai, T. Chem. Pharm. Bull. 45: 532 (1997). 8. Szejtli, J. Cyclodextrin Technology. Kluwer Academic Publishers, Dordrecht, The Netherlands (1988). 9. Rees, D. A. J. Chem. Soc. B: 877 (1970). 10. Saenger, W., Noltemeyer, M., Manor, P.C., Hingerty, B., and Klar, B. Bioorg. Chem. 5: 187 (1976). 11. Chatjigakis, A. K, Donzt5,C., and Coleman, A. W. Anal.Chem. 1632 (1992). 12. Cyclodextrin News (Szejtli, J., ed.) 331 (1993). 61 13. Pharr, D. Y., Fu, Z. S., Smith, T. K, and Hinze, W. L.Anal. Chem. (1989). 14. BuvBri, A., and Barcza, L.J. Incl. Phen. 379 (1989). 15. Wiedenhof, N., and Larnmers, J. N. J. J. Carbohydr. Res. 1 (1968). 16. Schonberger, B. P., Jansen, A. C. A., and Janssen, L. H. M. Proceedings of the FourthInternationalSymposium on Cyclodextrins, Dordrecht, The Netherlands, p. 61 (1988). 17. Wiedenhog, N. J. Chromatogr. 15: 100 (1964). 18. Tekeo, K, and Kondo, Y. Agr. Biol. Chem. 34: 954 (1970). 19. Suzuki, Y., Shima, A., Kichi, T., Kato, T., Misawa, F., Okimoto, M., and Saito, N. (Teijin). Ger. Offen. 2,532,051, Jpn. Kokai 76, 12, 941 (1975). 20. Lammers, J. N.J. J. Starch 19: 70 (1967). 21. Lammers, J. N. J. J. J. Chromatogr. 41: 462 (1969). 22. Zsadon, B., Szilasi, M,, Szejtli, J., Seres, G., and Tiidos, F. Starch, 3 0 276 (1978). 23. Kitahata, S., Okada, S., and Misaki, A. Agric. Biol. Chem. 43: 151 (1979). 24. Unpublished data (Cerestar USA, Inc.). 25. Armbmster, F. C. Proceedings of the Fourth International Symposiumon Cyclodextrins, Dordrecht, The Netherlands, pp. 33-39 (1988). 26. Schmid, G., Huberz, 0. S., and Eberle, H. J., Proceedings of the FourthZnternationalSymposiumCyclodextr+ns, Dordrecht, The Netherlands, pp.87-92 (1988). 27a. Shieh, W.U.S. Pat. 5,550,222(1996). 2%. Rendleman, J. A.,Jr. Carbohydr. Res. 223-237 (1993). 28. Croft, A. P., and Bartsch, R.A. Tetrahedron 39: 1417 (1983). 29.Wenz, G. Angew. Chem Int. Ed. Engl. 33: 803 (1994). 30. Compr.Suprumol. Chem J. Szejtli, T. Osa (eds), PergamonElsevier, OxfordNew York-Tokyo, 1996, Vol. 3. 92 (1951). 31. Schlenk, W., Jr. Fortschr.Chem.Forsch.

Gelb, R. I., Schwarta, L. M., Johnson, R. F., and Laufer, B. A. J. Am. Chern. Soc. 101: Pitha, J. US.Put. Boger, J., Corcoran, R. J., and Lehn, J.-M. Helv. Chin Acta 61: Lewis, L. N., and Sumpter, C. A. U S . Put. 5,025,073 Qi, Z. H., and Hedges, A. H. Use of cyclodextrins for flavors. In Ho, C.-T., Tan, C.-T., and Tong, C.-H., eds., Flavor Technology,ACS Symposium Series American Chemical Society, Washington, D.C. Over dozens of patents have been published, which include: (a) Mentink, L., and Serpelloni, M. US.Put. (b) Maffnnd, J.-P., and Courregelongue,J. U.S.Put. (c)Courregelongue, J., and Maffrand, J.-P. US. Put. (d) Award, A., Hedges, A., Shieh, W., Sikorski, C., and Smith, D. M. WO 9629893 AOPAB:961111 Schlenk, W. US.Put. Asahi Denka Kogyo KK. Jupun Kokai, Toda, J., Misaki, M., Konno, A., Wada, T., and Yasumatsu, K. In: Charalambous, G., and Ingle, G., eds., The Quality of Foods and Beverages. Academic Press, New York, p. Konno, A., Miyawaki, M., Miaski, M., and Yasumatsu, K. Agric. Biol. Chern. House Food Ind. K.K., Jupun Kokai,

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7 Starch-Lipid Interactions Ratnqjothi Hoover Memorial University Newfoundland St. John’s, Navfoundlanct, Canada

Lipids associated with isolated starch granules have been found to occur on the surface as well as inside the granule [1,2]. The surface lipids are mainly triglycerides PG), followed by free fatty acids glycolipids (GL), and phospholipids (PL), and they include those that may have been present on the granule surface in situ within the plant tissue and the nonstarch lipids, which are absorbed into the surface layer of starch granules during isolation procedures [1,3]. The nonstarch lipids occur as spherosomes and as components of membranes and organelles associated with storage protein [l]. The internal lipids are predominantly monoacyllipids [1,4,5],with the major components being lysophospholipids (LPL) and It is likely, that both free and internal lipids may be present in the free state as well as bound to starch components, either in the form of amylose inclusion complexes [6] or linked via ionic or hydrogen bonding to hydroxyl groups of the starch components [5]. Selective binary solvent extraction techniques [5,7 have shown that amylose-complexed lipids are more abundant in cereal than in legume and tuber starches (Table 1).

II. A.

Structure

Amylose is found with molecular weights ranging from lo5 to lo6 and with the number of glucose residues per molecule (DP) ranging from500 to

Hoover

1 Content Amylose Chains Complexed by Native Starch Lipids in Cereal, Legume, and Tuber Starches Amylose complexed with lipids Total native amylose

("/.l

12.1 7.8 13.3 15.2 21.1 11.3 13.9

Starch source Cereals WheaP OatSd Maize"" Rice"." (long grain) Wild rice' Pearl millet' Barleyg Legumes LentiP 5.6-12.4 Mungbean" Field pea" Black bean' Anto bean' Green arrow pea' Tuber and Root Potato" Cassava'

("/.l 27.3 19.4-22.7 27.2-27.5 19.5-28.3 29.4 28.8 29.2-32.7

22.7-25 14-15.3 18.7-22.2 19.4-30.2 28.2 14.6 22.0-27.2

35-39 45.3 43.2 41.2 44.7 85.6 24.7 21.5

'Vasanthan and Hoover [5]. %orrison and Laignelet [loo]. Woover and Vasanthan [loll. dHooverand Senanayake 'Unpublished results. 'Hoover et al. *Morrison et al.

[3]. Hizukuri et al. [S] and Takeda et al.[g] have demonstrated thatamylose from differentsources contains, on average, 2-8 branch points per molecule, the side chains ranging in chain length from to over 100 glucose units. The conformation of amylose in solution is still the subject of extensive investigation and speculation. However, there seems little doubt that coil+helix transitions are induced upon addition of complexing agents in an aqueous amylose solution [lo]. The complexing agent has been found to reside within the helix [ll-131. Lagendijk and Pennings [l21 showed that the structure of the amylose molecule in suspension can be represented as a chain with 10-12 helices, each containing a mean of 12 windings of six glucose units (length of winding: 8 A). It follows that every helix binds at

least one and, in manycases, two molecules of glycerol monopalmitate (GMP) during complex formation.The length of the GMP molecule is about 22 A. If a helix with 12 windings forms an inclusion compound with 2 moles GMP, the free space is 50 A (Fig. 1) and, consequently, consists of about six windings, or about 35 glucose units [12]. Jane and Robyt [l41 and Biliaderis and Galloway [l51 have shown that amylose forms single-chain V, helices (diameter 4.5A) enclosing single acyl chains of lipid, interspersed with shortlipid-free regions with a more random conformation that are about one-seventh of the total amylose [16]. In the helical form, the interior surface of amylose is built up by CH groups and glycosidic oxygen atoms forming a lipophilic core, while all the polar hydroxyl groups are positioned on the outer surface of the helix [ll].In the arnylose-lipid inclusion complex, the hydrocarbon chain of the lipid is inside the lipophilic core of the amylose helix, while the polar group of the lipid molecule is located near the entrance (Fig. 2) of the helix cavity [13,17-191. Godet et al. [l91 have shown by molecular modeling studies that the inclusion of a fatty acid aliphatic chain inside the amylose helix is possible only when the aliphatic chain and the helix axes are superimposed and when the aliphatic chain is in a trans conformation. Carlson et al. [l31 have shown by Raman spectroscopy that the hydrocarbon chain conformation inside the helix seems to be ordered as in the crystalline state. Amylose-monoglyceride complexes of different chain length exhibit the same X-ray diffraction pattern [15,20,21]as described by %bel [22] for

Schematic diagram of a glycerol monopalmitate/amylose complex. “free” space. (Reprinted with permission from Ref. 12.)

=

2 Monostearin-amylose complex, with the whole chain inside space. (Reprinted with permission from Ref. 13.)

the helical

the called “V” form of amylose, indicating that all monoglycerides, irrespective of chain length, the same type of inclusion compound with amylose. However, the intensity of the X-ray pattern is influenced by the monoglyceride chain length [21] (Table 2). The V,, (V-hydrate) [l51 pattern of amylose-lipid complexes exhibits reflection peaks [15,19,21,22] corresponding to Bragg angles 20 = 7-7.9 12.1-13.3 19.8-20.1 A. X-ray diffraction patterns of amylose-fatty acid complexes show a pseudohexagonal orthorhombic unit cell (dimensions = &b= c = 8.05 A) with left-handed, sixfold helices, which have a fiber repeat

distance of 8 A and pack antiparallel. Electron and X-ray diffractionstudies have shown that V-amylose complexes form lamellarlike crystals (lamellar with lamellar folding lengths of about 100 A for amthickness 75-100 ylose helices [25-271. Biliaderis and Galloway [l51 and Biliaderis and Seniviratne [28] have shown by dserential scanning calorimetry (DSc), X-ray,and structural analysis data that amylose-lipid complexes, in the solid state, exist mainly as two morphologically distinct (forms I and II) supermolecular metastable structures, depending on the crystallization conditions (temperature, type of ligand,etc.).Form is akineticallypreferredpolymorphthat exhibits a diffused two-line “V” diffraction pattern (Fig. whereas form I1 exhibits

e”) Figure 3 X-raydiffractiondiagramsofwetamylose-monoglyceridecomplexes obtained at various crystallization temperatures. (form 11, “V” pattern); B: 1-C16/6OoC (form I); C 1-C14/9OoC(form 11, “V” pattern); D: 1-C14/ (form 1). (Reprinted with permission from Ref. 15.)

Starch-Lipld Interactions

233

Figure Generalized mechanism for amylose-lipid complex formation from dilute solutions, which can account for the properties and postulated morphological features of forms I and 11. (Reprinted with permission from Ref. 15.)

awell-definedthree-line “V” diffraction pattern(Fig. Biliaderis and Galloway [l51 postulated a generalized mechanism for amylose-lipid complex formation from dilute solutions (Fig. According to this mechanism, at low crystallization temperatures (TJ, formation of complex I is the favored process. In the presence of strong complexing agents, and low T,, the nucleation rate is high, thereby causing a rapid “freezing” of helicalchain segments with very little crystallographic register (form I) throughout the structure. As such, interhelical amorphous chain regions would be under considerable strain, and it is likely that they would exhibitan elevated glass transition temperature (T,) very close to the melting temperature (T,,,I)of the helices of form 1. This suggests the existence of a high-energy bamer between forms I and 11. Thus, unless the system is partially melted, it would remain practically unchanged at T < T,,,I.On the other hand, at high T , the nucleation density would be much lower, and thus complex formation is

Hoover

allowed to progress as a conventional crystallizationprocess, therebyleading to a partially crystalline structure (form 11).

B. Occurrence Naturally occurring “V” structures have not been observed in starches with less than 30%amylose 1291. However, “V” structures can be found in native starches grown under the control of recessive genes such as the sugary (m), dull and amylose extender genotypes in single or multiple combinations [29]. The evidence for or against the existence of “V” complexes in native starches has been inconclusive, mainly because complexes could be formed during gelatinization [30-341 and heat-moisture treatment [35]. The absence of a “V” pattern in native starches does not prove the absence of the amylose-lipid complex; it merely proves that the complex is not arranged in crystalline domains that can be detected by X-ray diffraction techniques [36]. Recently, Morrison et al. [37-381 have shown, by means of ’3C-cross-polarization/magic-anglespinning NMR spectroscopy,X-ray diffraction, and DSC measurements, the existence of amylose-lipid complexes in native barley, maize, rice, and oat starches. On this basis, those authors postulated that amylose in cereal starches occur as two fractions, lipid amylose (GAM) and free amylose (F-AM),that are separate types of molecules rather than molecules with mixed character.

C.ComplexFormation Riisom et al. [39] showed that when monoglycerides are added as powders or as a melt, monoelaidate showed the highest complexing index (Table 3). The authors postulated that the observed differences in complexing ability may be due in part to differences in molecular structure, since the acid chains of monoelaidate and monopalmitate can easily form the extended zigzag conformation, whereas the cis-unsaturated monoglycerides have one or more kinks in their chain. Larsson [40] reported that the optimal conditions for formation of the amylose-lipid complex are directly related to the lipid monomer concentration. The most effective state of the lipid is the micellar solution (Fig. 5A), since the monomer concentrationin equilibrium with a micellar solution is higher than that in equilibrium with a liquid crystalline or crystalline phase [41]. Furthermore, the kinetics of supplying monomers from micelles to the solution are far superior to those of any other association state of the lipids. Many researchers [20,39,41,42] have shown that the lamellar liquid crystalline phase (Fig. 5A) is superior to the

3 Complex Formation Between Monoglycerides and Amylose in Aqueous Solution at 30°C

Amylose complex index (ACT

Na-cholate mesb dispersionsb Monoglyceride Monopalmitate Monoelaidate Monooleate Monoliioleate

Aqueous 33

40

71

81 98

93

16

T h e absorbance of the iodine-amylose complex was measured at nm using an iodine solution of the same concentration as the blank. Theamylosesolutionwith added monoglycerideserved as the control. The results are given as the amylose complexing index according to the following relationship: ACI =

absorbance

control - absorbance absorbance control

sample

loo

b10mg monoglyceride/40 mg amylose. Reproduced with permission from Ref. 39.

other liquid crystalline phases because of their ability to form free dispersions. The amylose complexing ability of monoglycerides in water at 60°C (Table has been shown [39] to increase in the following order: monopalmitate (forms a lamellar phase) > monoelaidate (forms a phase that is in the border region between the viscous cubic and the lamellar phases) > monooleate (forms a cubic phase) > monolinoleate (forms a cubic phase). However, when monoglycerides were reacted with amylose at 30°C in the form of liposomes (Fig. 5B) [droplets consisting of concentric spheres of alternating lamellar layers and water],the cis unsaturated compounds precipitated the amylose completely, whereas monopalmitate andmonoelaidate exhibited only slightly higher amylose complexing ability (Table when added as liposomes rather than as powders (since acyl chains of these compounds are crystalline partially crystalline at 30°C, rather than in the active lamellar form) [39]. However, when the reaction was carried out at 60°C (Table the amylose complexing ability of each monoglyceride was close to 100. When this same experiment (at 60°C) was repeated with reduced amounts of monoglycerides [39] (Table 3), the cis unsaturated compounds appeared to be more effective complexing agents than the trans unsaturated mono-

Figure Binary lipid-water system characteristic of polar lipids that form micellar solutions. The horizonta! axis defines the composition; the vertical axis defines the temperature. The phase diagram thus illustrates the temperature range and composition range of existence of the phases. In the indication of structures, each molecule is illustrated by the polar head (a circle) and one attached chain tail. B: Structure of a liposomal dispersion in water. The liposomes are multilamellar particles. (Reproduced with permission from Ref.

elaidate and the saturated monopalmitate. This was rather since the presence of cis double bonds theoretically should have decreased the complexing ability of monoglycerides with the amylose helix However, in starch,naturally occurring amylose-lipid complexes are formed from lysolecithin which is rich in cis-cis unsaturated linoleic acid.

Starch-Lipid Interactions Table Complex Formation Between Monoglycerides and Amylose in Aqueous Solution at 60°C Amylose complex index (ACI)" Aqueous Liposomes Na-cholate dispersions Monoglyceride mg Monoplamitate Monoelaidate Monooleate Monolinoleate

10 mg

55

33

71 16

2.5 mg

81 98 93

50 85

83

'See footnote in Table 3. Reproduced with permission from Ref. 39.

Thermal Formation of amylose lipid complexes alters swelling, solubility, and the gelatinization characteristics of starches. In general, granule swelling is delayedandamyloseleaching(Fig. 6) is reduced in the presence of lipidcomplexed amylose molecules [45-491. However, gelatinization temperatures starches may or may not be changed [21,33,50-551. Formation of amylose-lipid complexes has been shown [50,56] (Table 5) to decrease the gelatinization enthalpy (LW>. This was attributed exothermic formation amylose-lipid complexes during gelatinization. Upon gelatinization, amylose molecules become more accessible to interact with lipids. In this respect, the lipid molecules induce helicity inthe amylose chains and thereby crystallization [56] (exothermic effect).The amylose-lipid complex melting been shown to occur in the range 85-130°C transition (T=) has [10,21,30,50,56,57]. The transition is reversible, as evidenced by an exotherm on the DSC cooling curves [10,57] (Fig. 7).T, is influenced by moisture content (Fig. hydrocarbon chain length (Fig. 8, Table 6), complex concentration (Fig. 9), and crystallization temperature (Table 6). Biliaderis et al. [lo] observed that at water concentrations greater than 80%, a single symmetrical endothermis shown by amylose-lipid complexes (Fig. S), indicating that melting becomes highly cooperative at high moisture contents. Furthermore,the melting temperature(Ta) remains constantat high water levels [lo]. At moisture contents greater than the T, AM (amylose)-C12, AM-Cl6, and AM-lysolecithin complexes has been reported to be 96", 97", and 104"C, respectively [46,58,59]. At moisture con-

Hoover

a: SEM micrograph of a potato starch grain heated at 80°C for 10 min. b: SEM The interlaced network is the leached-out amylose. Magnification micrograph of a potatostarch grain heated at 80°Cfor 30 min. Magnification c: SEM micrograph of a potato starch grain treated as in 6b, but in the presence of 0.25% C16-l-monoglyceride.Note the absence of leached amylose. d: SEM micrograph of potato starch grain treated as in 6b, but in the presence of 1.0% C,,-type monoglyceride. Note that the amount of leached amylose is higher in 6c. (Reproduced with permission from Ref.

tents below 509i,amylose-lipid complexes exhibit two melting endotherms separated by an exothermic peak (Fig. 8). A mechanism of partial melting followed by recrystallization and final melting has been proposed to account for such nonequilibrium melting.The possibility ofchanges in lamella thickness as a result of such structural transformations was further suggested to account for the increase in melting temperature of the recrystallized material. showed that an increase in chain length of monoEliasson and Krog glycerides from C12 in glycerol monolaurin to C18 in glycerol monostearin increased T, of potato amylose from 85.1" to 103.5"C (Table The T, of trans unsaturated C18:l monoglyceride was found to be similar to that of

"-7 r

.

40

:

-

T

g

e0

e

V

a-,

Temperature

(.:E

Flgure DSC thermal curves of A"1-C16 at various water contents (90% to 30%). Weight of complex from top to bottom (mg): 1.01,2.01,2.44,2.64, 5.55, Heating rate mh". AU data files were normalized to a constant sample weight of 5.00 mg. (Reprinted with permission from Ref. 10.)

the saturated C18:O analog, whereas the cis unsaturated monoglyceride was for C18:2ciscis (Table less stable, with a T, of 97.0"C for C18:1dscis and 90.3"C Stute andKonieczny-Janda[58]alsoobserved similar transitions for amylose-fatty acid complexes. The melting enthalpies (AH,) of amylose-lipid complexes are usually in therange6.2-8.1callg,dependingonthecomplexingagentandthe conditions employed for complex formation [10,30,57-611.

holing

\

holing K)

G ,MS

Temperature

Differential scanning calorimetry thermograms of amylose complexesformed(a) with saturated monoglycerides:monolaurin(GML),monomyristin (G"), monopalmitin(GMP), and monostearin(GMS), and @) with monostearin(GMS), and theunsaturatedmonoglycerides:monolinolein(GMLi), with permissionfrom monoolein(GMO),andmonoelaidin(GME).(Reproduced Ref. 57.)

Amylose-lipid complexes have been shown[by heating the complexes in DSC pans with an excess amount of water (80% w/w) to approach zero entropyproductionmeltingconditions] to exist in the solid state as two morphologically distinct (forms I and 11) supermolecular metastable structures [15,28] (Table 6, Fig. Form I consists of helical chain segments of very little crystallographic register with one another (Table 6, Fig. It is formed at low crystallization temperatures (high rates of nucleation) and in the presence of strong complexing agents. Form I is considered to be a separate thermodynamic state with internal energy and entropy intermediate between those of a melt andof a classical crystalline system. Form11, which

.E!

180,

140

-

l30

-

120

:

.

Q0

I

0

I

10

30

40

COMPLEX

so

CONC

60

70

80

(i3

Melting peak temperatures (“C)of (a) AM-lydin, (b) AM-l-Cl6and (c) A”C12 as a function complex concentration. (Reproduced with permissionfrom Ref. 10.)

is formed at high crystallization temperatures (low rates of nucleation), exhibits a well-defined “V” X-ray pattern (Table 6, Fig. 3) typical of this particular crystalline structures. It has been suggested [15,28] that a prerequisite for the conversion form I + form I1 is the partial melting of its structure, which fosters crystallite formation (Fig. and thickening by chain diffusion. Forms I and I1 are present in many heat-processed foods, depending on the processing conditions, and they influence the textural properties and enzyme digestibility of these foods [34].

E. DigestibilityStudies Several researchers [57,62-671 have shown that lipid-complexed amylose is more resistant to hydrolysis by a-amylase and glucoamylase than is the uncomplexed amylose.Amylose complexed with lysolecithin(palmitic) and

7 Tkansition Temperatures and Enthalpies for Amylose-Monoglyceride Complexes

Cooling

Heating Monoglyceride complex in GML"." GMM~~ GMP" Dimodan PM"' GMSg GME~ GMO~.' GMLibJ

T (T)

(caVg dry

complex)

T (T)

(caVg dry

complex)

t

?

t

? ?

t t

?

&

t

?

2

?

? ?

t

&

?

t

?

t 1.5 t

t

?

t

?

? ? ?

? &

'Excess monoglycerides extracted with chloroform. correction made for uncomplexed lipids. %lycerol monolaurin. dGlycerol monomyristin. 'Glycerol monopalmitric. 'Distilled monoglyceride based on fully hydrogenated lard (c. 30% palmitic acid and 65% stearic acid) containing 92% l-monoglycerides. gGlycerol monostearin. hGlycerol monoelaidin. 'Glycerol monoolein. 'Glycerol monolinolein. Reproduced with permission from Ref. 57.

oleic acid has been shown to display substantially reduced (lysolecithin > oleic) susceptibility to a-amylase in vitro. However, in the presence of a large excess of a-amylase the same complexes are completely hydrolyzed (Figs. lOA, B). EliassonandKrog reportedthat amylose complexed with saturated monoglycerides was more resistant to enzymic breakdown than was amylose complexed with unsaturated monoglycerides. The stability of amylose-saturated monoglyceridecomplexes against enzyme hydrolysis was found to increase in the following order: monolaurin monomyristin < monopalmitin < monostearin. For unsaturated monoglyceridesin the complex, the stability was found to increase in the order monolinolein monoolein < monoelaidin. Seneviratne and Biliaderis showedthat the rate andextent of hydrolysis of helical inclusion complexes (forms Iand 11) amylose by Bacillus subtilis and porcine pancreatic a-amylases was higher in I. This ranking corroborated the structural features of these forms regarding

I

a-Amylolysis curve. Amylose (%) degraded to maltose equivalents,. when varying the degree of saturation of the lipid and enzyme concentration. free amylose p/g amylose); amylose-oleic acid complex; U,amylose-lysolecithin (palmitic acid) complex at two different enzyme concentrations. B: Amylose (% degraded to maltose equivalents versus time when exposed to a large excess of pancreatin (200 mg/g amylose). 0,Free amylose; amylose-lysolecithin (palmitic acid) complex. (Reproduced permission from Ref.

the degree of organization and perfection of ordereddomains of aggregated chains; i.e., form I (Fig. 4) with the less ordered structure (more open) in the solid state exhibits the highest susceptibility to a-amylase. This suggests that the supermolecular structure of “V”-amylose complexes influence the accessibility of a-amylases to the solid substrate andtherebycontrol its digestion kinetics. The same authors [68] also showed that at high concentrations, form Il is completely hydrolyzed in 72 h to soluble oligosaccharides. This suggests that even crystalline forms of the amylose complexes could be fully degraded under prolonged reaction time and high enzyme levels. Amyloselysolecithin complexes has been shown to be completely absorbed in the small levels were intestine of rats [65]. However, the plasma glucose and insulin significantly lower than those after intake of free solubilized amylose [65]. Seneviratne and Biliaderis [68] have postulated that the extent to which “V” amylose is hydrolyzed in vivo would depend on its supermolecular structure (as it forms during thermal processing of foods), the physical form of the foodmatrix,andthereactiontimewith the salivaryandpancreatic aamylases.

Amylopectin is a highly ramified structure containing 94-96% alpha-(l4)-glucosidic linkages and4-6% alpha-(l-6)-glucosidic linkages at the branch points. The average chain lengths of the amylopectin from A-, B-, andC-type starches are in the ranges of 23-29, 30-44, and26-29,respectively. The molecular weight of amylopectin is on the order of 107-108 1691. The cluster structure model contains three different types of linear chains, designated as B, and C chains. The A chain is linked to the rest of the molecule only through its reducing end group; the B chain is linked to an A chain, but also carries other A and/or B chains at one or more of its primary hydroxylgroups (i.e., branch points); the Cchain is unsubstituted at its reducing end group; thus, there is only one C chain per molecule. The A and B chains are of different lengths, and their degree of polymerization (DP) can vary. The linear A and B chains, in most amylopectins, have average DP values of 15 and 45, respectively. B chains form the backbone of the amylopectin cluster model and extend over two or more clusters. Each cluster contains two to four closely associated A chains.

Starch-Lipid Interactions

Complex Formation A number of studies with greatly conflicting results have investigated the extent to whichlipids interact with amylopectin. Gray andSchoch [47] demonstrated that the presence of polyoxyethylene monostearate, stearic acid, and mixtures of monoglycerides decreasedthe swelling power andsolubility of waxy sorghum starch. They postulated that amylopectin may form a complex with some of its outer branches and the fatty acids of the monoglyceride. Lagendijk and Pennings [l21 demonstrated complex formation between potato amylopectin and various monoglycerides. The amount of complexation was found to increaselinearlywithincreasedmonoglyceride chain length, showing values of 1.3, 1.6, and 3.0 m-mol X lo-* per g of AMP, respectively, for chain lengths of 12, 14, 16, 18, and 20. However, Krog [20] and Krog and Nybo-Jensen [42] reported no complex formation between amylopectin and monoglyceride. Batres and White [70] showed that potato amylopectin complexed to the greatest extent with glycerol monopalmitate (GMP), followed by glycerol monomyristate (GMM) and glycerol monostearate (GMS). The same authors showed (by iodine affinity studies) that roughly5-22% of the original amylopectin and between 4.1% and 37.6% of the original monoglycerides were tied up in the complex, with GMP showing the greatest complexing capacity. Evans [71] showed by DSC studies (moisture content >30%) the presence of two endotherms on the addition (2% level) of sodium stearoyl lactylate (SSL) to waxy maize starch. These endotherms corresponded to chain melting of SSL (30-40°C) and starch crystallization (55-80°C). However, no endotherms suggestive of an amylopectin-lipid complex were present at temperatures above 90°C. Furthermore, AH remained unaltered in the presence of SSL. Slade and Levine [72] showed that SSL and waxy maize starch, when heated at 120°C and then cooled for 24 h (prior to DSC measurements), exhibit anewendothermcenteredat 70°C. These authors attributed this endotherm to complex formation betweenSSL and waxy maize starch. This was based on the observation that SSL alone melts at 50°C and also on the absence of an endothermic transition for native waxy maize starch. Hahn and Hood [73] showed by equilibrium dialysis studies that the amount of stearic acid bound to waxy maize starch and amylose was 0.76 g/g amylopectin and 5.25 g/g amylose, respectively. Gudmundsson and Eliasson [74] showed the presence of a transition above 100°C for DSC thermograms obtained for 100% amylopectin with added surfactant/emulsifier. X-ray diffraction analysis of 100% amylopectin samples with added sodium dodecylsulfate (SDS) showed a mixture of “B” and “V” patterns. These experiments led the four authors [73,74] to speculate that complex formation

may have occurred between amylopectin and surfactant/emulsifier. Huang and White E751 reported that the percentage of complex composedof monoglycerides complexed with waxy corn were much lower for all monoglycerides [glycerol monolaurate (3.2%)GMM,(3.8%),GMP(3.8%),GMS (3.4%)] than in the study by Batres and White [70] on isolated potato amylopectin. Huang and White [75] have explained the conflicting information in the literature with respect to amylopectin-lipid interactions as follows: (1) The source of amylopectin varies among research studies and is not always reported; (2) conditions for preparing the complexes are different among the reported studies; lipids interact ‘more efficiently with amylopectin when it is isolated from the granule. Gudmundsson and Eliasson [74] also reported that complexformation is restrained when amylopectinis present in the native granule.

Starch granules when heated in excess water undergo an order-disorder phase transition called gelatinization. On cooling,the starch chains (amylose and amylopectin) in the gelatinized paste associate, leading to the formation of a more ordered structure. These molecular interactions are collectively termed retrogradation. Retrogradation is accompanied by increases in the degree of crystallinity, gel firmness, turbidity, and the appearance a “B” X-ray diffraction pattern [76]. Studies on the retrogradation of starch gels [77] using X-ray diffraction, shear modulus, and DSC have shown that the short-term development of gel structure and crystallinity in starch gels is dominated by irreversible (T 100’C) gelation and crystallization within the amylose matrix, whereas long-term increases in the modulus of starch gels were linked to a reversible crystallization (within gelatinized granules) involving amylopectin. Gel formation and retrogradation have important textural implications (mashed potatoes [45,78,79], gluten-free Chinese noodles [81], parboiled rice [82], and bread staling [go]) and dietary implications (resistant starch formation). Many researchers [74,75,84,87] have demonstrated that lipids retard retrogradation. However, the exact mechanism remains unclear. It has been suggestedthatcomplex formation between lipids and starch components and/or surface adhesion of the lipids on amylopectin chains [12,52] or on the starch granule surface [88] could hinder crystallization during gel storage. However, it is also possible that reduced retrogradation in the presence of lipids could also correspond to a decrease in the mobility (due to waterwater interaction and/or to an increase in the local viscosity of the starch

suspension) of those starch chains that are involved in double-helix formationand lateral associations during recrystallization.Gudmundssonand Eliasson [74] analyzed the retrogradation of mixtures with different amylose/ amylopectin ratios and 50%(w/w) water content in the absence and presence of surfactants/emulsifiers. The results showed that the relationship between the melting enthalpy of recrystallized amylopectin and the proportion of amylopectin was not linear (Fig. 11A). Mixtures with less than 50% amylopectin showed ahigher melting enthalpythanexpected. The effect of surfactants/emulsifiers on thatrelationship was to decrease the retrogradation of all the mixtures (due to complex formation), but they had their greatest effect on 100% amylopectin samples and their least effect on mixtures with less than 90% amylopectin (Fig. 11B, C). Biliaderis andTonogai [84] monitored the development of storage modulus (G') at 0.2 Hz and 18°C with time (20 h) for rice, defatted rice, and pea starch gels (35% w/w), alone and in the presence of lysophosphatidylcholine (LPC) added'at 4% w/wonadry starch basis. The results revealed a biphasic gelation process: an initial rapid rise in G' (due to amylose gelation) followed by- aphase of slower G' development (due to amylopectin crystallization). LPC retarded the firming process of all starch gels, particularly with respect to amylopectin recrystallization. The above authors corroborated these observations by DSC analysis of the gels over a longer storage time (up to h). Muira et al. [S51 have shown that the ability to reduce retrogradation of nonglutinous rice starch gels decreased in the order glycerol monopalmitate > glycerol monomyristate > sucrose esters of palmitic acid > sucrose esters of stearic acid > sodium stearoyl lactylate. Huang and White [75] also showed (on waxy corn starch gels) that the ability of monoglycerides to inhibit retrogradation increases with a decrease in chain length (Table In a DSC study, Ward et al. [86] showed that the extent of retrogradation of corn an& wheat amylopectin was not significantly affected by the addition of SSL. However, the onset temperatures (To) were decreased. When SSL was adided to starches, neither enthalpy values nor To were affected. The results showed that SSL does not affect the extent of recrystallization (amount of crystallites formed), but does affect the nature of the crystallites formed when amylose is not present. Gudmundsson [S71 studied the effect of added cetyltrimethylammonium bromide (CTAB)-amylosecomplex on the retrogradation ofwaxy maize, maize, and potato starches and on potato amylopectin by DSc. The starches and amylopectin samples with CTAB-amylose complex were subjected to four different heat treatments that either melted the complex or left the complex intact. The DSC measurements showed that intact CTAB-amylose complex had much less effect on decreasing the retrogradation of the starches and the amylopectin than samples with melted complex prior to

measurements (Table These findings demonstrate that lipids can interact directly with amylopectin and in that way decrease the retrogradation.

The complex formation between monoglycerides and amylose has found extensiveapplication in theproductionofdehydratedmashedpotatoes

Relation between the melting enthalpy and the proportion of amylopectin in the mixtures. ( . theoretical , line, calculated from the enthalpy of 100% amylopectin; measured values). ( A H c values are obtained from the mean of measurements at l, 2, 4, and 14 days of storage.) B: Retrogradation, measured as melting enthalpy versus time for mixtures with 90% and amylopectin with and without monoglycerides (2.9 mg/100mg mixture). (0,100% amylopectin without monoglycerides; 90% amylopectin without monoglycerides; 0,100% amylopectin with added monoglycerides; 0,10% amylopectin with added monoglycerides.) C Retrogradation, measured as melting enthalpy versus time for mixtures with 10% and 50% amylopectin with and without monoglycerides (2.9 mg/100 mg mixture). amylopectin without monoglycerides; 50% amylopectin with added monoglycerides; , 10% amylopectin without monoglycerides, 0, 10% amylopectin with added monoglycerides.) (Reproduced with permission from Ref. 74.)

+,

+

.,

Interactions -l

S

e-

Continued

8 Effect Corn Starch"

Monoglycerides on Gelatinization and Retrogradation

Waxy

Temperature' (T) &Gd

Additiveb

TOG'

Control GML GMM GMP GMS

66.7 a 65.7 c 66.2 b 66.1 b 66.7 a

42.5 a 43.4 a a

m= ND

ROC

RR' matter)dry (caVg

8.9 a 8.3 a 8.3 a 8.9 a 8.3 a

13.3 a 15.5 a 17.8 a

ND ND

4.1 a 3.5 b 3.7 ab 3.5 b 3.5 b

2.2 a 1.2 c 1.0 c 1.5 b 2.1 a

"Values are the means of four replicate determinations. Common letters indicate significance (p .05) among treatments in each column. bGML, monolaurin; GMM, monomyristin; GMP, monopalmitin; GMS, monostearin. T m ,onset temperature of gelatinization; onset temperature of retrogradation; R,, temperature range of gelatinization; RR,temperature range of retrogradation. dEnthalpiesof gelatinization and retrogradation, respectively. "Not determined. Melting of GMP and GMS coincided with the retrogradation peak, so it was not possible to determine TOR RR. Reproduced with permission from Ref. 75.

In potato granule processing, the significance of this complex formation is in the possibility of partially removing the free starch from potato cell-binding matrix into the clathrate complex using monoglyceride alone or in combination. Hoover and Hadziyev showed that glycerol monostearate was more effective than other monoglycerides in preventing stickiness in the manufacture of instant mashed potato granules. The complex-forming ability of amylose with monoglycerides and related surfaceactive monoacyl lipids has also been exploited in bread making to retard staling of bread either directly, by preventing the amylose from crystallizing or indirectly, by changing the water distribution in the bread and in extruded starch-containing products to control texture Russel et al. have shown that retrogradationof amylose-containing starches include the formation of an enzyme-resistant starch (RS) fraction (undigestible in vivo and in vitro) that comprises short-chain linear a-glucans. RS is found in food products processed by methods using relatively high moisture contents, such as cooking, baking, and autoclaving Sievert and Pomeranz haveshownthat RS is produced as a result of interaction between amylose chains. Czuchajowska et al. and Szczodrak and Pomeranz have shown that the yield (amount of crystallized amylose) of RS from autoclaved high-amylose maize and barley starches decreases on the addition of lipids. These authors postulated that RS formation

is competitively affected bythe complexation of amylose with added lipids. Mercier et al. [64] have shown the formation of amylose-lipid complexes during twin-screw extrusion cooking of cassava starch. Addition of fatty acids or monoglycerides decreased the water solubility of extruded cassava starches [64], whereas triacylglycerol had no influence. Biliaderis et al. C991 have demonstratedthe formation form 11amylose-lipid complexes during parboiling of rice. This complex was shown to decrease the loss of soluble amylose and starch during cooking of parboiled rice, and was also found to impart a firmer texture for some pressure-parboiled rice samples. The same authors postulated that amylose-lipid helices of the form I1 polymorph are thermally stable at temperatures over 100°C and can, thus, act as physical crosslinks to stabilize the rice grains during cooking in boiling water.

It is evident from this review that a greatdeal of attention has been focused on the influence of added lipids on the gelatinization, retrogradation, and digestibility properties of starches and their isolated components. However, there is a dearth of information on the interactions of monoglycerides with physically modified starches. Physical modification such as heat-moisture treatment [35], annealing [104], and defatting [5,105] has been shown to alter the structural arrangement of the starch chains within the amorphous and crystalline regions of the granule. Therefore a comparative study of the physicochemical properties of native and physically modifiedstarches in the presence of lipids may provide deeper insight into the relationship between starch molecular structure and the extent of lipid binding. Research should also be directed toward understanding the reactivity of isolated amylose and amylopectins of different chain lengths toward fatty acids and monoglycerides.

1. Morrison, W. R. Starch 33: (1981). 2. Vasanthan, T., andHoover, R.Food Chem. 43: 19 (1992). 3. Galliard, T., and Bowler, P. (1987). Morphology and composition of starch. In: Galliard, T.,ed., Starch: Properties and Potential, vol. 13. Wiley, New York, p. 55. Hargin, K. D., and Morrison, W. R.J. Sci. Food Agric. 31: 877 (1980). 5. Vasanthan, T., and Hoover, R. Food Chem. 337 (1992). Acker, L. Fette, Sei$enAnstrichmittel79: 1 (1977). 7. Morrison, W. R.,and Coventry, A. M. Starch 37: 83 (1985).

8. Hizukuri, S., Takeda, Y., and Yasuda, M. Carbohydr. Res. 94: Takeda, Y., Shirasaka, K, and Hizukuri, S. Carbohydr. Res. 132: Biliaderis, C. G., Page, C.M., Slade, L., and Sirett, R.R. Carbohydr. Polymers Banks, W., and Greenwood, C. T. Carbohydr. Res. 21: Lagendijk, J., and Pennings, H. J. Cereal Sci Today 15: Carlson, T. L. G.,Larsson, K, Dinh-Nguyen, N., andKrog, N. Starch 31: Jane. J. L., and Robyt, J. F. Carbohydr. Res. 132: Biliaderis, C. G., and Galloway, G. Carbohydr. Res. 189 Karkalas,J.,andRaphaelides, S. Carbohydr.Res. 157 Takeo, K., and Kuge, T. Agric. Bwl. Chem. 33: Pearce, L. E., Davis, E. A., Gordon, J., and Miller, W. G. Cereal Chem. Godet, M. C., Tran, V., Delage, M. M., and Bulbon, A. Znt. J. Biol. Mucromol. 15: Krog, N. Starch 23: Hoover, R., and Hadziyev, D. Starch 33: Zobel,H. F. Starch 40: Mikus, F. F., Hixon, R.M.,and Rundle, R. E. J. Am. Chern. Soc. 68: Takeo, K., Takamura, and Kuge, T. Starch 25: Manley, R. St. J. J. Polymer Sci A2: Yamashita, Y. J. Polymer Sci. A3: Yamashita, Y., Ryugo,J.,andMonobe, K. J. ElectronMicroscopy 22: Biliaderis, C. G., and Seneviratne, H. D. Carbohydr. Polymers 13: Zobel,H. F. Starch granulestructure.In:Alexander, R., andZobel, H. F., eds., Developments in Carbohydrate Chemistry.American Association of Cereal Chemists. St. Paul, MN, p. Kugimiya, M., Donovan, J. W., and Wong, R. Y. Starch 32: Eberstein, K, Hopeke, R., Konieczny-Janda, G., and Stute, R. Starch 32: Kugimiya, M., and Donovan, J. W. J. Food Sci. 4 6 van Lonkhuysen, H., and Blankestijn, J. Starch 26: Biliaderis, C. G. Can. J. Physiol. Pharmacol. 69: Hoover, R., and Vasanthan, T. Carbohydr. Res. 252: Eliasson, C., and Larsson, K. Physicochemical behavior the components wheat flour. In: Cereal in Breadmaking. Marcel Dekker, New York, p. Morrison, W. R., Tester, R. F., Snape, C. E., Law, R., and Gidley, M. J. Chem. 70: Momson, W. R., Law, R. W., and Snape, C. E. J. Cereal Sci 18: Riisom, T., Krog, N., and Eriksen, J. Cereal Sci. 2: Larsson, K. Starch 32:

Cereal

41. Larsson, K Physical state of lipids and their technical effects in baking. In: (Barnes, P. J., ed.), Lipids in Cereal Technology.Academic Press, New York, p. 237 (1983). 42. Krog, N.,and Nybo-Jensen, B. J. Food Technol. 5 77 (1970). 43. Zobel, H. F., French, A. D., and Hinkle, M. E. Biopolymers 5: 837 (1967). 29 (1976). 44. Momson, W. R., Bakers Digest 45. Hoover, R., and Hadziyev, D. Starch 33: 346 (1981). 198 (1964). 46. Yasumatsu, K., and Moritaka, S. J. Food Sci. 47. Gray, V. M.,and T. J. Schoch, Starch 14: 239 (1962). Cereal Chem. 81 48. Ghiasi, K., Hoseney, R. C., and Vamano-Marston, E. (1982). 49. Eliasson, A. C. Starch 3 7 411 (1985). 50. Eliasson, A. C . Carbohydr. Polymers 6: 463 (1986). 51. Eliasson, A. C. Thermochimica Acta 95: 369 (1985). 52. Osman, E. M., and Dix, M. R. Cereal Chem. 3 7 464 (1960). 53. Eliasson, A. C., Larsson, K., and Miezis, Y. Starch 33: 231 (1981). 54. Eliasson, A. C., Carlson, T. L. G., Larsson, K., and Miezis, Y. Starch 33: 130 (1981). 55. Bulpin, P. V., Welsh, E. J., and Moms, E. R. Starch 34: 335 (1982). 56. Biliaderis, C. G., Page, C. M., Slade, L., and Sirrett, R. Carbohydr. Polymers 5: 367 (1985). 57. Eliasson, A. C., and Krog, N. J. Cereal Sci. 3: 239 (1985). 58. Stute, V.R., and Konieczny-Janda, G. Starch 35: 340 (1983). 59. Kowblansky, M. Macromolecules 18: 1776 (1985). 502 (1981). 60. Biliaderis, C. A., Grant, D. R., and Vose, J. R. Cereal Chem. Page, C . M., Maurice, T. J., and Juliano, B. J. Agric. 61. Biliaderis, C. Food Chem. 3 4 6 (1986). 62. Larsson, K., and Miezis, Y. Starch 31: 301 (1979). 63. Kim, Y. J., and Robertson, R. J. Starch 31: 293 (1979). 64. Mercier, C . R., Charbonniere, R., and de la Gueriviere, J. F. Cereal Chem. 5 7 4 (1980). 65. Holm, J., Bjorck, I., Ostrowska, S., Eliasson, C., Asp, N. G., Larsson, K., and Lundquist, I. Starch 35: 294 (1983). 697 (1975). 66. Hanna, T. G., and Lelievre, J. Cereal Chem. 67. Acker, L. Getreide-Mehl. Boot 36: 291 (1982). 68. Seneviratne, H. D., and Biliaderis, C. G. J. Cereal Sci. 13: 129 (1991). 69. Hizukuri, S. Carbohydr. Res. 141: 295 (1985). 70. Batres, L. V., and White, P. J. J. Am. Oil Chem. Soc. 63: 1537 (1986). 71. Evans, I. D. Starch 38: 227 (1986). 72. Slade, L.,and Levine, H. Recent advances instarch retrogradation. In: Stivala, S. S., Crescenzi, V., and Dea, I. C. M., eds., Industrial Polysaccharides: The Impact Biotechnology and Advanced Methodologies. Gordon & Breach, New York, p. 387 (1987). 73* Hahn, D. E., and Hood, L. F. Cereal Chem. 64: 77 (1987). 74. Gudmundsson, M., and Eliasson, A. C. Carbohydr. Polymers 13: 295 (1990).

Hoover Huang, J. J., and White, P.J. Cereal Chem. 70: Hoover, R. Food Reviews Int. 11: Miles, M. J., Moms, V. J., Orford, P. D., andRing, S. G. Carbohydr. Res. 135: Harrington, W. O., Olson, R. L., and Nutting, M.D.Am. Potato J. 3 7 Ooraikul, B., and Hadziyev, D. Can. Inst. Food Sci Technol.J. 7 Cam,N. O., Norman, W. R., and Frazier, P. J. Crit. Reviews Food Sci. Nu& 31: Lii, C. Y., and Chang, S. M. J. Food Sci Bhattacharya, K R. In: Juliano, B. O., ed., Rice: Chemistry and Technology M C C , St. Paul, MN, p. Englyst, H. N., Anderson, V., and Cummings, J. H. J. Sci. Food Agric. 34:

85.

Biliaderis, C. G., and Tonogai, J. R. J. Agric. Food Chem. 39: Miura, M., Nishimura, A., and Katsuta, K Food Structure 11: Ward, K E.J., Hoseney, R. C., and Seib, P. A. Cereal Chem. 71: Gudmundsson, M. Carbohydr. Polymers 1 7 Germani, R., Ciacco, C. F., and Rodriquez, D.B. Starch 35: Krog, N., Olsen, S. K., Toemas, H., andJoensson, T. Cereal Foods World 34: Rao, P.M., Nussinovitch, A., and Chinachoti, P. Cereal Chem 69: Rogers, D. E.,Zeleznak, K. J., Lai, C. S., and Hoseney, R. C. Cereal Chem 65: Zeleznak, K J., and Hoseney, R. C. Cereal Chem 63: Pisesookbuntemg, N., and D’Appolonia, B. L.Cereal Chem. 60: Launay, B., and Lisch, J. M. J. Food Eng. 2: Russel, P. L., Berry, C. S., and Greenwell, P. J. Cereal Sci. 9: Sievert, D., and Pomeranz, Y. Cereal Chem. 66: Czuchajowska, Z., Sievert, D., andPomeranz, Y. Cereal Chem. 68: Szczodrak, J., and Pomeranz, Y. Cereal Chem. 69: Biliaderis, C., Tonogai, J. R., Perez, C. M., and Juliano, B. Cereal Chem. 70: Momson, W. R., and Laignelet, B. J. Cereal Sci l: Hoover, R., and Vasanthan, T. Carbohydr. Polymers 19: Hoover, R., and Senanayake, S. P. J. N. Food Research International 2 9 Hoover, R., Swamidas, G., Kok, L. S., andVasanthan, T. Food Chem. 56 Hoover, R., and Vasanthan, T. J. Food Biochem 1 7 Hoover, R., Swamidas, G., andVasanthan, T. Carbohydr. Res. 246:

8 Interactions in whey Protein/ PolysaccharideMixtures atpH 7 P. B. Fernandes* Nestec Ltd., Nest16 Research Centre Lausanne, Switzerland

INTRODUCTION Foods are complex mixtures of a wide variety of different components. It is important to understand how these various components interact witheach other to be able (1) to make cost-efficient use of functional ingredients and to control, predict, and improve textural properties of fabricated foods. Globular proteins (whey proteins, egg white, etc.) and polysaccharides are being increasingly used as functional ingredients in the food industry. Thermally set induced gels can be producedindividually from both globular proteins and polysaccharides. Whey protein isolates (WPI) are being used in food products to increase viscosity, to form gels, to stabilize emulsions or foams, or as water-binding agents (Kinsella and Whitehead, 1989). The globular proteins P-lactoglobulin, a-lactalbumin, and bovine serum albumin are the principal components ofwhey. The gelation of WPI is usually achieved upon heating above their denaturation temperature. Upon heating, the globular proteins are partly unfolded, and some of the amino acid residues that were originally located in the interior of the protein are exposed. Protein molecules then associate and aggregate because of hydrophobic in*Current affiliation: Friskies R&D Center Amiens, Amiens, France. Part this work was presentedatthe Food Hydrocolloids Conference, September 6-10, 1994, the Ohio State University, Columbus, Ohio.

teractions, disulfite bond formation, or hydrogen bonding between exposed amino acid residues on different molecules (Clark and Lee-Tuffnell, 1986). This phenomenon of heat-induced gelation is important because it contributes to mechanical properties, shapes the product, holds other food components in its rigid matrix, and retains water in the product. Considerable work has been carried out on the rheology of P-lactoglobulin and WPI systems (Paulsson et al., 1986; Stading and Hermansson, 1990; Kuhn and Foegeding, 1991; Hsiehetal., 1993; Mlekoet al., 1994; Fernandes,1994). Recently, Clark and Lee-Tuffnell (1986), Doi and Kitabatake (1989), and Smith (1994) have reviewed research on the gelation of globular proteins. Polysaccharide gums can be classified according to specific criteria, e.g., natural or synthetic, and ionic or nonionic (Glicksman, 1979). These polysaccharides provide awide range of structural characteristics, molecular shape and size, conformations in solution.Therefore, systems containing polysaccharides displaya wide range ofrheologicalproperties. Morris (1986) has reviewed research on the physicochemical andgelling properties of several types of polysaccharides. In real food gels, proteins and polysaccharides coexist in the biopolymernetworksandaffect the overall gel properties. Tolstoguzov(1986)andLedward(1994)reviewedresearch on the functional properties of protein-polysaccharide mixtures. Interactions between polysaccharides and proteins may have three consequences: their COsolubility, incompatibiIity, and complexing (Tolstoguzov, 1986). At neutral pH, weak attractive interactions may occur between anionic polysaccharides and proteins carrying a net negative charge. Even if both biopolymers present the same net charge, there is still a possibility for electrostatic interactions between the local positive charges on the protein and the polysaccharide. These interactions may be exploited to produce mixed gels with useful properties (Stansby, 1980). In concentrated proteidpolysaccharide mixtures with no association between the biopolymers, the system may separate into two distinct phases, one rich in polysaccharide and the other rich in protein. This phenomenon be described as thermodynamic phase separation between protein and polysaccharide molecules. These proteidpolysaccharide interactions are very sensitive to variables such as temperature, pH, andionic strength. Thus, any change in these variables may lead to a change from thermodynamic phase separation to protein/polysaccharide complex formation (or vice versa). Any change toward protein/polysaccharide phase separation in emulsified food will promote the creaming and flocculation phenomena of oil droplets, since proteinandpolysaccharide molecules are driven into separate phases. Thus, it is of great interest to know howproteins

behave in the presence of polysaccharides when these mixed systems are submitted to heating or cooling. proteirdpolysaccharide interactions became of Recently, the study great interest (Bema1 et al., 1987; Kato. et al., 1989; Dickinson and Euston, 1991; Burova et al., 1992). Gelation of polysaccharides (e.g., kappa-carrageenan, agar-agar, etc.) is usually achieved by lowering temperature (Fernandes et al., 1992), while in the case of globular proteins (e.g., whey proteins, egg white, etc.) gels are obtained upon heating above their denaturation temperature (Bottomley et al., 1990). If a gelling polysaccharide is used in a globular proteidpolysaccharide mixture, gelation can be achieved either by increasing or by decreasing temperature. The aim of the present chapter is to provide a description of gel formation whey proteins/polysaccharide mixtures at pH 7 upon decreasing and increasing temperature and to compare the data to those of pure whey protein systems.

II. A.

AND METHODS Materials

A commercial whey protein isolate (Le Seur Isolates, Le Seur, MN, USA) was used as the protein source. The total protein content was 95%, and it contained approximately 2% ash (Ca < 0.185, K < 0.15%, and Na < 0.38%). The polysaccharides were kappa-carrageenan, under K' form, from SBI 0, locust bean gum and guar gum from SBI, xanthan gum from Xanthan Jungbunzlauer (A), modified waxy starch Colflo 67 from National Starch (US), andmaltodextrin Cerestar 1906 [dextrose equivalent (D.E.) = 2-51 from Cerestar (F). NaCl was purchased from Merck.

B. Preparation of Solutions The whey protein isolate was dissolved in distilled water at room temperature, and pH was adjusted to 7.0 with 0.1 M HC1 or 0.1 M KOH. In the case of the addition of NaC1, the whey proteins were dissolved in solutions of the indicated salt. The polysaccharides kappa-carrageenan, xanthan gum, guar gum, and locust bean gum were first dispersed in water under moderate agitation for 1 h, at room temperature, and then heated at 90°C for 30 min while stirring. The starch and maltodextrin samples were first dispersed in water under moderate agitation for 1h, at room temperature, and then heated to 96°C while stirring. The whey protein isolate/polysaccharide mixedsystems were prepared by mixing at room temperature the solutions of whey protein and polysac-

charides at the desired ratio and total biopolymer concentration. The pH of mixtures was also controlled in order to be 7.0.

C. 1. Rheological Measurements Dynamicexperiments were performed using a Carri-Med CSGlOO controlled-stress rheometer fitted with parallel plate device (gap 2 mm; plate diameter 6 cm) with radial grooves in order to avoid gel slippage. The WPI and WPVpolysaccharide solutions were poured onto the plate of the rheometer at 25°C and covered with light oil to prevent dehydration. (a) Temperature-Increase Experiments. The WPI and the WPVpolysaccharide solution were heated gradually from 25°C to 80°C within 10 min (5.5"C/min),maintainedat80°C for 20 minandrapidlycooledto25°C (controlled bythe rheometer). Their gelation kinetics were monitored during the holdingperiodat80°Candafter cooling at25°Cbymeasuring the storage modulus G' and the loss modulus G" at a fixed frequency of 1.0 Hz. The system was allowed to set for approximately 5 h, and then the viscoelastic behavior of the gels was characterized by the frequency dependence of G' and G'' moduli. Since the and G"(o)curves were almost independent of frequency and G' was always significantly higher than G",only the final values of G', at the fixed frequency of 1.0 H z , for WPI alone and WPI/polysaccharide mixtures are presented. (b) Temperature-DecreaseExperiments. The G' and G" moduli were obtained from temperature sweep by cooling the systems from 40°C to 5"C, maintaining them at 5°C for 5 h and then heating up to 55°C at a rate of l"C/min. The sol-gel transition temperature was defined by the crossover of the G' and G" moduli, as described for physical gels (Lin et al., 1991; Carnali, 1992). The strain amplitude was fixed at 0.01.

2. DifferentialScanningCalorimetryExperiments DSC experiments were performed on 12% WPI and 12% WPI/O.l% kappacarrageenan or guar gum systems to obtain the denaturation curve. The instrument used was a Setaram Micro-DSc. All samples were heated from 25°C up to 95°C at a rate of l"C/min.

3.

Microscopy

The gels of WPI and WPI/polysaccharidemixtures were prepared by heating themat90°C during minutes. Small cubes of the gels were fixedin

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261

phosphate-buffered 2.5% glutaraldehyde, postfixed in2% osmium tetroxide, dehydrated in a graded ethanol series, and embedded in a low-viscosity resin (spurr). A Reichert ultramicrotomeOMU-3 was used to obtain semithin sections (1 Fm) that were examined by phase contrast light microscopy.

RESULTSANDDISCUSSION A. Effect of Temperature Increase on Whey Protein Isolate/Polysaccharide Mixed Systems The viscoelastic properties of whey proteins as a function of protein concentrationupon heating were previouslydemonstrated(Fernandes, 1994) using similar experimental conditions as those described in this chapter.The gel point was observed at concentration of 11%. The interactions between the WPI and polysaccharides were studied by adding increasing amounts of polysaccharides to the 12% WPI gel. The final values of the G’ modulus of mixed gels composed of 12% WPI plus increasing amounts of various polysaccharides are shown in Fig. 1. Polysaccharides were kappa-carrageenan, xanthan gum, locust bean gum, guar gum, starch, and maltodextrin. The values of the G‘ modulus of WPI alone are also given for comparison (Fernandes, 1994). The mixed gels of WPI/kappa-carrageenan, xanthan, locust bean gum, and guar gum were opaque, while the mixed gels of WPVstarch and maltodextrinwere transparent. It should be noted that the pure WPI gels withoutaddition of ions were also transparent. The addition of small amounts of kappa-carrageenan and xanthan gum up to 0.1-12% WPI increased strongly the concentration dependence of G’. The addition of galactomannans to WPI had the same effect, but this increase was much less pronounced than with WPI/kappa-carrageenan or xanthan-gum mixed gels. Starch addition did not significantly change the concentration dependence of G’, while maltodextrin lowered it drastically. The microstructures of WPI/polysaccharide mixed gels are presented in Fig. 2. The WPI/maltodextrin gel is not shown because with this sample we did not find any organized structure distinct from the gel network of pure WPI. In all other WPI/polysaccharide-mixed gels, organized structures dispersed within the WPI gel network were observed. We believe that these structures represent phase-separated polysaccharides. In the case of kappacarrageenan, guar gum, and locust bean gum, these structures are globules with a diameter of about 1-5 Fm. They may be designated as microgels. It is interesting to note that the WPI/kappa-carrageenan gel shows a larger number of microgels per volume unit thando those gels of WPI/guar gum or locust bean gum mixtures. Thus, each microgelof kappa-carrageenan should have fewer molecules of this polysaccharide than the microgels of

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[12%WPI + polysaccharide] concentration (“h) Final values of the G‘ modulus mixed gels composed of 12% W P I plus increasing amounts of polysaccharides. Polysaccharides were kappacarrageenan,xanthangum,guar gum locustbeangum,starch,andmaltodextrin. Values of the G’ modulus of W1 alone are displayed for comparison. Frequency: 1.0 Hz, temperature:

the galactomannans. Since gel formation of kappa-carrageenan is possible above the transition temperature of helical conformation (Plashchina et al., the kappa-carrageenan system may contribute to a higher gel rigidity (active filler). This result is in agreement withthe rheological measurements presented in Fig. 1. The texturizing effect of kappa-carrageenan in the WPI gel is considerably higher than that of galactomannans. This comparison is also valid in terms of synergistic interactions. In the case of WPI/xanthan mixed gels,we also observed the presence of microgels; however, theseseem to aggregatehoalescence, possibly before gelation andto form larger structures. This occurredwithoutand in the presence of NaC1, which does not seem to affect the final gel network. In the case of WPI/starch mixed gel, few starch granules dispersed in WPI gel network can be observed. Whether aspecific polysaccharide forms a heat-induced mixedgel with a globular protein depends on its ability to interact with the protein. The

Phase contrast light microscopy micrograph

showing networkstructure

12%WPI/O.l% polysaccharide mixed gels atpH 7 after heat treatment (30 min at 90°C).

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chemical structure, molecular weight, and rheological properties of the individual polysaccharides determine the final rheological characteristics and structure of WPI/polysaccharidemixed gels. The synergistic interactions observed in the WPI/polysaccharidemixed gels can be ascribedmainly to volume exclusion effects originating from incompatibility between unlike biopolymers. The phase separation phenomenon leads to an increase in the effective concentration of WPI and consequently to an increase in gel strength. For starch granules this effect is less pronounced. It seems that the starch granules somehow replace the missing protein molecules, having in the WPI/ starch mixed gel a similar viscoelastic behavior to that of WPI gel at the same total polymer concentration. Maltodextrin addition does not present any synergistic effects with WPI. Apparentlymaltodextrins disturb the structure of a WPI gel network. The weakening effect displayed by the maltodextrin in WPI/maltodextrin blends compared to that of WPI/starch mixture is related to its low molecular weight. DSC experiments (heating at a constant rate) were conducted on 12% WPI/O.l% kappa-carrageenan or guar gum mixtures in order to compare the effect of a charged to that of a neutral polysaccharide on the WPI denaturation process (Fig. 3). The same effect was observed with both polysaccharides. The WPI denaturation temperature did not change in the presence of the polysaccharides, but a slight decrease (about 16%) of the enthalpy was observed (Table 1).When the evolution of the heat flow vs. temperature for the pure WPI is compared to that of the WPI/polysaccharide mixtures it is evident that denaturation commences earlier in the case of the WPI/polysaccharide blends (at 30°C vs. for the pure WPI solution). This means that the polysaccharides somehow promote a slight partial denaturation (increase of the hydrodynamic volume occupied by the protein molecules) of WPI at low temperature (-30°C). This assumption is also supported by the

Table Effect of NaClConcentration on the Thermal Behavior and Gel Structure of Whey Protein/Kappa-CarrageenanMixed

Systems Tm (“C)

G’ at 5°C

(“C) 7

28

10

15 20

35

50

Tg NaCl, M

30

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(Pa)

lower enthalpy values obtainedby DSC for the WPVpolysaccharide mixtures. It is interesting to note that the sulfate groups of kappa-carrageenan did not show any peculiar effect on the protein denaturation.

B. Effect of Temperature Decreaseon Whey Protein Isolate/Polysaccharide Mixed Systems The gelation ability of mixtures of WPI (12%) with kappa-carrageenan, xanthan, locust bean gum, guar gum, starch, and maltodextrin (polysaccharide concentration 0.1%) with temperature decrease was initially investigated (by oscillatory shear experiments-data not shown). With the exception of the WPIkappa-carrageenan blend, all the other WPI/polysaccharide mixtures did notshow any gelation.Therefore, only the viscoelastic behavior of WPI/ kappa-carrageenan mixtures was further studied by means of temperature sweep measurements. The evolution of the G' and G" moduli with decrease and increase of temperature for the 12% WPI/O.l%kappa-carrageenanmixture without NaCl additionis presented in Fig. The sol-gel transition occurred at -7°C (gelation temperature, T,). Then, at 5°C the G' modulus after 5 h attained a steady value of about 10 Pa. The melting temperature was -28°C. These values are presented in Table 1. The transition temperature of kappa-carrageenan at a concentration of 0.1% is in the same range (6-8°C without thermal hysteresis and no gel formation) (Rochas and Rinaudo, 1980; Fernandes et al., 1991). This indicates that kappa-carrageenan is the primary agent responsible for the gel network formation in the mixed system. The presence of WPI induces gelation and the appearance of a significant thermal hysteresis (T,,, - TB 20°C). The appearance of a thermal hysteresis in a kappa-carrageenan gel is directly related to the formation of carrageenan aggregates (Rochas and Rinaudo, 1980). Obviously,the presence of WPI in the 0.1% kappa-carrageenan solution promoted aggregation of carrageenan molecules. The synergistic interactions just described for the WPIkappa-carrageenan mixed systems can be interpreted as a stabilizing effect of kappacarrageenan aggregates by whey protein molecules. This may be explained by phase separation (volume exclusion phenomena) between globular proteins and the polysaccharides, and also possibly by the presence of local links of the type protein.NH:-"SO,.carrageenan. The effect ofNaClconcentrationonWPVkappa-carrageenanmixed systems is presented in Table 1. The evolution of the G' and G' moduli with decrease and increase of temperature for the 12% WPI/O.l% kappa-carrageenan mixture in 0.1 M NaCl as example is presented in Fig. The gel strength increased with the presence of NaCl. The gelation temperature (T,)

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oteinPoiysaccharide MixturesWhey

269

and melting temperatures (T,,,) also increased in the presence of NaCl. The effect of NaCl addition on the thermal behavior and gel structure of W V kappa-carrageenan mixed systems should be explained by the fact that the sodium cations are gel-inducing ions of kappa-carrageenan systems (Rochas and Rinaudo, and also because the increase of the ionic strength has a strong influence in the intermolecular interactions between globular proGault and Korolezuk, teins (Gault and Fauquant,

On heating, the WPIkappa-carrageenan, xanthan gum, guar gum, and locust bean gum gels presented synergistic interactions, while the WVstarch and maltodextrin blends did not show synergistic effects. The charged polysaccharides (kappa-carrageenan andxanthan gum) presentedthe most important synergistic interactions. Apparentlythere is a thermodynamicincompatibility between whey proteins and these polysaccharides. This seems to be supported by their microstructure as observed by phase contrast light microscopy. On cooling, the WIkappa-carrageenan mixture presented important synergistic effects that led to gel formation and the appearance of a thermal hysteresis. This effect increased in the presence of NaC1.

Financial support is gratefully acknowledgedfrom the European Community (FLAIR program). I acknowledge Mr. Alois Raemy for technical assistance in the DSC experiments and Ms. M.-L. Dillmann for the microscopy observations and the help of Professor V. B. Tolstoguzov and of Dr. F. Dannenberg for fruitful discussions.

Bernal, V. M., Smajda, C. H., Smith,J. L., and Stanley, D. W. Interactions in proteid polysaccharide/calciumgels. J. ofFood Sci. 1121-1125 (1987). Bottomley, R. C., Evans, M. T. A., and Parkinson, C. J. Whey proteins. In:Hamsed, P., ed., Food Gels. Elsevier Science Publishers, pp. (1990).

270

Fernandes

Burova, T. V., Grinberg, N. V., Grinberg, V. Y., Leontiev, A. L., and Tolstoguzov, V. B. Effect of polysaccharides upon the functional properties of 11s globulin broad beans. Carbohydr. Polym. 18: 101-108 (1992). Camali, J. 0. Gelation in physically associating biopolymer systems. Rheologica Acta 31: 399-412 (1992). Clark, J. E., and Lee-Tufhell, C. D. Gelation of globular proteins. In: Mitchell, J. R., and Ledward, D. A., eds., Functional Properties of Food Macromolecules. Elsevier Applied Science, New York, pp. 203-272 (1986). Clark, A. H., Richardson, R. K, Robinson, G., Ross-Murphy, S. B., and Weaver, A. C. Structure and mechanical properties of agar/BSA co-gels. Prog. Food Nu& Sci. 6 149-160 (1982). Dickinson, E., and Euston, S. R. Stability of food emulsions containing both protein and polysaccharide. In: Dickinson, E., ed., Food Polymers, Gels and Colloids. Royal Society of Chemistry, Cambridge, England, pp. 132-146 (1991). Doi, E., and Kitabatabe, N. Structure of glicin and ovalbumin gels. Food Hydrocoll. 327-337 (1989). Femandes, P. B. Viscoelastic characteristics of whey protein systems at neutral pH. Food Hydrocoll. 277-285 (1994). Femandes, P. B., Gonplves, M.P., and Doublier, J. L. Phase diagrams in kappacarrageenanAocust bean gum systems. Food Hydrocol2. 5(1/2):71-73 (1991). Femandes, P. B., GonGalves,M. P., and Doublier, J. L. Effectof galactomannan addition on the thermal behaviour of kappa-carrageenan gels. Carbohydr. Polym. 1 9 261-269 (1992). Femandes, P. B., Gonplves, M. P., and Doublier, J. L. Influence of locust bean gum on the rheological properties of kappa-carrageenan systems in the vicinity of gel point. Carbohydr. Polym. 22: 99-106 (1993). Gault, P., and Fauquant,J. Aptitude B la gtlification thermique de la P-lactoglobuline: Influence du pH, de l’environment ionique et de la presence des autres prottines du lactoserum Lait 72: 491-510 (1992). Gault, P., and Korolezuk, J. Effects of physim-chemical parameters on heat-induced gel firmness and water-holding capacityof whey protein concentrate. In: Visser, H., ed., Protein Interactions.V.C.H. Publishers,.New York, pp. 193-207 (1992). Glicksman, M. Polysaccharide gums. In: Blanshard, J. V. M., and Mitchell, J. R., eds., Polysaccharides in Foods. Butterworths, London, pp. 185-204 (1979). Hsieh, Y. L., Regenstein, J. M., and Rao, M. A. Gel point whey and egg proteins using dynamic rheologicaldata. J. Food Sci. 116-119 (1993). Kato, A., Sato, T., and Kobayashi, K Emulsifying properties protein-polysaccharide complexes and hybrids. Agric. Biol. Chem. 53(8): 2147-2152 (1989). KinselIa, J. E., and Whitehead, D.M. Proteins inwhey:Chemical, physical and functional properties. In: Kinsela, J. E., ed., Advances in Food and Nutrition Research. Academic Press, New York, pp. 343-438 (1989). Kuhn, P. R., and Foegeding, E. A. Factors influencing whey protein gel rheology: Dialysis and calcium chelation. J. Food Sci. 56(3): 789-791 (1991). Ledward, D. A. Protein-polysaccharideinteractions. In: Navam, S. H., and Ziegler, G. R., eds., Protein Functionality in Food Systems. Marcel Dekker, New York, pp. 225-259 (1994).

Lin, Y. G., Mallin, D. T., Chien, J. C. W., and Winter, H. H. Dynamic mechanical measurement crystallization-induced gelation in thermoplastic elastomeric poly@ropylene). Macromolecules Mleko, S., Achremowicz, B., and Foegeding, E. A. Effect of protein concentration on the rheological properties of whey protein concentrate gels. Milchwissenschafr 4 9 0 : Moms, V. J. Gelation of polysaccharides. In: Mitchell, J. R., and Ledward, D. A., eds., Functional Properties of Food Macromolecules. Elsevier Applied Science, New York, pp. Paulsson, M., Hegg, P. O., and Castberg, H. B. Heat-induced gelation of individual whey proteins. A dynamic rheological study. J. Food Sci. 51: Plashchina, I. G., Muratalieva, I. R., Braudo, E. E., and Tolstoguzov, V. B. Studies of the gel formation of kappa-carrageenan above the coil-helix transition temperature. Carbohydr. Polym. Rochas, C., and Rinaudo, M. Activity coefficients of counterions and conformation in kappa-carrageenan systems. Biopolymers 19: Smith, D. M. Protein interaction in gels: Protein-protein interactions. In: Navam, S. H., and Ziegler, G. R., eds., Protein Functionality in Food Systems.Marcel Dekker, New York, pp. Stading, M., and Hermansson, A. M. Vlscoelastic behaviour of P-lactoglobulin gel structures. Food Hydrocoll. Stansby, G. Proteinaceous gelling systems and their comolexes with polysaccharides. Food Chem. Tolstoguzov, V. B. Functional properties of protein-polysaccharide mixtures. In: Mitchell, J. R., and Ledward, D.A., eds., Functional Properties of Food Macromolecules. Elsevier Applied Science, London, pp.

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Polysaccharide-Metal Interactions Monique A. V. Axelos Institut National de

,

Recherche Agronomique Nantes, France

During the last 30 years, polysaccharide-metal interactions have attracted considerable interest from researchers in scientific, technological, and medical fields as well as from the industrial community. Under suitable conditions, the binding of metal ions to polymer leads to gel formation. The gels are used traditionally in food [l-31 and more recently in fields Phase separation and precipitation mayalso be obtained, and these maybe adapted to removing heavy metals from aqueous solution [5] and to human prophylaxis substances against poisoning by toxic metals [6]. To a first approximation, such applications are based on the high selectivity shown by polyuronatestowardmultivalent cations and by sulfated polyions toward monovalentcations. Several models have been proposed to explain the polyion/cation interactions, including delocalized and cooperative interactions and site binding [7-lo]. None of the models gives satisfactory predictions on the number of bound cations, which highlights the complexity of this problem. Metal cation binding depends on a large number of parameters, of which charge density and polymer concentration are the most relevant. But the nature of the charged groups, their neighborhood, and the cations seem also to playa role. Binding processes may be studied experimentally in polymer solutions by determining the metal ion activity or the free metal ion concentration. Different methods, viz., equilibrium dialysis, selective ion electrodes, dye-binding spectrophotometry, electron spin resonance PR),

Axelos and nuclear magnetic resonance (NMR) depend on the nature of the cation. From the binding isotherms thus measured, a quantification of the polymer/ cation affinity may be done and the influence of ionic strength, pH, temperature, etc., evaluated. As already mentioned, polysaccharide/cation interactions lead to two transitions: sol-gel transition and phase separation. The study of these two phenomena is of considerable practical interest. Below the sol-gel transition concentration and above the transition temperature, viscous solutions develop. Polysaccharide viscous fluids are used as thickeners, and viscoelastic fluids are used as spreads. Above the sol-gel transition concentrations and below the sol-gel transition temperature, viscoelasticity develops. Generally, a high cation concentration is conducive to precipitation of a polymer- and cation-rich phase and a relatively pure solvent phase.

II. A number of methods have been developed for measuring metal ion activities. Commercially available, ion-selective electrodes allow the determination of some monovalent and divalent cation activities ma+, K+,Ag+, Pb2+,Cd”, Ca2+)and some anion activities (Cl-, Br-, CN-, SCN-, S’-, F-, NO;). The linear response domain is between 1 M and 10-3-10-5 M, depending on the ion [ll-131. The major problem contributing to erroneous results arises from significant interference by other cations and halides that are veryoftenpresentin the polymer solution. The equilibrium dialysis method often suffers from relatively large errors, especially at low metal ion concentrations or when the amount of metal ion bound is small [14]. The dye spectrophotometricmethod uses a relatively weak dye-metal ion equilibrium as an activity probe for the metal ion [15]. By working at very low dye concentrations relative to the polyionand metal concentrations, the polyion-metal ion binding is not disturbed bythe dye. The two-wavelengths ratio method is suitable at M concentrations. This method is easy to perform and gives reliable results, but it needs a careful selection of the dye, depending on the salt and solvent conditions: for example, eriochrome black T is recommended for Mg” at pH 8, and tetramethylmurexide for Ca2+ and Zn2+ at pH The ESR method is, of course, devoted to metal ions with unpaired electrons,such as Mn2+[l61 and Cuz+[17]. Results from ESR on C u 2 ’ give information about the local structure of the bound cation and the coordination number. NMR spectroscopy may be used to evaluate the selective binding of monovalent ions such as Na’, K+, Rb+, CS+, and I- to polysaccharides [B-191. The formation of complexes be-

nteractions Poiysaccharide-Metal

275

tween borate and hydroxycompounds have also been identified by"B-NMR spectroscopy [20]. In contrast to the potentiometric and equilibrium dialysis methods, electron spin resonance (ESR) and NMR enable determination of the number of bound cations, even in the absence of added salts.

B. Binding Isotherms and Theoretical Approaches The mean ionic activity obtained from the methods just described permits the calculation of the fraction of free (M,)and bound (Mb)metal ions. Data are very often expressed as binding isotherms, such as the bound cations per mole of polyion versus the total amount of cations added per mole of polyion [13-211 or the bound cation per mole of polyion versus the free cation concentration [12]. Another very much used representation is the Scatchard plot, which is the plot of the moles of cation bound per mole of macromolecule (P) divided by the moles of free cation versus the moles of cation bound per mole of macromolecule. In the simple case of identical independent sites, this plot follows the following linear equation:

The ordinate intercept is equal to the number of sites n dividedby the polymer/cation dissociation constant k; the abscissa intercept is n, and the slope is -k". In many cases, however, the plot is nonlinear but displays a concave or a convex curvature. Concavity indicates the existence of either different independent sites on the macromolecule or that k is inconstant but increases with the number of bound cations. This nonlinear interaction is called anticooperativizy [12,13,21]. If cations are boundmoreandmore strongly as binding proceeds, a convex curvature is obtained, which is an indication of cooperativity [13,22,23]. Other representations depend on the theoretical interpretation given [8]. Ion binding is normally analyzed by the Manning theory of condensation phenomena in solutions of linear polyelectrolytes [24]. This theory predicts that counterions of charge number Z will becondensedon the polyion until the charge density parameter 5 is reduced to the critical value 1/Z (5 = e*/&kTb),where is the proton charge, E is the dielectric constant of the solvent, T is the absolute temperature, b is the average spacing betweencharged groups along the axis of the polyionchain,and k is the Boltzmann constant. Within the context of this theory, counterion binding to polyions is a purely electrostatic process independent of the nature of anionic sites and counterions. The other mechanism proposed toexplain complexation betweensmall ions and polyelectrolytes is specific interaction. This complexation is a mon-

omer propertydescribable phenomenologically byan effective complexation constant. However, macromolecular systems exhibit some very particular features arising from the fact that ligands are connected to the polymer chain, which greatly limits the analogy with classical complexation between small molecules [25,26]. More recently, a theory of the complexation of polyanions by divalent cations has been proposed, whereby the formation of mono- and dicomplexes is possible [27]. Monocomplexation locally inverts the charge of the polyelectrolyte and transforms it to a polyampholyte. The formation of a dicomplex creates a bridge between charged monomers.The complexation constants for mono-anddicomplexation strongly decrease at high ionic strengths. If the complexation constants are large enough, the polyelectrolyte precipitates atmoderate salt concentrations. If monocomplexation is favored, the polymer redissolves by strong charge inversion at moderate salt concentrations. Thus, successive polymer precipitation and redissolution occur as the divalent salt concentration is increased. If dicomplexation is favored, precipitation only results. Redissolution of the dicomplex occurs at high ionic strengths, where the complexation is inefficient.

THE PERTINENT ION-BINDING PARAMETERS A. Polysaccharide Charge Density and Distribution Charge density is the key parameter of the counterion condensation theory. The higher the charge density, the stronger is the interaction of cations with anionic groups. For a large number polysaccharides, the activity coefficient and transport parameter for monovalent cations, obtained from conductimetry and potentiometry,were found to decrease when the charge den[28-291. However, sity increased, in good agreement with Manning’s theory some discrepancies were obtained for polymers with high charge densities, like pectate; these are characterized by higher values than predicted in the presence of Na+ [29]. Large deviations between the experimental and the calculated values for the conductivity have also been reported for carboxymethylcellulose in m C 1 solutions [30]. Results obtained for divalent cations in the case of polyuronates were also in serious disagreement with Manning’s theory; for example, experimental values obtained for the system, polygalacturonate-Ca2+or Sr2+were considerably lower than predicted, especially when the mean charge density increased or when a blockwisearrangement of thecarboxyl groups was tested [21,29]. The same results were obtained for carboxymethylcellulose in the presence Caz+and BaZ+[30]. All these data suggest that there must be specific effects for each counterion owing to its strong interactions with the polymer anion. In contrast, no specificity was found for Mgz+ or any

nteractlons Polysaccharide-Metal

divalent cation by sulfated polyions like dextran sulfate and carrageenan [21-311. The general trend emerging from these results is that the manning theory provides a qualitatively correct description of the equivalent conduction of rodlike polyelectrolytes; but when counterions strongly interact with a polyion, specific effects are a function of the ion.

Metal Ion The conformation of sugar rings also plays a role in the polysaccharidecation interaction, as demonstrated a long time ago by Kohn and Larsen [32] with calcium salts of different polyuronic acids. They found that the activity coefficients of calcium polyguluronate and calcium pectate were very close to each other and several times lower than that of calcium polymannuronate. The major effect was identified with the cation. Many studies report the determination of an affinity series for a given polysaccharide. It is instructive to distinguish between three types of polysaccharides, viz., polysaccharides containing uronic acid, polysaccharides containing sulfate, and polysaccharides containing a primary amine group. Members of the first class, e.g., alginate and pectin, display nonspecific electrostatic interactions with monovalent salts but show a highselectivity toward divalent cations. The following decreasing scale of selectivity has been measured for pectins with different degrees of methylation, irrespective of botanical origin: Pb" Cu2+>> Zn2+> Cd" > Ba" > Ni" > Caz+ > CO" Mn2+ >> Mg2+ [22,23,29,33-351. A slightly different affinity series was obtained for alginates C u 2 + > Cd" > Ba2+> Ni2+> Ca" > CO'+ > Mn"[36-381. These sequences are obviously due to the electronic structure of the cations rather than to their size. In contrast to alginate and pectin, the microbial polysaccharide gellan, bearing a glucuronic acid, displays a lack of such specificitywith the alkaline-earthcations;i.e., Pb2+ > > Zn2+ > Ba2+ r~ Sf2+ Caz+ r~ MgZ+.Furthermore, gellan gum is sensitive to monovalent salts that are able to induce gelation, with a gel strength increasing the order Li' < Na' < K' < CS' < H+ [39]. Specific interactions initiating gelation have also been reportedfor xanthan in the presence of trivalent chromic ions 1401. The most striking feature of sulfated polysaccharides (e.g.,K-carrageenan) is their specific interactions with some monovalent cations. These gelling polysaccharidesundergo a very cation-sensitive coil-helix transition, following the order Rb+ > > K+ > [41-421. For N(CH3);, Na+, and Li', the interactions are predominantly of a nonspecific electrostatic nature [43]. The same nonspecilicity was reported for divalent cations; nevertheless, with respect to their helix-promoting efficiency, the following

-

-

T

C S +

m+

M

A

+

Axelos

order was found:Ba" > C a z + > S?' > M C > Zn" > CO" [41]. Furcellaran displays the same sensitivity toward monovalent salts as K-carrageenans [44 -451. On the contrary, it has been found that pure L-carrageenans are sensitive not to monovalent cations but to divalent cations. Calcium, for example, has a strong effect on the contormationof L-carrageenan [46]; such an effect may be predicted from nonspecific electrostatic interactions alone [47]. Polysaccharides like chitinandchitosan, bearing aprimaryamine group, are positively charged in acidic media. Their potential in nonfood applications comes nevertheless from their ability to form complexes with many of the transition metals: for example, chitosan strongly interacts with Cu2+to form [ C u NH2(0H)2]0[48] and with Fe2+,HgZ+,and Pb2+to form similar adducts

C. Cooperativity The binding properties of cations to polyuronic acids and their oligomeric fragments havebeen the subject ofmany experimental investigations, in order to explain the excess of cation binding observed, compared to what is expected from purely coulombic interactions.Evidence for cooperativity, suggested by the shape of the Scatchard plots or the determination of the empirical Hillparameter, was generally ascribed to binding in "egg-box cavities." The analysis of much published dataon calcium binding to pectin reveals some contradictions. An anticooperative effect was observed at low pectate concentration in 0.01 M NaCl [21] or at any ionic strength between 0.02 and 1 M NaCl [12]. Braudo et al. [50] showed in 0.1 M NaCl that the binding is cooperative and that the higher the charge density, the more cooperative are the interactions. Garnier et al. [l31 discovered an anticooperative effect associated with the electrostatic interactions of counterions and polyelectrolytes in water, and a net cooperativity in 0.1 M NaC1. From the parametrization of binding isotherms, it is difficult to really discriminate between long-range electrostatic and short-range specific modes of interactions; other independent techniques must be employed, e.g., NMR and EPR spectroscopy. From the analysis of 13C-NMR spectra and relaxation times, Wangetal. [51] havefoundthatin the calcium-alginatesystems,there exists a specific interaction characterized by a strong cooperative bond, involving all functional groups, between guluronate residues and Ca". On the other hand, in transition metal (Cu, CO, Mn)-alginate systems, complexation occurs with bothmannuronateand guluronate residues: other functional groups, such as hydroxyl, do not participate. Recently it has been observed on the excited proton resonance spectra of cooper complexes that the distribution of copper between mannuronate and guluronate depends onsolvent conditions [52].

Polysaccharide-Metal Interactions

Cosolutes added in aqueous solutions often have dramatic effects on the system properties.Among them, sequestering agents, e.g., phosphate,citrate and ethylenediamine tetraacetic acid, may reduce or completely inhibit the polysaccharide-cation interaction by forming stable complexes with the cations. Control of the release of cations in the polymer solution is then obtained, for example, by changing the pH. A mixture of the sequestrant and slowly hydrolyzing D-glucono-&lactone,for example, has been used to procure homogeneous alginate gels by internal liberation of Ca2+ It has already been stated that polysaccharides subject to conformational transition, e.g., galactan, are very cation sensitive. An anion-specific effect on ionic polysaccharides such as xanthan and succinoglycan and on neutral polysaccharides such as agarose has also been observed at concentrations of salt higher than 0.2 M. For a given calcium salt concentration, the transition temperature of succinoglycan increased in the order I- Br< ClFor agarose, sodium salts destabilize the gel in the order NaBr NaN03 NaSCN The same sequence, reversed, has been found for K-carrageenan The effects coincide with the well-known Hofmeister, or lyotropic, series. The molecular interpretation of this phenomenon is still the subject of debate. Results from the nuclear spin relaxation of anions on and K-carrageenan have been interpreted in terms of ion agarose binding; the explanation is unclear. Norton et al. proposed a bulk effect leading to a change in the solvent structure. Temperature is yet another key influence on polysaccharide-metal interactions. Metal binding normally decreases with increasing temperature.

Many biopolymers dispersedin aqueous mediaseparate into liquid and solid phases by the addition of salts to the sol,or, as we havelearned,may alternatively gel. A precipitate (distinguished from a gel) may result from phase separation. Syneresis may be a consequence. These occurrences and the conditions inducing them, being of considerable practical importance, delineated by salt concentration are best studied with phase diagrams versus polymer concentration. The phase diagrams usually contain two main regions separated by a demixing line below which a homogeneous, transparent solution or gel is observed. Above the line, two phases are in evidence: a transparentphase low in polymer concentration in equilibrium with a polymer-richphase appearing as a precipitate, an aggregate,or a uniformly

Axelos

turbid dense gel. Gelation may be determined visually by slowly tilting test tubes or more precisely by rheology.

B. PhaseDiagrams Three major types of phase diagrams are distinguishable (Fig. 1).In Type I phase diagrams, high salt concentration is required to induce the polymer to precipitate at a critical concentration (Csalt*) that is almost independent of the polymerconcentration.TypeIphase diagrams correspond to the called type-H system introduced by Ikegami and Imai [60]. In the example of high-methoxyl pectins = 0.37 M NaCl was required for precipitation, but more than 1 M NaCl was necessary to precipitate alginates (5 = 1.6) [59]. Type I1 phase diagrams are characterized by very low C&,* that increases linearly with the polymerconcentration. The slope of the demixing line increases with 5. Type I1 phase diagrams correspond to the Type L system of Ikegami and Imai [60] obtained for pectin, for example, in the presence of copper [61]. Type I11 phase diagrams show a concave shape of the demixing line, and opposite to Type 11, the higher the 5, the lower is C&,*. Type I11 behavior is intermediate between weak cationpolyion interactions, corresponding to Type H, and strong interactions, corresponding to Type L. These last phase diagrams were observed for pectin in the presence of Ca”and for alginate with copper in the presence of monovalent salts [59]. Generally, for a given cation a system evolves toward Type I, then 111, thenType 11, as 5 increases.Fora given polymer, with a constantcharge density the same evolution is noticed when the added cation changes from monovalent to divalent, and within the divalent group, affinity series obtained in this way from Mg2’ to Mn2+to Ca” to is in agreement with direct measurements of the number of bound cations. The same Type 111 phasediagrams were obtained for kappa-and iotacarrageenans with monovalent and divalent salts [62].

C. Gelation Figure 1 shows that subdivisions of the polymer-concentration graph may appear in Type I1 and I11 phase diagrams. Indeed, the sol-gel transition is characterized by the emergency elastic properties in the system, which can thus be followed by mechanical measurements [63-661. The transition is quite sensitive to heat. elevated concentration of cations is required for gelation at higher temperatures than at lower temperatures, andthe area

*c is the charge density parameter, defined earlier.

TYPE

I

2 phases sol

TYPE

2 phases

pq TYPE In

---"""_

Polymer Concentration

Schematicrepresentation of thethreemajor types phasediagrams: concentration cation added against polymer concentration; demixing line (-), sol-gel transition (-----).

on the phase diagram the gel phase thus decreases The area of the gel phasedepends also indirectly on the polymer-cationaffinity. Strong interactions lead very easily to precipitation; weak interactions do not yield a gel, as has been illustrated with pectin, in which it is possible to change easily the polymer-cation affinity by increasing the degree of methylation (DM). In Fig.2, it is seen in the presence of Ca2+that the gel phase increases as the DM increases. At low DM, the demixing line appears at very low metal concentrations. At high DM,there is no gelation, because not enough intermolecular crosslinks are formed, and the divalent cations act like monovalent cations, begetting precipitationathigh salt concentration('Qpe I phase diagram). Inthe presence copper vis-a-vis calcium, bothlines trend downwards and the gel phase with Cu" is very restricted

Axelos 0.01

0.006

2

0 10

-l 12

Pectin concentration

2 Phase diagram: molar concentration added calcium versus concentration pectin with different degreesof methylation (DM) in M sodium chloride. 0DM = DM = 28%.

In helix-formingbiopolymers, gelation is precededbyacoil-helix transition that is equally sensitive to temperature, solvent conditions, and the nature and concentrationof ions. The gelation temperature is customarily determined from a phase diagram constructed as a plot of the total ionic concentration against inverse temperature. Gel networks develop from isothermal and reversible aggregation of double helices when adding K’ by dialysis Despite considerable research effort the molecular mechanisms by which the helical carrageenans form a gel network’have not been satisfactorily established.

POTENTIALITIES A.Heavy-MetalBiosorption The potential of biopolymers for use in the removal of toxic metals from dilute aqueous solutions has long been recognized. Biosorption is an alternative to the traditional physicochemical methods of treating industrial efThis method utilizes natural products (agricultural and forestry fluents

Interactions Polysaccharide-Metal

wastes,microorganisms, humic substances, aquatic and marine algae) as adsorbents The method is assumed to be metabolism independent, inasmuch as the substrate is dead biomass. By this method, the substrate complexes with residual metal through active sites (e.g., carboxyl and sulfated groups), each having different strengths.The performance of the bioadsorbents is often enhanced by crosslinking (with epichlorohydrin, formaldehyde, or bis(etheny1)sulfone). Crosslinking leads to greater rigidity in a bioadsorbent, as a consequence of the loss of its ability to swell.

Biotechnology is a growing field in which is exploited the polysaccharidemetal interaction, to immobilize living microorganisms, for example. The polysaccharide stimulating the most interest is alginate in milli- and micrometer gel bead sizes Alginate gels may be formed rapidly andeasily around the micrborganismsbymixingaqueous sodium alginate with the Duetotheir substrate and dripping the mixtureinto a solution of 0”. extreme biocompatibility and high mechanical properties, alginates may also be used in medicineand agriculture tocontrol the release of drugsand Homogeneous alpesticides into a system containing metal ions ginate gels have been proposed to replace agar and agarose in traditional microbiology and in new biotechnological applications

Inasmuch as fiber has strong in vitro mineral-binding capacities,the addition of fiber to the human diet has raised questions about the possible negative effects on mineral bioavailability. Fiber is suspected of reducing human essential trace elements to critical levels. The main inhibitor of mineral absorption identified in plant foods seems to be phytic acid, a myoinositol Despite the large number of clinical studies dealing with hexaphosphate this issue, the subject is still controversial. From the literature it appears, under relevant physiological conditions of the intestine, that the interaction of many cations with the polysaccharides is very weak. The polysaccharide-metal interaction has been proposed for usein eliminating toxic cations from the human body.

Gel formation and phase separation induced by polysaccharide-metal interactions have been the subject of many investigations. This notwithstanding,

Axelos

the binding phenomena and the relevant parameters of the interactions are still veryoftennotadequatelyexplained. The structures initiated by the interactions are still very poorly resolved on an atomic scale, and many questions remain. This chapter is not intended to be an exhaustive discourse of the subject, but simply to point out the necessity of further research to advance understanding of the structure-function relationships between polythat the interaction may be adapted to new saccharides and metal ions food and nonfood processes.

1. Sime, W. J. Alginates. In: Harris, P., ed., Food Gels. New York Elsevier Applied Science, pp. 53-78 (1990). 2. Rolin, C., and De Vries, J. In: Hams, P.,ed., Food Gels. New York: Elsevier Applied Science, pp. 401-434 (1990). 3. Stanley, N. F. Carrageenans. In: Hams, P., ed., Food Gels. New York Elsevier Applied Science, pp. 79-120 (1990). 4. Stokke, B. T., Elgsaeter, A., Smidsord, O., and Christensen, B. E. Carboxylation of scleroglucan for controlled crosslinking by heavy metal ions. Carbohydr. Polym. 5-11 (1995). 5. Volesky, B. Biosorbents for metal recovery. Tibtech. 96-101 (1987). 6. Kohn, R., and Hirsch, J. Binding of calcium, lead, and copper (11)cations to galactaric and 2,5-furandicarboxylic acids and to D-galacturonic acid and its derivatives. Coll. Czech. Chem. Comm. 51: 1150-1159 (1986). 7. Manning, G. S. Limiting law and counterion condensation in polyelectrolyte solutions. I. Colligative properties. J. Chem. Phys. 51: 924-933 (1969). 8. Schwarz, G.Cooperative binding to linear biopolymers. I. Fundamental static and dynamics properties. Eur. J. Biochem. 442-453 (1970). 9. Oosawa, F. Polyelectrolytes. New York Marcel Dekker (1971). 10. Gueron, M., and Weisbuch, G. Polyelectrolyte theory. I. Counterion accumulation, site binding and their insensitivity to polyelectrolyte shapein solutions containing finite salt concentrations. Biopolymers 1 9 353-382 (1980). 11. Manzini, G., Cesaro, A., Delben, F., Paoletti, S., and Reisenhofer, E. Copper (11) binding by natural ionic polysaccharides. Part I. Potentiometric and spec443-454 (1984). troscopic data. Bioelectrochem. Bioenerg. 12. Lips, A., Clark, A. H., Cutler, N., and Durand, D. Measurements of cooperativity of binding of calcium to neutral sodium pectate. Food Hydrocoll. 87-99 (1991). 13. Gamier, C., Axelos, M. A. V., and Thibault, J.-F. Selectivity and cooperativity in the bindingof calcium ionsby pectins. Curbohydr.Res. 71-81 (1994). 14. Schlemmer, U. Studies of the binding of copper, zinc and calcium to pectin, alginate, carrageenan and gum guar in HC03-CO: buffer. Food Chem. 32: 223-234 (1989).

Interactions Polysaccharide-Metal Kwak, J. C. T., and Joshi, Y. M. The binding of divalent metal ions to polyelectrolytes in mixed counterion systems. I. The dye spectrophotometric method. Biophys. Chem. 13: Tsutsumi, A.,Ya, D., Hiraoki, T., Mochiku, H., Yamaguchi, R., and Takahashi, N. ESR studies of Mn(I1) binding to gellan and carrageenan gels. Food Hydrocoll. Debongnie, P., Mestdagh, M., and Rinaudo, M. An EPR and potentiometric study of the complexation of copper ions by galacturonic acid and galacturonans. Carbohydr. Res. 170: Smidsrod, O., and Grasdalen, H. Some physical properties of carrageenan in solution and gel state. Carbohydr. Polym. Belton, P. S., Morris, V. J., and Tanner, S. F. 39Kand "Rb NMR study of chemical shift effects in and K-carrageenan gels. Macromolecules 19: Gey, C., Noble, O., Perez, S., and Taravel, F. Complexes of borate ions with guar D-galacto-D-mannan polymer and related model compounds. Carbohydr. Polym. 173: Mattai, J., and Kwak, J. C. T. Divalent metal ion binding to polyelectrolytes with different polyion structure and functional groups. Macromolecules 19: Braudo, E. E. Polyuronide interactions with polycoordinative metal ions. Food Hydrocoll. 5: Dronnet, V. M., Renard, C.M.G.C., Axelos, M. A. V., and Thibault, J. F. Characterization and selectivity of divalent metal ions binding by citrus and sugar-beet pectins. Carbohydr. Polym. 30: Manning, G. S. The molecular theory of polyelectrolyte solutions with applications to the electrostatic properties of polynucleotides. Quart. Rew. Biophys. I I: 25. Pezron, E., Liebler, L., Ricard, A., Lafuma, F., and Audebert, R. Complex formation in polymerion solutions. I. Polymer concentration effects. Macromolecules Pezron E., Liebler, L., and Lafuma, F. Complex formation in polymer-ion lutions. Polyelectrolyte effects. Macromolecules Wittmer, J., Johner, A., and Joanny, J. F. Precipitation of polyelectrolytes in the presence of multivalent salts. J. Phys. IZ France 5: Joshi, Y. M., and Kwak, J. C. T. Mean and single ion activity coefficients in aqueous mixtures of sodium chloride and sodium pectate, sodium pectinate and sodium carboxymethylcellulose.J. Phys. Chem. 83: Thibault, J. F., and Rinaudo, M. Interactions of mono and divalent counterions with alkali- and enzyme-deesterified pectins in slat free solutions. Biopolymers Bordi, F., and Cametti, C. Equivalent conductivity of carboxymethylcellulose aqueous solutions with divalent counterions. J. Phys. Chem. Piculell, L. Effects of ions on the disorder-order transitions of gel-forming polysaccharides. Food Hydrocoll. 5:

Kohn, R., and Larsen, B. Preparation of water-soluble polyuronic acids and their calcium salts, and the determination calcium ion activity in relation to the degree of polymerization. Acta Chem. Scand. Deiana, S., Micera, G., Muggiolu, G., Gessa, C., and Pusino, A. Interaction of transition-metal ions with polygalacturonic acid: A potentiometric study, Colloids Surf: 6 Kohn, R. Binding of divalent cations to oligomeric fragments of pectins. Carbohydr Res. 160: Debongnie, P. Ph.D. dissertation. Physico-Chimie de la complexation des cations multivalents par les pectates. Louvain la Neuve Haug, A. The affinity of some divalent metals to different type of alginates. Acta Chem. Scand. 1 5 Hassan, R., Makhlouf, M., Summan, A., and Awad, A. Influence of frequency on specific conductance of polyelectrolyte gels with special correlation between strength chelation and stability divalent metal alginate ionotropic gels. Eur. Polym. J. Ouwerx, C., Velings, N., Mestdagh,M. M., and Axelos, M. A. V. Polymer Gels and Networks. In press. Grasdalen, H., and Smidsrod, 0. Gelation of gellan gum. Carbohydr. Polym. Hansen, E. W., and Lund, T. Gelation of xanthan in the presence of trivalent chromic ions monitored by 'H NMR spin-lattice relaxation. A kinetic study. J. Phys. Chem. 99: Rochas, C., and Rinaudo, M. Activity coefficients of counterions and conformation in Kappacarrageenan systems. Biopolymers 19: Moms, E. R., Rees, D. A., and Robinson, G. Cation-specific aggregation of carrageenan helices: Domain model of polymer gel structure. J. Mol. Biol. 138: Piculell, L. Gelling carrageenans. In: Stephen, A. M., ed., Food Polysaccharides and TheirApplications. Marcel Dekker, New York, pp. Tanner, S. F., Morris, V. J., and Belton, P. Multinuclear N.M.R. studies of group I cations in furcellaran gels. Int. J. Biol. Macromol. 12: Zhang, W., Piculell, L., andNilsson, S. Macromolecules 25: Rinaudo, M., Kariman, A., and Milas, M. Polyelectrolyte behavior of carrageenans in aqueous solutions. Biopolymers 18: Nilsson, S., and Jonsson, B. Helix-coil transition ionic polysaccharides analyzed within the Poisson-Boltzmann cell model. Effects of polyion concentration and counterion valency. Macromolecules Domard,A. pH andc.d.measurements on a fully deacetylatedchitosan: Application to Cu" polymerinteractions. Int. J. Biol. Macromol. Winterowd, J. G., and Sandford, P. A. Chitin and chitosan. In: Stephen, A. M., ed., Food Polysaccharides and Their Applications. Marcel Dekker, New York, pp.

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Braudo, E.E., Soshinsky, A. A.,Yurvey, V. P., and Tostoguzov, V. B. The interaction of polyuronides with calcium ions. 1. Binding isotherms of calcium ions with pectic substances. Carbohydr. Polym. 18: 165-169 (1992). Wang,Z.-Y., Zhang, Q.-Z., Konno, M,, and Saito, S. Sol-gel transition of alginate solution by additions of various divalent cations: ”C-NMR spectroscopy study. Biopolymers 33: 703-711 (1993). Velings, N. Proprietes physico-chimiques des billes d’alginates et leur influence sur l’incorporation ou le relargage de molecules bioactives. Ph.D. dissertation. Universite catholique de Louvain la Neuve, Belgium (1997). Draget, K. I., Bstgaard, K., and Smidsrod, 0. Homogeneous alginate gels: A technical approach. Carbohydr. Polym. 14: 159-178 (1991). Clarke-Sturman, A. J., Pedley, J. B., and Sturla, P. L. Influence of anions on the properties of microbial polysaccharides in solution. Int. J. Biol. Macromol. 8: 355-360 (1986). Piculell, L., and Nilsson, S. Anion-specific effects in aqueous agarose systems. 1. Effects on the coil-helix transition and gelation of agarose. J. Phys. Chem. 93: 5596-5601 (1989). Piculell, L., and Nilsson, S. Anion-specific effects in aqueous agarose systems. 2. Nuclear spin relaxation of ions in agarose gels and solutions. J. Phys. Chem. 93: 5602-5611 (1989). Norton, I. T., Moms, E. R., and Rees, D. A. Lyotropic effects of simple anions on the conformation and interactions of kappa-carrageenan. Carbohydr. Res. 134: 89-101 (1984). Pezron, E., Liebler, L., Ricard, A., and Audebert, R. Reversible gel formation induced by ion complexation. 2. Phase diagrams. Macromolecules 21: 11261131 (1988). Axelos, M. A. V., Mestdagh, M. M., and Francpis, J. Phase diagrams of aqueous solutions of polycarboxylates in the presence of divalent cations. Macromolecules 6594-6602 (1994). Ikegami, A., and Imai, N. Precipitation of polyelectrolytes by salts. J. Polymer. Sci. 56: 133-152 (1962). Axelos, M.A.V., Gamier, C., Renard, C. M. G. C., and Thibault, J.-F. Interactions of pectins with multivalent cations: Phase diagrams and structural aspects. In: Visser, J., and Voragen, A. G. J., eds., Pectins and Pectinases. Elsevier, Amsterdam, pp. 35-45 (1996). Michel, A. S., Mestdagh, M. M., and Axelos, M. A. V. Physico-chemical properties of carrageenan gels in presence of various cations. Int. J. Biol. Macromol. In press. Axelos, M. A. V., and Kolb, M. Crosslinked biopolymers: Experimental evidence for scaler percolation theory. Phys. Rev. Lett. 1457-1470 (1990). Cuvelier, G., and Launay, B. Frequency dependence of viscoelastic properties of some physical gels near the gel point. Macromol. Chem. Macromol. Symp. 40:23-31 (1990). Wang,Z.-Y., Zhang, Q.-Z., Konno, M., and Saito, S. Sol-gel transition of alginate solution by additions of various divalent cations: critical behavior of relative viscosity. Chem. Phys. Lett. 186 463-466 (1991).

Axelos Carnali, J. 0. Gelation in physically associating biopolymer systems. Rheol. Acta 31: Gamier, C., Axelos, M. A. V., and Thibault, J. F. Phase diagrams of pectincalcium systems: Influence of pH, ionic strength and temperature on the gelation of pectins with different degree of methylation. Carbohydr. Res. 240: Viebke, C., Piculell, L., and Nilsson, S. On the mechanism of gelation of helixforming biopolymers. Macromolecules 27: Volesky, B. Biosorption of Heavy Metals. CRC Press, Boca Raton, Ha. Brady, D., Stoll, A., and Duncan, J. R. Biosorption of heavy metals cations by nonviable biomass. Environ. Technol. 15: Holan, Z. R., Volesky, B., and Prasetyo, I. Biosorption of cadmium by biomass of marine algae. Biotechnol. Bioeng. 41: Kumar, P., and Dara, S. S. Utilization of agricultural wastes for decontaminating industriaVdomestic wastewaters from toxic metals. Agric. Wastes 4: Dronnet, V.M., Renard, C. M. G.C., Axelos, M. A. V., and Thibault, J. F. Binding of divalent metal cations by sugar-beet pulp. Carbohydr. Polym. In press. Martinsen, A., S&&-Brak, G., and Smidsrod, 0. Alginate as immobilization material: I. Correlation between chemical and physical properties of alginates beads. Biotechnol. Bioeng. 33: Lapasin, R., and Pricl, S. Rheology of Industrial Polysaccharides: Theory and Applications. Blackie Academic and Professional, London,pp. Skjgk-Brak, G., and Espevik, T.Application of alginate gels in biotechnology and biomedecine. Carbohydr. Eur. 14: Nair, B. M., Asp, N.-G., Nyman, M., and Persson, H. Binding of mineral elements by some dietary fiber components in vitro (I). FoodChem. 23: Schlemmer, U. Studies of the binding of copper, zinc, and calcium to pectin, alginate, carrageenan and gum guar in HC03-C02 buffer. Food Chem. 32: Rossander, L., Sandberg, A.-S., and Sandstrom, B. The influence of dietary fiber on mineral absorption and utilization. In: Schweizer, T. F., and Edwards, C. A., eds., Dietary fiber-A Component of Food. Springer-Verlag, London, pp. Idouraine, A., Hassani, B. Z., Claye, S. S., and Weber, C. W. In vitro binding capacity of various fiber sources for magnesium, zinc, and copper. J. Agric. Food Chem. 43: Sandberg, A.-S. Bioprocessing and bioavailability of iron and zinc.In: Schweizer, T.F., and Edwards, C.A.,eds., Dietary f i b e r 4 componentof food. Springer-Verlag, London, pp.

Rheology of Structured PolysaccharideFood Systems: Starch and Pectin Paul E. Okechukwu* and M. Anandha Rao

Cornell Universityseneva Geneva, New York

Most food systems consist of aqueous mixtures of biopolymers that interact in several ways to produce various attributes that impact on quality, texture, and stability. Polysaccharides constitute an important group of biopolymers three-dimensional networks, that interact under specific conditions to or structures, that bind the aqueous phase. These structures play important roles in the texture, flavor release, and stability of a wide range of food products, including dressings,desserts, jellies, and soups. Polysaccharide polymers, including, starches, pectins, xanthan, alginates, carrageenan, and locust bean gum, are widelyused as thickening, stabilizing, and gelling agents. They are able to accomplish this through their ability to swell and absorb water and to reorder their molecular configurationthrough.inter- and intrapolymer interactions and entanglements. As structure inducindaltering agents, they may modify flow behavior and properties to increase viscosity and yield stress. As gelling agents, they transform the viscous behavior of food with high moisture content to solidlike elastic behavior. Thus with the use of these polysaccharides, individually or in combination with others, it

*Current affiliation: The Federal Polytechnic, Oko, Nigeria.

Rao

is possible to fabricate products with rheological properties ranging from viscous to elastic. Rheologicalproperties involving the flow and deformation characteristics of materials under stress are important in understanding these structures andin the handling, processing, mastication, and utilization of foods. Because of the large number of polysaccharides presently used to provide structure in foods, an exhaustive treatise of the subject is not considered. Rather, efforts will be focused on the basic rheological principles employed in the description and analysis of these structured systems, and on a concise review of the role of these structured polysaccharides as thickening and gelling agents, with emphasis on starches and pectins. The thickening and gelling characteristics of other biopolymers may be found elsewhere Hams, (Glicksman,

II. Rheological properties are based on flow and deformationresponses of foods when subjected to stress. In a Cartesian system of coordinates, astress tensor or force imposed on unit surface area of a test material, canbe resolved in terms of nine components (uij),three normal and six tangential stresses. In viscometric flows of present-day rheometers, uI2= uZ1; the other four tangential components, ~ 1 3 , ~ 3 1 ,and ~ 3 3 2 ,are equal to zero, the stress tensor may be written as:

Rheological properties of food materials over a wide range of phase behavior can be expressed in terms of viscous (or viscometric), elastic, and viscoelastic functions that relate some components of the stress tensor to specific components of the strain or shear rate response. In terms of fluid and solid phases, viscometric functions are generally used to relate stress with shear rate in liquid systems, while elastic functions are related to the appropriate stress function to strain in solids. Viscoelastic properties cover the intermediate phase, where the material exhibits bothviscousand elastic properties.

Three material functions-the viscosity function q and the first and second normal stress coefficients JI1 and &-can be evaluated from the stress tensor

Rheology ofPolysaccharides Structured

291

using Eqs. (2-4), which relate specific stress components uijto the shear rate p:

Steady-state experiments employing rotational, capillary, and tube flow viscometers are commonly used to generate data for evaluation of these material properties. Reportedvalues of the viscometric coefficients indicate that q and JI1 are large and positive, whereas +z is small and negative (Macosko, 1994). While the first normal stress difference is known to be responsible for the climbing film phenomenon often referred to as the Weissenberg eflect, for extrudate swell, and for normal force pump, the second normal stress difference determines where the free surface of a fluid flowing downa trough would be convex (Barnes et al., 1989; Macosko, 1994). Most rheological studies have been concentrated on the viscosity function and dynamic viscoelastic properties, and havefocused much less on normal stress differences (Rao, 1992), apparently on account of the high cost of rheometers capable of measuring normal stresses and their limited practical application.

1. Viscosity-ShearRateRelationships At high concentrations, most shear-thinning biopolymer solutions exhibit a similar three-stage viscous response when sheared over a wide shear rate range (Fig. 1):At low shear rates, they show Newtonian properties, with a constant zero shear viscosity (qo)over a limited initial shear range followed by a shear-thinning range where solution viscosity decreases in accordance with the power law relationship to a limiting infinite shear-viscosity (q-) and a constant q.. range. The apparent viscosity (qap)of the solution can be correlated with shear rate (p) using the Cross equation (Eq. or the Carreau equation (Eq. 6), respectively:

where a, and A, are time constants related to the relaxation times of the polymer insolution and m and N are dimensionless exponents.These models described well the shear dependence of aqueous dispersions of high-meth-

Rao

log (shear rate) At lowshearrates,foodpolymersolutionsshowNewtonianbehavior, with a constant zero-shear viscosity(qo)over a shear rate range followedby a shearthinning range where solution viscosity decreasesin accordance with the power law relationship to a limiting value (q..). A second Newtonian region, at high shear rates with the infinite-shear viscosity (q-), can be encountered.

oxy1 pectins and locust bean gum (Lopes da Silva et al., 1992), konjac flour gum (Jacon et al., 1993), and mesquite gum solution et 1994). In addition to predicting zero-shear viscosities of locust bean gum dispersions, the time constant in the Carreau model was related to entanglement time derived from Rouse's theory (Lopes da Silva et al., 1992). For small values of qm,the Cross exponent m tends to a value (1 n), where n is the power law flow behavior index (Launay et al., 1986; Giboreau et al., 1994). For the shear rate (qc),where qsp= (qo qJ2, the Cross time constant a,= l/-$,. Generally, p, gives an order of magnitude of the critical shear rate marking the end of the zero-shear rate Newtonian plateau or the onset of the shear-thinning region.It is therefore important to recognize the shear rate dependence of the rheological behavior of polysaccharide polymers in solution and the difficulty involved in obtaining experimental data over the applicable shear rate range of 10-6-104S-' (Barnes et al., 1989). The low shear rate region of 10-4-100 is often used for the characterization and differentiation of structures in polysaccharide systems through the use of stress-controlled creep andnondestructiveoscillatory tests. The range of10°-104 falls within the operational domain of most commercial rheometers and can only be effectively covered by a combination of procedures and measuring instruments.

+

For alarge number of random coil biopolymers, adimensionlessrelationship (Fig. 2) can be found between log((qo vs. log where c is

-0.5

0.0

0.5

1.0

2.0

1113 For many polymer dispersions,a dimensionless relationship can be found betweenlog(q, - qs)/qsvs.log c[q], where c isconcentration, isintrinsic viscosity, and is solvent viscosity. (From Morris etal., 1981.) concentration andqsis solvent viscosity (Morris et al., 1981). The magnitude c [ q ] is called the coil overlap parameter. We note the link between zero shear viscosity and molecular weight via intrinsic viscosity, [q],and, equally importantly, the zero shear viscosity of a random coil polymer dispersion can be predicted from knowledge of [ q ] .The concentration at the transition from dilute to concentration regimes is denoted as the coil overlap concentration c*. In starch dispersions, Evans and Haisman (1979) pointed out that the mass fraction of granules plays an important role in the magnitudes of rheological properties. Recently, Okechukwu and Rao (1995) showed that the mean diameter of the granules and the size distribution of the granules influence the magnitude of the consistency index and the flow behavior (shearthinning or shear-thickening), respectively.

3. ShearStress-ShearRateRelationships Most fluid and semisolid foods exhibit behavior that is either only shearthinning or shear-thinning with a yield stress. The power law (Eq. 7) model and the Herschel-Bulkley (Eq. 8) model continue to be popular to describe flow properties of foods, with the former being used extensively in engineering applications:

and Rao

where is shear rate (S-’), is shear stress (pa), n and n,, represent the flow behavior index, and K and KHrepresent the consistency index. However, it is important to relate magnitudes of model parameters to the structure/composition of foods and to establish widely applicable relationships. The Casson model (Eq. is a structure-based model that, although developed for characterizing printing inks originally, has been used for a number of dispersions:

+

= &, K,(j)O.’

The Casson yield stress uk = and the Casson plastic viscosity qca= (K,)’.The latter can be used as the infinite shear viscosity (q-) (Metz et al., A general model for shear rate-shear stress data that under specific assumptions reduces to specific models was presented by Ofoli et al. =

+ q&p

where n, and n2 are constants and q- is the infinite-shear viscosity. It is important to note that one model may be applicable at low shear rates and another at high shear rates (Dervisoglu and Kokini,

Properties In the measurement of flow properties, it is important to recognize that the dimensions of the measuring geometry-diameter of a capillary, diameter of concentric cylinders, and diameter of parallel plates-play an important role in the calculation of shear rates; only with cone-plate geometry, the shear rate is independent of the diameter of the rotating cone. The effect of slip at walls may influence magnitudes of flowproperties of some foods that are dispersions,anditcanbeinvestigatedquantitatively as described in and Ma and Barbosa Chnovas Grikshtas and Rao Biaxial extensional viscosity of semisolid foods is a technique that is increasing in popularity, primarily because universal testing machines can be employed to obtain the experimental data and wall slip is not a hindrance Campanella et al., Nuclear magbut an asset (Chatraei et al., netic resonance tube flow velocity spectrum has been shown to be a useful on-line technique for obtaining rheologicalparameters(Seymour et al., On another positive note, the ready availability of powerful desktop computers has simplified data collection and analyses, one can focus on studying the role of food composition on flow properties. In addition, con-

Rheology ofPolysaccharides Structured

295

trolled-stress and low-friction (via compressedair bearings) rheometers have become commercially available.

5. YieldStressMeasurement Although the concept of yield stress was questioned recently (Barnes and Walters, 1985), within the time schedules of most food processes it is useful in food process design, sensory assessment, and modeling. One characteristic of materials with yield stress may be explained by structural deformation and breakage of network bonds (Keentok, 1982). On this basis, yield stress can be characterized in two ways:static and dynamic. Briefly, the yield stress is an undisturbed sample is the static yield stress, while that of a sample whose structure has been completelybrokendown is the dynamic yield stress (Steffe, 1992). The vane method has been employed for determining the yield stress of foods in the controlled shear rate mode (Qiu and Rao, 1988; Tung et al., 1990; Steffe, 1992) and in thecontrolled shear stress mode (James et al., 1987; Wilson et al., 1993; Yo0 et al., 1995). Yieldstress can be calculated from the maximum value of torque recorded at low rotational speeds with a controlledshear rate viscometer. The maximum torque (T,,,)value recorded with a controlledshear rate viscometer, andthe diameter (DJ and the height (Z9) of the vane are used to calculate the yield stress (0;) using Eq. (ll),which was derived by conducting a torque balance on the surface the impeller @my and Boger, 1983):

C. Measurement of Viscoelastic Properties A detailed discussion of the apparatus and data analysis of the various techniques can be found in earlier reviews (Mitchell, 1984; Rao, 1992). Here, only the oscillatory (dynamic) and quasistatic tests, which are two important categories among the rheological procedures used to elucidate viscoelastic properties of foods, are discussed. With the availability of automated rheometers, these tests are being used increasingly. However, care should be exercised in ascertaining that the tests are conducted inthe linear viscoelastic range. With respect to notation,in general, the moduli obtained using linear and shear strains are denoted by E and G, respectively.

1. DynamicRheologicalProperties In the oscillatory (dynamic) tests, a food

sample is subiected to a small.

and Rao

where yo is the strain amplitude and o is the angular frequency. The applied strain generates two stress components in the viscoelastic material: an elastic component in line with the strain and an out-of-phase viscous component. Differentiation of Eq. (12) yields Eq. (13), which shows the strain rate for evaluating the viscous component to be w/2 radians out of phase with the strain: =

COS

ot

(13)

For deformation within the linear viscoelastic range, Eq. (14) expresses the generated stress (ao)in terms of an elastic, or storage, modulus G' and a viscous, or loss, modulus G": = G'yo sin wt

+ G"y0 COS

(14)

For a viscoelastic material, the resultant stress is also sinusoidal but shows a phase lag of radians when compared with the strain. The phase angle covers the range of - w/2 as the viscous component increases. Equation (15) expresses the sinusoidal variation of the resultant stress: = a, sin

+

(15)

Combining Eqs.(14) and (15), the following viscometric expressions emerge that define viscoelastic behavior:

];[

G' =

tan

(16)

G" G'

=-

where G' is the storage modulus, G" is the loss modulus, and tan is the loss tangent. The storage modulus G' expresses the magnitude of the stress that is stored in the material (or recoverable per cycle of deformation). G is a measure of the energy lost as viscous dissipation per cycle of deformation. G' and G" are influencedbyfrequency,concentration,temperature,and strain. For strain values within the linear range of deformation, G' and G" are independent of strain. The loss tangent is the ratio of the energy dissipated to that stored per cycle of deformation. These viscoelastic functions have important roles in the rheology of structured polysaccharides.

Evaluation of G' and G" as a function of frequency, time, and temperature provide useful information on the phenomenon of gelation and the melting characteristics of thermoreversible gels.Inthermally irreversible gels,themoduli are generallyobserved to decreasewithan increase in temperature, apparentlyas a result of a reduction in networklinkages (Clark, 1992). The general increase of gel strength with concentration has been the focus of many investigations for a wide range of gelling products. Clark and Ross-Murphy (1987) suggested that at high concentration, G' would vary with tan is also dependent on frequency and increases with frequency. However, the frequency sensitivity appears to decrease with an increase in the elastic component of the viscoelastic material. Based on the relative magnitudes of G' and G" in a frequency sweep experiment within the linear viscoelastic strain range, one can classify the rheological behavior of foods into three types (Ross-Murphy, 1984): 1. Biopolymer solution (e.g., guar gum sulution): At low frequency, G" is always greater than G'; further, at the lowest frequency, G' o2and G" As frequency or concentration is increased, G' still increases faster than G" andthere is a crossover; i.e., the predominantly liquidlike behavior at low frequencies changes to a solidlike response at higher frequencies. Also, G' and G" show a less pronounced dependence on frequency. The value ofG' at high frequencies is known as the plateau modulus. 2. Weak gel: G' and G' would be almost independent of frequency, and possibly G"/G' would be about 10". 3. For a true gel, G"/G' would be about lo-'. The last two criteria are based on the observation that G' is independent of o and at least one order of magnitude greater than G". It has also been suggested that the temperature at which there is a rapid decline in tan is indicative of gelation onset (Hansen et al., 1990, 1991; Ikkala, 1986). In the course of sol-gel transition, G' shows greater sensitivity to frequency and increases faster than G. The crossover point, or when G' first equals G', is generally recognized as the gel point (RossMurphy, 1991). Another criterion of gel point,advancedbyWinterand Chambon (1986), is when G' and G" are proportional to or when plots of log G' and G" against log o are parallel over a wide range of frequency (0)-

Simple relationships appear to exist between steadyshear and dynamic rheological properties of some structured polysaccharides when corresponding shear rates and frequencies are the same. At large shear rates, the CoxMerz rule (Cox and Men, 1958), expressed in Eq. (19), has been found to apply to a number of hydrocolloid gums: guar, locust bean, mesquite seed,

Rao

and konjac flour (Mills and Kokini,

Jacon et al.,

Yo0 et al.,

=

G*/o),o is the frequency of oscillation (rad S"), and G* = Deviation from the Cox-Men rule reflects the gel-like nature of gelatinized starch and other polysaccharide dispersions (Ross-Murphy, The complex viscosity can befurther expressed in terms of real and imaginary parts:

rl* = q' -

(20)

The Cox-Merz relationship offers an alternative procedure for obtaining steady-shear data for materials that present difficulties such as slippage and outward migrationof sample in the more classical methods using geometries such as the cone and plate. Time scales associated with dynamic tests are often too short for observations of rheological events. With longer observation times, transient (quasistatic) tests sometimes provideusefulinformationon viscoelastic behavior.

2. Quasistatic Tests Mechanical models consisting of an array of elements consisting of a spring and a dashpot can be used to simulate elastic and viscous components in a viscoelastic material subjected to shear or deformation in the linear viscoelastic range. The Maxwell element, consisting of a spring serially connected with a dashpot, and the Kelvin-Voigt unit, in which a spring is arranged in parallel with a dashpot (Fig. represent the simplest combinationsof elastic and viscous components in food systems. The properties of the spring, which obeys Hooke's law, and the dashpot's behaving as a Newtonian fluid permit a rational analysis of a viscoelastic response, provided the material is strained within its linear range. At constant stress, a solid will attain a stable deformation and will recover completely on removal of the stress. Further, at constant deformation, the stress does not relax to zero. Stress relaxation and creep compliance with recovery are two important transient procedures in which materials are stressed and deformed within the linear viscoelastic range. (U) Relaxation. In stress relaxation, the material is instantaneously subjected to a constant deformation (E), and the stress decay is monitored as a function of time ( t ) (Fig. A generalized mechanical modelconsisting of n Maxwell elements with independent elastic modulus (Ei) and viscosity (q)arranged in parallel with a residual spring has formed the basis of the

Force

Force

3 The Maxwell element, consisting of a spring serially connected with a dashpot, and the Kelvin-Voigt unit, in which a spring is arranged in parallel with a dashpot, represent the simplest combinations of elastic and viscous components food systems.

Relaxation

t*

Time (S) In stress relaxation, the material is instantaneously subjected to a constant deformation and the stress decay is monitored as a function of time (9.

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300

analysis of most stress relaxationstudies. The time-dependent relaxation modulus, E(t), is given in Eq.

+ E2e-1'rz+

E(t) =

- +E

+ En-l

where E = T~ = qi/Ei,and En-, is the elasticity of the residual spring. Shear and uniaxial deformation are possible in stress relaxation studies. Shear deformation is normally performed in rheometers with the parallelplate, cone-and-plate, andcoaxial geometries, while the uniaxial deformation appears to be dominatedby uniaxial compressionusing the Instron Universal Testing Machineor similarly operating instruments. Mechanicalmodels consisting of binary and single Maxwell elements have been used to describe the viscoelastic behavior of pectinand agarose gels (Gross et al., Winwoodetal., Costelet Combyet al., In their study on cellulose derivative and BSA, Castelain et al. observed the presence of residual stress in the stress relaxation of the polysaccharide protein system and described the viscoelastic behavior in terms of a two Maxwell elements in parallel with a residual spring. Evidence seems to show that sample geometry, presence of frictional forces, compression rates, and deformational level have significant effects on the stress relaxation curves (Doublier et al., (b) Creep Compliance. In a creep experiment,anundeformed sample is suddenly subjected to a constant shearing stress, that is in the linear viscoelastic region. As shown in Fig. 5, the strain will increase with time and approach a steady state where the strain rate is constant. The data are analyzed in terms of creep compliance:

Y J(t, uC)= V C

A typical creep-compliance curve can be divided into three principal regions (Sherman, A regional (A-B) of instantaneous compliance J , in which the bonds between the different structural units are stretched elastically. In this region, if the stress were removed, the structure of the sample would recover completely. We note that

Jo = G O

where Go is the instantaneous elastic modulus. The magnitudes of G, and of G' at a low frequency (- Hz) were found to be of the same order of magnitude for a gel-like modified starch paste (Giboreau et al., and for a tomato concentrate and Rao,

D

JR

B """-"""""

JO

Time Ina creep experiment, an undeformedsample is suddenlysubjected to aconstantshearingstress, that is in thelinear viscoelastic region. The creep compliance, J(t) = ?/U=,is a function time, describes the viscoelastic behavior, and can be expressed in terms mechanical models.

2. Region B-C corresponds to a time-dependent retarded elastic region with a compliance JR. In this region the bonds break and reform, but all of them do not break and reform at the same rate. The equation for this part, using mean values for the parameters, is JR = Jm( 1 - exp

<)

In Eq. J,,, is the mean compliance of all the bonds and T,,,is the mean retardation time; T, equals J,q,,, where q,,,is the mean viscosity associated with elasticity. One can replace the mean quantities with the spectrum of retarded elastic moduli (Gi) and the viscosities (q),where Ji = l/Gi. n p i ally, one or two Kelvin-Voigt elements can be used todescribe the retarded elastic region. Region C-D is a linear region of Newtonian compliance in which the units as a result of rupture of the bonds flow past one another. The compliance JN and the viscosity are related by

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The entire creep-compliance curve, from A to D, of ice cream mix (Sherman, 1966) and tomato concentrates and Rao, 1996) has been described by a six-parameter model: J(t) = Jo

+ J,(1 -

t + J2(1 - e-".') + r(,

Inaddition,when the stress is suddenlyremovedat D, the pattern consists of an elastic recovery (D-E) followed by a retardedelastic recovery (E-F). Because bonds between structural units are broken in region C-D, a part of the structure is not recovered. For values of time after the applied stress is removed, the recovered strain approaches a maximum value, S,, called recoverable shear (Dealy, 1982): S, = UJ:

(27)

where is the applied stress and J is the value of the straight-line portion of the creep-compliance curve extrapolated to zero time. A shear modulus, G,, can be defined as the reciprocal of the steady-state compliance, JS: = G,S,

(28)

This equation is called Hooke's law of shear.

RHEOLOGICALCHANGESDURING STARCH GELATINIZATION When an aqueous suspension of starch is heated above the gelatinization onset temperature, the granules absorb large amounts of water and swell considerably to impart a substantial increase to the viscosity of the suspension. As granule swelling progresses, the suspension is transformed into a paste that exhibits rheological properties very different from those of the initial suspension. The swelling of granules is also accompanied by leaching, or solubilization, of granular constituents, mainly amylose, into the continuous suspending matrix. Swelling of granules attains a maximum value at elevated temperatures and is subsequently followed by granular disruption and exudation of granule contents into the suspension matrix. Thus gelatinized starch suspension may be regarded as a composite material consisting of swollen granules and granular fragments dispersed in a continuous bio1990; polymer matrix (Evans and Haisman, 1979; Eliasson, 1986; Noel et al., 1993). It is logical that the properties of the dispersed phase, the continuous phase, and interactions between the components are three important groups of considerations for measurements that provide insights into the rheology of gelatinized starch suspensions. It is also important to rec-

Rheology of Polysaccharides Structured

303

ognize that starch gelatinization studied by various techniques, including viscometry and particle size measurement by laser diffraction, is essentially a first-order kinetic process whose extent is determined principally by pastLund, Okechukwu et ing temperature and time (Kubota et al., al., Okechukwu and Rao, Temperature and heating rates are important variables during the sol-gel transition period.A good appreciation of the transition phase is vital to a full understanding the rheology of gelatinized starches.

A. Viscous Property Changes During Gelatinization of Starches Studies using laser diffraction show that granule swelling during starch gelatinization progresses in a manner that increases the granule size distribution, when measured by the standard deviation granules in suspension (Okechukwu and Rao, This change in the polydispersityof the starch granules introduces important rheological consequences on the suspension behavior. Its impact, though not fully understood, is presently assessed on the flow behavior low-starch suspensions. Dolan et al. developed a model(Eqs. and to describe apparent viscosity as a function of time during starch gelatinization under nonisothermal conditions. It contains an exponential function of the temperature-time history and the Arrhenius equation to describe the gelatinization reaction. The special form of the model for constant shear rate and starch concentration is:

-

where q. is apparent viscosity (Pa S), qdim is dimensionless apparent viscosity, is ungelatinized apparent viscosity(Pa S), is the highest magnitude of q. during gelatinization, $ is the integral of the time-temperature history, E, is the activation energy of gelatinization (J/mole), R is a gas constant J/mol K),and T is the temperature of the fluid (K). The model development was based on earlier work on the viscosity of protein doughs by Morgan et al. and additional .discussion on its application to starch dispersions can be found in Dolan and Steffe Because of the difference in structure of protein doughs and starch dispersions, the rheological behavior of the latter will be much different, and it may be possible to develop models that are relatively more functional.

-

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B. Viscous and Viscoelastic Properties of Gelatinized Starch Dispersions A number of factors, which include concentration, volume fraction and size of swollen granules, temperature, and heating rate, have been reported to affect the rheological behavior of gelatinized starch suspensions. Mild thixotropy has been observed in gelatinized wheat starch dispersions characterized at low temperatures such as 25°C but not at 60°C and above (Doublier, 1981, Harrod, 1989; Bagley and Christianson, 1982). The time-dependent rheological behavior shown by starch is attributed to structure formation by amylose leached into the suspending matrix on cooling to low gelling temperatures (Ellis et al., 1989). Gelatinized starch suspensions exhibit yield stresses at low shear rates (Evans and Haisman, 1979; Kubota et al., 1979; Doublier, 1981; Christianson and Bagley, 1984; Colas, 1986; Harrod, 1989; Giboreau et al., 1994). At high shear rates, the shear responses follow the power law relations, prompting the use of the Hershel Bulkey model and the power law model for the analysis and description of the rheological behavior of starch suspensions. Yield stresses in gelatinized corn starch suspensions at a sediment volume fraction of about 0.6 were shown to increase with granule swelling (Christianson and Bagley, 1984). At low starch concentration, most authors have usedthe simple power law model without a yield stress for describing shear responses over a wide range of shear.The flow behavior index n and the consistency coefficientK are useful parameters for describing the flow behaviorof starch suspensions. Gelatinized starch pastes preheated to temperatures of about 90°C are generally reported to be shear-thinning (pseudoplastic) fluids with values of n considerably less than 1.0 (Evans and Haisman,1979; Doublier, 1981; Colas, 1986; Ellis et al., 1989; Noel et al., 1993). This value was also reported by Colas (1986) to decrease linearly with increasing starch concentration in crosslinked corn starches. This observation appears consistent with those of Evans and Haisman (1979),who showed that in suspensions of corn, potato, modified corn, and tapioca starches as well as wheat flour, the index decreasedrapidly from unityandapproacheda constant valueathigh concentration. However, several studies on starch gelatinization have indicated that some starches, notablywheat,corn,waxymaize,andcross-linkedwaxy maize,may show shear-thickening(dilatant) behavior with values of n greater than 1.0 during the early stages of gelatinization (Bagley and tianson,1982;ChristiansonandBagley, 1983; Dail andSteffe,1990a,b; Okechukwu and Rao, 1995). Particle size measurements of gelatinizinggranules seem to suggest a relationship between n and the change in polydis-

Rheology of Polysaccharides Structured

305

persity of granules during the sol-gel transition process. The flow behavior index appears to decrease linearlywith an increase in the standard deviation (SD) of heated corn starch granules (Okechukwu and Rao, 1995). With an R' of 0.979, Eq. (31), which is independent of pasting temperature, provides the basis for resolving the changes in the value of n observed at various stages of starch gelatinization:

n = 2.17 - 0.0968SD

(31)

Equation (31) suggests a maximumn value of about 2.2 for corn starch. The minimum values of n, usually within the pseudoplastic range, would depend in part on the particle size distribution of the gelatinizing granules, which increases as gelatinization progresses. By arbitrarily choosing gelatinization to be complete at 95"C, it was shown that a range of values of n can be obtained, depending on the extent of granule swelling of corn starch (Fig. 6). Apparently, the different values of n found in the literature for suspensions of the same starch pasted under various temperature-time profiles reflect various extents of starch granule swelling. Early investigations on the rheology of raw starch suspensions reveal that dispersions of corn starch in water and aqueous mixtures of ethylene glycol and glycerin exhibited dilatant behavior with values of n in the range 1.2-2.5 (Griskey and Green, 1971). It would therefore appear that gelatinization may be described as a

Extent of granule growth Figure Assuming granule growth to be complete at 95"C, various values of the behavior index n of a 2.6%corn starch dispersion were obtained, depending on the extent of starch granule growth. (From Okechukwu and Rao, 1995.)

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transition process that transforms the rheological behavior of starch suspensions from dilatant to pseudoplastic.

1.

Effect of StarchConcentration,GranuleVolume Fraction, and Size

Power law relations of the form shown in Eq. (32) have been used to describe the influence of starch concentration on the viscosity of gelatinized wheat starch suspensions over the shear rate range 20-200 S-': K = K,cb

(32)

where K is in Pa. S" and starch concentration was defined in mg/mL (Doublier, 1981). For gelatinized starch characterized at 70°C, K, = 8.74 and b = 3.88 for starch concentration, in the range 2 . 5 4 % . Evans and Haisman (1979) reported that the apparent viscosity at any shear rate qu raised to the power was linearly correlated with concentrationfor potato, tapioca, and corn starches within the shear rates 10-103 S-': (.I+)',>"^= K"(c -

(33)

C,,)

where is the concentration at whichthe viscosity extrapolated to zero and K" is a proportionality constant. Thus at various shear rates it is possible to construct linear plots relating apparent viscosity with starch concentration. Plots of apparent viscosity as a function of starch concentration were later begreatlyaffectedby the shown by BagleyandChristianson (1982) pasting temperature and time. However, they obtained a temperature-independent master plot that correlated the viscosity ratio (q/cQ) with the mass fraction of swollen starch granules in gelatinized wheat starch suspension, cQ. A similar master plot was also obtained by plotting the ratio K/CQ against the volume fraction of swollen corn starch granules, since at low starch concentrations volume fraction approximates mass fraction (Okec h u m and Rao, 1995); for a 2.6%starch suspension the plot can be represented by Eq. (34): K

- = 0.48 exp 16.94cQ CQ

R2 = 0.852

(34)

There is empirical evidence that suggests thatsuspensionviscosity increases with the volume fraction occupied by the starch granules. At low volume fraction, the granules are relativelyapart,with little interaction. However, as volume fraction increases, intergranular interactions become important, with suspension viscosity showing a greater dependence on volume fraction. Comparison of experimental results with predictions from the Krieger-Dougherty(Krieger, 1985) model for rigid spheres (Eq. 35) re-

vealed a poor agreement, attributed to granule geometry’s being different from spherical, polydispersity, deformability, and granular interaction (Ellis et al., Noel et al.,

The intrinsic viscosity term, [q], in Eq. was higher than the theoretical value of even for rigid sterically stabilized polymethylmethacrylate spheres (Choi and Krieger, Starch granules are generally not spherical and tend to show a range of particle size distribution. At a fixed volume fraction, the polydisperse granules will produce a higher suspension viscosity in comparison with rigid spheres. Studies employing laser diffraction techniques to measure the sizes of granule have revealed that gelatinization involves essentially two stages of water absorption: an initial stage of swelling, characterized by a steady increase in mean granular size and particle standard deviation, as well as a later granular disruption stage. The later stage shows a reduction in mean granule size as well as a decrease in standard deviation. During the initial stage of gelatinization, the consistency index K of a corn starch dispersion was correlated with the mean granule diameter, D (Fig. 7), and described by Eq. (Okechukwu and Rao,

E ,

25

40

45

50

Mean diameter (um) Duringtheinitial stage of gelatinization,therelationshipbetweenthe cornstarchsuspensionconsistencyindex, K, andthemeangranulediameter, D, followed Eq. (36). (From Okechukwu and Rao, 1995.)

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K = I?, exp

R2= 0.989

(36)

where I?, and are empirical constantswith estimates of2.071 X lo-' (mPa. S") and 0.369 (pm-'), respectively. During the later stage of gelatinization involving the bursting of granules, a progressive reduction in suspension viscosity takes place, and Eq. (36) does not describe the suspension viscosity data.

The viscosity of gelatinized starch suspensions generally decreases with an increase in temperature. This rheological response preheated starches has been shown to follow an h h e n i u s dependency of the consistency coefficient on temperature, as shown in (37):

K = Km exp

(g)

(37)

where Ea is the activation energy and Km is an empirical constant. For a wheat starch dispersion heated to 96"C, values of E, = 5.13 kcal/mole and Km = 1.51 X Pa were reported(Doublier,1981).

Dynamic experiments employing small-strain oscillatory shear are generally used in elucidating the viscoelastic properties of gelatinizing starch suspensions. In these studies, conducted at strain values within the linear viscoelastic range, the rheological responses are resolved into elastic and viscous components that provide bases for evaluating the storage modulus (Gf), the loss modulus (G"), and the loss tangent (tan The linear viscoelastic range for polymer systems with fillers such as starches have been shown to be very low (EvansandHaisman, 1979; Eliasson, 1986; Miles et al., 1985; Svegmark and Hermansson, 1991b). Table 1 suggests that variations in the linear viscoelastic range due to starch concentration, shear treatment, measurement temperature, and botanical source are small over the wide range the two main polymer constituents of starch: amylose and amylopectin. Evaluation of the rheological parameters as a function of temperature, heating rate, and concentration of starch and other constituents has been rigid or elastic used to provide useful information on the development structures in starch dispersions during the heating(Eliasson, 1986; Okechukwuetal., 1991; Hansen et al., 1991). Generally, G' and G" increase sharply from temperatures close to the starch gelatinization onset to peak values, and thereafter decrease as temperature increases. The peak temperature appears to be close to the concludinggelatinization temperature. While

Rheology of Structured Polysaccharides Table

Rheological Properties of Amylose and Amylopectin

Index

Amylose

Minimum conc. for gelation (co) Linear viscoelastic range Concentration dependence of G' Gel strength development

Reversibility

Temperature dependence of G' c* =

Amylopectin

-0.9% ( w h ) and <(c*

(w/w) but

-

strain

G'

c" (power relation)

a

- 3-7

Fast and exponential maximum gel strength developed in less than h Irreversible at temperatures <1OO"C but reversible at temp. Weak

-

>(C*

strain G'

c

(linear relation)

Slow initially and sigmoidal with respect to time; max. strength attained in days Reversible at temperatures
coil overlap concentration

the increase in the moduli is attributed to granule swelling, the decrease beyond the peak temperature may be linked to granule softening and disintegration (Autio, 1990; Biliaderis, 1992) or coleaching of amylopectin into the continuous matrix (Hansen et al., 1991). In wheat and maize starches, this initial peak is followed by a second peak when heated to 95°C. Associated with the rise of G' up to the peak value is a steady fall in tan to a fairly constant low level that appearsto be insensitive to starch concentration for most starches. However, wheat starch seems to behave differently as tan 8 is reported to rise to a peak value of almost 2.0 after an initial fall (Eliasson, 1986). Rheological studies on starch gels cooled after heating indicate that the decrease in G' after the peak value was also reflected in the strength of the gel on cooling (Autio, 1990; Hansen et al., 1991; Okechukwu et al., 1991). Apparently,the terminal heating temperature, referred to as endpoint temperature byHansenetal.(1991), is an importantcontributorto gel strength.

4.

Effect of Gums and Other Hydrocolloids

In solution, hydrocolloids such as locust bean gum, xanthan, mesquite seed gum, and guar gum behave as Newtonian fluids at low shear rates, showing

31

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a constant viscosity with variations in shear rate. As shear rate increases beyond a critical value, the solution exhibits pseudoplastic behavior, with a decrease in viscosity as shear rate increases (Giboreau et al., Jacon et al., Yo0 et al., Most hydrocolloids, particularly the randomcoil galactomannans, do not show the mild thixotropy exhibited by most gelatinized starch suspensions (Alloncle etal., Addition of small amounts of hydrocolloids to starch suspensions has been reported toincrease significantly the viscosity and to modify the flow behavior of heated starch Sajjan and Rao, Alloncle etal., dispersions (Raymentetal., The hydrocolloids reduced the yield stresses exhibited by gelatinized starch, apparently as a result of the restriction of granule swelling imposed by the hydrocolloids in solution (Liu and Lelievre, In addition, the gums were reported to increase the pseudoplasticity of starch suspensions. The ability of the gums to increase starch suspension viscosity has been attributed to the increase in concentration of the hydrocolloid in the continuous matrix as granule swelling progresses (Alloncle and Doublier,

Starch gels are composites in which swollen granules, intact and ruptured, are embedded in a continuous gel network consisting predominantly of amylose. Heating of starch dispersions to temperatures above their gelatinization temperatures is required to swell the granules and more importantly to solubilize the amylose. The rheological properties of a starch gel depends on those of the continuous matrix, the volume fraction, shape, and deformability of the filler granular material, and the filler-matrix interaction (EliasMiles et al., Morris, Hansen et al., The conson, tinuous matrix may be composed of amylose or a mixture of amylose and amylopectin, depending on the type of starch and on heating conditions Langton and Hermansson, Autio, Amylose (Miles et al., appears to be the major component of starch, whose concentration in the continuous phase determines whether a heated starch dispersion would gel or not. It is leached out as granule swelling progresses; and in most suspensions heated above granule rupture under shear may lead to appreciable coleaching of amylopectin, as observed for wheat (Doublier, oat (Doublier et al., potato (Langton and Hermansson, Svegmark and Hermansson, and corn starches (Hansen et al., Next, a brief description of the rheological behavior of amylose and amylopectin gels is given, for a better appreciationof the mechanical responses ofstarch gels.

Rheology of Structured Polysaccharides

A.

Rheological Behavlorof Amylose and Amylopectin Gels

The critical concentration (co) of amylose below which a gel cannot form is about 0.8-1.1% and appears to be independent of molecular weight and the origin of the biopolymer (Ellis and Ring,1985; Clark et al., 1989; Biliaderis, 1992). This lower magnitude of c, compared to the molecular-weight-dependent overlap concentration (c* = 1.5%) obtained through viscometry suggests that molecularentanglements are not primarilyresponsible for amylose gel formation. During sol-gel transition, amylose undergoes conformational ordering resulting in aggregation and association the polymer chains to form athree-dimensionalnetwork. Amylose gel formation progresses through stages: (1) molecular aggregation, presumably by double-helix formation and elongation at the tip, and (2) lateral association of the helical regions (Morris, 1990; Biliaderis, 1992). The sequence of the stages appears to be influenced by molecular weight or the degree polymerization (DP). While aggregation is favored by a high DP, lateral association is favored by low-molecular-weight amylose molecules (Kitamura et al., 1994). Amylose gels are generally stiff, with values of G’ showing no variation with frequency.The gels are irreversible when heated up to 100°C and develop full gel strength within 2-4 h of aging (Miles et al., 1985; Zobel, 1988). G’ is highly dependent on amylose concentration, which often can be described by a power relationship (Clark et al., 1989; Ring, 1985; Biliaderis, 1992; Svegmark and Hermansson, 1993). Rheological and thermal data are also available on the gelation properties of amylopectin (Ring et al., 1987; Biliaderis and Zawistowski, 1990; Biliaderis, 1992). Amylopectin solutions require high concentrations of the polymer (10% and above), usually higher thanthe overlap concentration (c* 0.9), and low temperatures for gelation. Development ofelastic structures, from measurements of G’ as a function of time, is initially slow and approaches a limiting value after following a sigmoid pattern. At concentration, G‘ and G” of amylopectin gels show strong frequency dependence during early stages of curing. However, as gel strength approaches the limiting value, G’ increases faster than G” and shows a linear dependence on concentration and a decreasing influence of frequency. Unlike amylose gels, amylopectin gels are thermoreversible, with endothermic transition energies below 100°C. concise comparison important rheological properties of amylose and amylopectin gels is given in Table 1.

-

B. StarchGels

As a hot gelatinized starch dispersion is cooled, G’ begins to rise at a temperature designated as gel temperature (Tge,).This temperature, which marks

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the onset of structure development in the hot paste, also coincides with the peak temperature on atan S-temperature plot (Hansen et al., 1990). Studies with gelatinized cornstarch dispersions in water (Hansen et al., 1991) and potato starch in amylose solution (Svegmark and Hermansson,1993) showed that Tgelincreases with starch concentration and the terminal heating temperature or endpoint temperature. At high starch concentration, the onset of gelation may occur at a temperature as high as 75"C, as observed for 10% potato starch in 2% amylose solution. When hot gelatinized dispersions of starch are rapidly quenched and aged isothermally, structure development, measured by oscillatory shear on a time scale, follows a biphasic pattern (Fig. 8). The initial rise in G' marks the beginning of the short-term developmentin gel strength, attributable to amylose networkformationand growth in the dispersionmatrix. This view is supportedby comparative studies on G' development in dispersions of smooth pea starch and amylose in water quenched from 90" to 26°C (Miles et al., 1985) and G* growth in suspensions of potato starch in amylose and amylopectin raised to 75°C and subsequently cooled to 30°C (Svegmark and Hermansson,1993). In starches, such as that of potato, with large swelling ability and hindered solubilization of amylose into the continuous matrix, the initial gel strength development

600

I

7

Time (min) -

600

700

Aging time (min) When hot gelatinized dispersions of starch (5% and 9% cowpea starch) were rapidly quenched to 20°C and aged isothermally, structure development, measured by oscillatory shear on a time scale, followed a biphasic pattern; inset shows variation of tan

Rheology of Structured Polysaccharides

313

is poor, due to low levels of amylose in the suspending matrix. The latter stage in gel strength is the contribution due to amylopectin recrystallization.

1.

.

Thermodynamic incompatibility exhibited by amylose and amylopectin in solution often produces a phase separation in which one phase is enriched with amylose and the other contains mostly amylopectin (Kalichevsky and Ring,1987). The ratio of amylose to amylopectin (r) in the system has considerable effect on the strength of the resultant gel. The phase inversion point is defined as the critical ratio of amylose to amylopectin (r*) at which amylose replaces amylopectin in the continuous phase as amylose is progressivelyadded to a solution ofamylopectin.For ratios of r < r*, gel characteristicswere similar to thatof amylopectin gels; but for r > r*, strong gels were obtained that were reminiscent of amylose gels. The critical ratio has been shown tobe close to 0.43 in studies involving uniaxialrampression (Leloup et al., 1991) and close to 0.18 using small-amplitude oscillatory shear (Doublier and Llamas, 1993). This inversion of rheological behavior has been used to explain the lower gel strength obtained for potato starch whencompared with cereal starches (SvegmarkandHermansson, 1990, 1991b, 1993). According to these authors, a more efficient redistribution of amylose andamylopectin in the continuous anddispersedphases,respectively, is responsible for the stronger gel from cereal starches.Inwheat starch, most of the amylose was leached out of the granules on heating to 95"C, whereas only anegligible amount was found outside the potato grancited as ules. Coleaching of amylopectin into the continuous matrix is responsible for the weakening in the strength of starch gels (Hansen et al., 1991).

Generally G' and G" of starch gels tend to increase with frequency which can be represented by power functions:

(W),

G' = G'*(w)" G" = G"*(w)P

where G'*,G"*, a,and are empirical constants. For starch gels, G" shows a greater frequency dependence than G' (Fig. 9). At high starch concentration (greater than the close packing concentration), the G' of most starch gels at full strength is almost flat, with a slight variation with frequency over the range 0.1-20 H z , with G' > 10G" (Alloncle and Doublier, 1991; Biliaderis and Tonogai, 1991). With decreasing starch concentration, both

o-*

loo

Frequency (Hz)

For cornstarch gels, from frequency sweep data at greater frequency dependence than G'.

lo2 G'' showed a

G' and G" increase with greater sensitivity to frequency (Eliasson, 1986). Generally, the complexviscosity q* for most starch gels decreases with frequency.

3. ConcentrationDependence of GelStrength Results from studies on the gelation of starches from various botanical sources indicate that the elastic modulus increases with starch concentration. Potato and pea starches have shown a linear dependence of G' with concentration over the range 6-30% starch solids (Ring, 1985; Evans and Haisman, 1979), while corn starch exhibited a linear dependence of log G' with concentration over the range 5-10% (Hansen et al., 1990). However, when potato starch was dispersed in 2% amylose, G' increased exponentially with starch concentration within 1-10% (Svegmark and Hermansson, 1993). Exponential relationships have also been used to describe the variation G' with concentration of wheat starch suspensions over the range 2-6% (Wong and Lelivre, 1981). Powerlaw functions have also been used todescribe the influence of starch concentration (c) on G':

G' cc

.

(40)

Published values of the index a include 2.3 and 2.4 for garbanzo bean and rice starches, respectively, over the range 8-40% (Biliaderis, 1992) and 4.0

Rheology of Structured Polysaccharides

315

for wheat flour (Evans and Haisman, 1979). Differencesin gel strength have been reportedfor starches from rice cultivars with different amylose content. While G' increasedexponentially,tan decreasedlinearlywith amylose content (Biliaderis, 1992).

Influence of Lipids and Emulsifiers on Starch Gels The interaction of fatty compounds with starch involves the inclusion of fatty adjuncts into the helical structures of the amylose component of the starch. The formation of the inclusion compounds requiring temperatures 250°C affects the swelling, solubility, and viscous properties of the starch in various ways, depending on the type of starch and the hydrocarbon residue of the fatty adjunct (Leach et al.,1959; Gray and Schoch, 1962; Osman and Dix, 1960; Osman, 1975; Krog, 1973). Generally,compounds with large hydrocarbon residues, such as stearic acid, glycerol monostearate, (GMS) and sodium stearoyl lactylate (SSL) tend to restrict granule swelling and repress amylose solubilization (GrayandSchoch, 1962; Eliasson, 1986). Restriction of swelling ability by the fatty materials was also supported by DSC studies that show a decrease in the endothermic enthalpy on addition of most lipids to starches (Biliaderis and Tonogai, 1991). Addition of longchain fatty acids to starch componentsreduced the viscosity of amylose solutions butincreasedslightlythat of amylopectin(Raphaelides,1992). Only the effect on amylose was dependent on the concentration of the fatty acid. Addition of stearic acid to mixtures of amylose and amylopectin producedviscosity changes ranging from an increase to a considerable reduction. Factors such as fatty acid chain length and concentration, amylose/ amylopectin ratio, and starch concentration play important roles in the rheology of starch suspensions treated with fats and emulsifiers. In a study in which GMS and SSL were added to various starches before heating, the fatty compounds delayed the onset or rise of the storage modulus during heating of 10% suspension of wheat, maize, and potato starches but did not influencewaxybarley starch. The fatty compounds also interferedwith structure formation in starch gels, as suggested by the increase in tan during gelatinization of starches treated with GMS andSSL (Eliasson, 1986). However, Biliaderis andTonogai(1991)reportedthat20-35% rice and wheat starch gels exhibited higher G' values when lipids were included. Similarly, addition of 1%soybean oil to wheat starch increased G' (Lindahl and Eliasson, 1986).

5.

Influence of Other Hydrocolloids on Starch Gels

Addition of guar, locust bean, and xanthan gums at 0.1-0.5% levels to gelatinized corn starch suspension resulted in an increase in G' and G" of

Rao

the hotpasteandaffected the frequency dependence of the two moduli (Alloncle and Doublier, 1991). The relationship between the increase in G’ and G” and the concentration of the hydrocolloid were also shown to be linear for xanthan gum but a complex function for the galactomannan guar gum and locust bean gum.While the galactomannans increasedthe variation of G‘ and G” with frequency, xanthan decreased the frequency dependence of G” to a “plateau” but did not alter that of G‘, suggesting that the galactomannans have a greater tendency to increase viscosity rather than gel strength. The hydrocolloids were also reported to affect the maximum gel strength and the gelation kinetics in various ways. The study suggests that the roles of galactomannans and xanthan gum in the continuous phase are differentandcanberesolved in the context of phaseseparation due to incompatibility between amylose and the hydrocolloid in solution. Smallstrain oscillatory shear measurements also demonstratedthat rice starch granules embedded in gellan-locust bean gum matrix reinforced the matrix, and the reinforcement increased if the starch was gelatinized (Liu and Lelievre, 1992a). From these studies it is apparent that the main effect of the starch granules was to increase the concentration of the hydrocolloid in the continuous phase and thereby to raise its modulus.

6.

Effect of Proteins on the Strength of Starch Gels

Proteins appear to show some selectivity in interactionwith starches, as shown by their rheological response to oscillatory shear. Addition of gluten to 6.5% (w/w) suspensions of different starches at 1%level, calculated on a dry starch basis, increased the G’ of wheat and rye starches, lowered that of maize starch, but did not affect the modulus of barley, triticale, andpotato starches (Lindahl and Eliasson, 1986). G’ was also found to increase with increasing amounts of gluten (1-4%) added to the wheat starch. In another study, zein, gliadin, glutelin, and glutenin were added to amioca (98% amylopectin) starch, Hylon V (50% amylose and 50% amylopectin), and potato starch (100% amylose) and sheared at temperature of64-100°C using a small-amplitude oscillatory test procedure (Chedid and Kokini, 1992; Madeka and Kokini, 1992). Gelatinization of amylopectin, and not amylose, was reported to favor a synergistic increase in viscosity with gliadin and glutenin, followed by zein and glutelin. In another study, in which gelatin at a 0.88-1.5% level was added to 1.5% highly crosslinked waxy maize starch (chosen to minimize leaching of amylose into the continuous phase) and heated to 80”C, the moduli of the gelatidstarch composite were consistently higher than those of the gelatin alone (Abdulmolaetal.,1995). It appears that the rheological behavior of starches is affected by proteins,

depending on the amylose/amylopectin ratio and starch.

the type of protein and

Pectins are polymers of polygalacturonic acid esterified with methanol to various degrees. Commercially, the hydrocolloid is obtained mostly from citrus peel and apple pomace, where it is present at levels of about 15-35% (Nelson et al., 1977; May, 1990). Extraction can be modified to yield two types of pectins, basedon the degree of methoxylation (DM). High-methoxyl pectins (HMP), with a DM greater than 50%, form gels at low pH in the presence of high amounts of sucrose or similar solids (>55% w/w). On the other hand, low-methoxyl pectins (LMP) have DM values less than 50% and require a divalent cation such as Ca2+for gel setting (Carr et al., 1995). Amidated pectins, which are also categorized as low-methoxyl pectins, are obtained by reacting pectins with ammonia to produce polymers with amide groups in addition to the ester and acid moieties (May, 1990).

In solution, pectins behave as random-coil polymers,with viscoelastic properties influenced by DM, pectin concentration, pH, temperature, and ionic concentration (Chou and Kokini, 1987; Hwang and Kokini, 1992; Lopes da Silva, 1994). Solutions of HMP in water and 0.1 M NaCl over the concentration range 1.5-3.93 g/dL were found to be shear-thinning at 25°C when sheared over the range 1-100 S-'. The apparent viscosity-shear rate data of the solutions were well described by the Cross and Carreau models,with estimates of zero-shear viscosities and time constants showing power law dependence on pectin concentration. At pH 7 and 0.1 M NaC1, estimates of the exponents for the variation of the time constants withHMP concentration were shownto be close to 2 and4 for the Cross andCarreaumodels, respectively (Lopes da Silva et al., 1992). Exponents for the zero-shear viscosities from the Cross and Carreaumodels versus pectin concentrations were within the range 4.2-4.8 (Lopes da Silva et al., 1992). These values appear to be higher than the generalization of 3.3 for entangled polysaccharides et al., 1981). Generally, the viscosity of pectin solutions increases with a decrease in pH, apparently with an increase in hydrophobic interaction and a decrease in electrostatic repulsion. At low pH levels of about 3.0, LMP solutions exhibit shear-thinning rheological behavior with characteristics similar to those of HMP. Solution viscosities were highly concentration dependent, with power law exponents

and Rao

31

for the variation of zero-shear viscosity and time constant, with pectin concentration being6.9and6.5,respectively. The study of Lopes da Silva et al. (1992) also showed that rheological behavior of LMP solutions are strongly influenced by pH. LMP solutions of the concentration range 0.130.92 g/dL exhibited Newtoniancharacteristics at pH 7 when sheared at 25°C over 0.1-100 S-'.

Gelation of At low water activity and high acidity, gelation by HMP proceeds through association of polymer chains to form junction zones strengthened by hydrophobic and hydrogen bonding interactions between methyl ester groups (Clark et al., 1990; Oakenfull,1991). At lowpH, electrostatic repulsion between pectin molecules is suppressed by the low ionization of the acid groups on the polymer chains. Gelation is therefore strongly dependent on pH, with gel strength and gelation temperature decreasing with an increase in pH(May, 1990). Other factors affecting HMP gelation include pectin source, molecular weight, DM, and presence of cosolutes. Pumpkin pectin gave weaker gels at concentrations greater than 1% and required considerably lower concentration for gelation compared with slow-set, medium-set, and rapid-set pectins obtained from citrus peels (Ptitchkina et al., 1994). Pectins with about 70% DM and above are used primarily as "rapid-set" pectins. Slow-set pectins with better gelation control are obtained by reducing the degree of methoxylation through hydrolyzing the ester groups. Sugars are usedin formulations requiring HMP gelation;they reduce water activity andstabilize the hydrophobic interactions required for gel formation. Generally, gel strength and setting temperature increase with sugar concentration to various levels, which depend on the type of sugar (Christensen, 1982). In addition to pH, pectin concentration,aging temperature, andcooling temperature are important considerations in gel formation and strength. Generally, small-strain oscillatory shear showed an increase in G' and G" with pectin concentration (c). G' was shown to be proportional to C"' for HM pectin in 60% fructose (Lopes da Silva and Rao, 1995) and in 60% sucrose gels (Lopes da Silva et al., 1995) for pectin concentration rangingfrom 0.5% to 2% at 10°C. Gelation with a slow-set HMP in 65% fructose showed that structure development rates (SDR) were higher at lower temperatures and higher pectin concentration and when the pectin was hydrated for 1h (Rao and Cooley, 1993). The study also revealed that the SDR did not show an Arrhenius dependence on temperature butfollowed the Flory-Weaver model or its modification for crystal growth.

Rheology of Structured Polysaccharides

319

C. Gelationof LMP The “egg-box” model has been usedfor studying the mechanism of gelation in low-methoxyl pectins (Morris et al., 1982; May, 1990). In the model, a section of two pectin chains that mustbe free of ester groups is held together by a number of Caz+ or other suitable divalent cation to form dimers that subsequently aggregate three dimensionally to produce a gel. Gel properties are dependent on pectin type and concentration, calcium/pectin ratio, temperature, and the presence of cosolutes such as sugars, citrates, sorbitol, and other calcium-sequestering agents. An increase in pectin concentration and Caz+/pectin ratio and a decrease in pH produced gels with significant increase in firmness inLMP formulations using different sweeteners. However, the Caz+ requirement for gelation depended on the type of sweetener. Optimum levels of Ca” required for gelation seem to depend on the DM of the pectin as well as the type of cosolutes present. A review of the variation of a gel mechanical property with Ca2+reveals that gel strength increases very rapidly as Ca2+ approaches the optimum level required for gelation (Hughes et al., 1980). As the DM of the pectin decreases, the calcium requirement decreases while the gel-setting temperature increases (Rolin and deVries, 1990). Unlike HMP, the gelation of LMP is not significantly affected by pH within the range of 5-7.0 (Hughes et al., 1980). The presence of cosolutes sucrose, glucose, fructose, and sorbitol significantly affected values of the storage modulus as a function of time in LMP gels at pH and pH 7; at pH 7, synerisis was manifested as first a steep drop and then a steep increase in the values of the modulus (Gross0 and Rao, 1998).

D. Gelation of Other Food Polysaccharides Other food polymers that are used to create food gels include agar, sodium alginate, gellan, and carrageenan. Their gelation properties have been discussed in Hams (1990) and will not be covered here. It is worth noting that the egg-box model is also used to understandgelation phenomena in sodium alginates.

c concentration, wlw coil overlap concentration, wlw c, concentrationwhenviscosityextrapolatedtozero,

c*

WIW CQ

starch granule mass fraction (SGM)

and Rao

G* =

+

diameter of the vane, m activation energy of gelatinization, J/mole elastic modulus, Pa storage modulus, Pa G" loss modulus, Pa (G")* complex modulus, Pa height of vane, m shear compliance, Pa" consistency index, Pa S" flow behavior index, dimensionless mass of heated starch granules per unit weight of dry starch gas constant, 8.314 J/mol K standard deviation of gelatinized starch granules temperature of fluid, "C, K maximum torque, N-m time constant, S deformation phase angle, rad time constant, S Casson yield stress, Pa Casson plastic viscosity, Pa S shear stress, Pa oscillating strain or deformation strain amplitude critical shear rate, S" shear rate, S-' viscosity function Pa S apparent viscosity, Pa S dimensionless apparent viscosity, Pa S ungelatinized apparent viscosity, Pa S highest magnitude of q. during gelatinization, infinite shear viscosity, Pa complex viscosity, Pa S zero shear viscosity, Pa S intrinsic viscosity, dL g" volume fraction, 4) = cQ maximum volume fraction tangential components of stress, Pa stress tensor time constant, S integral of time-temperature history, t-K

Rheology of Structured Polysaccharides

PI first normal stress coefficient, Pa ?Pz

S’

second normal stress coefficient, Pa oscillatoryfrequency,rad S-’

S’

Abdulmola, N., Hember, M. W. N., Richardson, R. K, and Moms, E. R. Composite rheology in mixed biopolymer systems. Paper presented at the 8th International Conference and Industrial Exhibition on Gums and Stabilizers for the Food Industry. Wrexham, Wales, UK Alloncle, M., and Doublier,J. L. Viscoelastic properties of maize starchhydrocolloid pastes and gels. Food Hydrocoll. Alloncle, M., Lefebvre, J., Llamas, G., and Doublier, J. L. A rheological characterization of cereal starch-galactomannanmixture. CerealChem. 66: Autio, K Rheological and microstructural changes of oat and barley starches during heating and cooling. Food Struc. P Bagley, E. B., andChristianson, D. D. Swelling capacity of starch and its relationship to suspension viscosity: Effect of cooking time, temperature and concentration. J. Texture Studies 13: Barnes, H. A. Shear-thickening (“dilatancy”) in suspensions of nonaggregating solid particles dispersed in Newtonian liquids. J. Rheol. 33: Barnes, H.A., and Walters, K. The yield stress myth? Rheol. Acta Barnes, H. A., Hutton, J. F., and Walters, K. An Introduction to Rheology. Elsevier Science, New York Biliaderis, C. G. Characterization of starch networks by small strain dynamic rheometry. In: Alexander, R. J., and Zobel, F., eds., Developments in Carbohydrate Chemistry. Am. Association of Cereal Chemists. St. Paul, Minn., pp. Biliaderis, S. G., and Tonogai,J. R. Influence oflipids on the thermal and mechanical properties of concentrated starch gels. J. A q . Food Chem. 39: Biliaderis, C. G., and Zawistowski, J. Viscoelastic behavior of aging starch gels: Effects of concentration, temperature, and starch hydrolysates on network properties. Cereal Chem. 67(3): Campanella, 0. H., Popplewell, L. M., Rosenau, J. R., and Peleg, M. Elongational viscosity measurements melting American process cheese. J. Food Sci Can, J. M., Sufferling, K., and Poppe, J. Hydrocolloids and their use in the confectionery industry. Food Technol. 49(7): Castelain, C., Concalves, M. P.,Doublier, J. L., and Lefebvre, J. Protein-polysaccharide interaction: Rheological evidence for aggregation process in aqueous

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Index Activity coefficient, Additive property, Agar, Agarose, Aggregation, Aging, Alginate(s), coprocessed with Avicel AC, MCC, Alginic acid, n-Alkylglucosides, Amorphism, Amphiphile(s), Amphiphilicity, a-Amylases, P-Amylase, Amylodextrin, retrograded, or-Amylolysis, Amylopectin, cluster structure model, conformation, gels, -lipid complexes, model for, phosphate ester groups in, precipitation, retrograded, theological properties, structure, Amylose,

[Amylose] amorphous, -amylopectin incompatibility, branching, coil-helix transition, conformation, DSC curves of, complexes, gels, helical form, -lipid complexes,

digestibility of, mechanism, retrograded, rheological properties, Anomers, Antagonistic ternary dispersion, Anticooperativity, Antinutitional aspects, Antiplasticization, Arabinoxylans, Area/volume ratio, Arrhenius equation, Association(s), cooperative, mechanics of, molecular, tendency, Attrition, Avicel AC, Avogadro’s number, Avrami equation,

330 Binding isotherms, 275 Biopolymer(s) random-coil, 292 solution, 297 Biosorption, 282 Biotechnology, 283 Bis(ethenyl)sulfone, 282 Branching, 3, 27, 30 Bread, 92, 129 antistaling role of lipids, 127 staling of, 123, 125, 131 staling retardation in, 252 Bridging flocculation, 9 Brownian activity, 4, 9 Carboxymethylcellulose (CMC), 173, 276 Carrageenan@), 3, 28, 30, 32, 33 aggregation by Na+, 50 chain flexibility of, 29 domain model of 31 double helix of 30 gelation requirement, 32 L-carrageenan, 29, 32, 278, 39, 43, 46 A-, 29, 38, 45, 46 29, 32, 38, 39, 43, 46, 48, 259, 261, 278, 279 domain model, 31 gel-forming ability, gel strength, 52 viscoelasticity, 266 specific ion effects, 32 synergism with WPI, 266 Carreau equation, 291 Casson model, plastic viscosity, yield stress, 294 Cation effect, 51, 52 Cellobiose, 23 Cellulose, 4, 16, 27, 170 alkylated, 3 crystalline structure, 28 gel, 172, 182 hydroxylated, 3 microdefibrillated,

Index Cellulose derivatives, 4 Charles-Boyle law, 9 Chitin, 278 Chitosan, 278 Coacervates, 6 Coacervation, Coefficient(s), 10 Huggins interaction, 11 Coil(s) non-free-draining, 5 overlap concentration,parameter, 293 random, 4 Collision diameter, 9 Complementarity, 6 Compliance, 301 Concentrated regime, 4 Configurations, 2, 22 Conformation(s), 4, 16 boat, 20, 21 chair, 20 energetically feasible, 25 ordered, 29 sterically regular, crystal-like, 28 Conformational mobility, 25 Consistency index, 293, 294 Contour length, 24 Cooperativity, 9, 278 Couchman-Karasz equation, 89, 90 Cox-Men. rule, 297 deviation from, 298 Creaming, 258 Creep compliance, 298, 300, 301, 302 Critical micelle concentration,4 Cross equation, 291 Crosslinking, 3, 29, 283 electrostatic, 5 Curdlan, 27, 29, 37, 38, 39, 43, 46, 48, 52 effect of alkali, 44 electron micrographs of, 49, 51 helical conformations, ordered, random-coil, 43, 44 specific rotation of, 48 triple helices, 44 X-ray diffraction patterns, 43,

Cycloamyloses, Cyclodextrin(s), analysis, applications, chemical modification of, -CO, complex, degree of substitution, from starch, inclusion complexes, nonsolvent process for, periodate oxidation of, physicochemical properties of, separations by selective binding, solvent process for, Cycloglucans, Deesteriiication, Degree of polymerization @P), Degree of substitution, Dextran, Dextrins, DP, Schardinger, Dielectric constant, Differential scanning calorimetry (DSc), Dilatancy, Dilute regime, Dimethylformamide (DMF), Dimethylsulfoxide (DMSO), Disordered assemblies, Dispersion antagonistic, dilute, Dispersion dynamics, Double-layer organization, DSC on WI/K-carrageenan, on WPI/guar, Egg-box structure, Elasticity, modulus of, 6

Elastic recovery, Electrostatic repulsion, Electroviscous region, Emulsion stabilization, Endpoint temperature, Entropy, configurational, conformational, loss of, Epichlorohydrin, Equilibrium dialysis, method, dye-metal, method, dynamic, monolayer concentration, polyanion-counterion, Equivalent hydrodynamic sphere@), Esterification, Etherification, Excluded volume, effects,

Fat mimetic, Fat replacement, Fiber, Films, Flory-Huggins interaction parameter, Flory-Weaver model, Flow behavior index, laminar shear, Fluid(s) elastic, pseudoplastic, shear-thinning, viscoelastic, Foam stability, Formaldehyde, Formic acid, Fox-Flory equation, Fractal dimension, Free volume, Functionality of MCC/CMC, Furcellaran,

332 Galactan, Galactomannans, Galactose, Gelatin, Gelatinization, Gelation, by alkali, by electrolytes, by neutralization, by synergism, domain model, effect of hydrocolloids, effect of ions, effect of lipids, HMP,

LMP, prerequisite for, protein, variables, Gel(s), amylopectin, amylose, emulsifiers, influences on starch, fractal dimension, hydro-, ionic, lipids, influences on starch, mechanical properties, mesomorphic, 5 micro-, microfibrillar differences, micrographs of, mixed, point, quaternary structure, rigidity, starch, phase separation, strength, concentration dependence, strong, thermally irreversible, reversible, true, weak,

[GeWl whey protein isolate, mixed, xero-, Gellan, Glucanase resistance, 44, Glucans, Glucose polymers, Gordon-Taylor equation, Guar gum, Gums, dispersed, effect of water, Heat shock, Heavy-metal biosorption, Hemiacetal, Herschel-Bulkley model, Heterogeneity, Hill parameter, Homogenization, Hooke’s equation, Huggins equation, Hydrocolloids, Hydrodynamic interaction, Hydrodynamic radius, 5 Hydrogel, Hydrogen bonds, Hydrophile-lipophile balance, Hysteresis, IJKI splution, Indulgem, Interaction(s), coefficients, distance, hydrodynamic, hydrophobic, 44, noncovalent, polysaccharide-cation, -metal, -polysaccharide, -protein, -water, solute-solute, specific ion,

333 [Interaction(s)] starch-lipid, in food processing, 250 van der Waals, 24 Interfacial tension, 7, 9 Ion activities, 274 Ion binding, 279 Ion-selective electrodes, 274

Junction zones, 4, 5, 33, 52, 63, 109

Kelvin-Voigt unit, 298 Kinetic equilibrium, 12 stability, 10 Konjac flour gum, 292, 297 Konjac glucomannan, 38, 39, 43, 46, 178 Krieger-Dougherty model, 306

Liquid crystals, 5 Lipids, 78, 126, 127, 227, 136, 315, 315 Locust bean gum, 38,48,52, 259, 261, 292,297, 309, 315 Iwcarrageenan gel, 52 Ixanthan sol, 52 Loss tangent, 296

Macromolecular assemblies, 45 Macromolecule(s), 2 adsorption of, Maltodextrin, 259, 265 Malto-oligosaccharides, 90 Maltose, 23 Maltotriose, 71 Manning theory, 275, 276 Maxwell element, 298 MCC/alginate, 182 MCCfiydrocolloid functionality, 179 heat-stability, 175

MCCEMC, 185 MCC in ice cream, 180 Mesophases, lyotropic, thermotropic, 5 Mesomorphs, 5 Mesquite seed gum, 292, 297, 309 Micelle, 3, 4 Microcrystalline cellulose (MCC), 170, 173, 180 Microgels, 261 Microscopy, 260 Microstructure of mixed gels, 261 Modulus (Moduli) and food rheological classification, 297 bulk, 6 loss, 296, 308 of elasticity, 6, 296, 298 plateau, 297 retarded elastic, 301 rigidity, 6 stress relaxation, 6 storage, 296, 308 of mixed gels, 261, 262 time-dependent relaxation, 300 Molar substitution, 212 Monosaccharide structures, 17 Mutarotation, 20, 21

NaCl, 265 Newtonian flow, 291, 292, 301 Newtonian fluids, 309 Novagelm, 182, 185

Oligosaccharides, 2, 210 Ordered polysaccharide assemblies, 4 Oscillatory tests, 295 Osmotic pressure, 9 concentration dependence, 10

Parallel orientations, 3 Parboiled rice, 136 Particle growth, 10

Index Pectin(s), high:methoxyl (HMP), gelation, low-methoxyl (LMP), gelation, rhamnose kink, slow-set, Peptization, Phase diagrams, Phase inversion point, Phase transition, of V-complexes, Phytic acid, Polyelectrolytes, Polymer characteristic ratio, flexibility, free-draining, non-free-draining, random-coil, 24 rigidity, topology, Polysaccharide(s), -anion sensitivity, aqueous, association tendency, block copolymers in, -cation interaction, cis-, free-draining, gelling, macromolecular assemblies, -metal complexes, metal specificity, microfibrillar differences, helical structures, micrographs of gel-forming, molecule, non-free-draining, nongelling, ordered assemblies, primary structure, -protein interactions, random-coil(s), 9, rodlike, secondary structure,

[Polysaccharide(s)] solation-gelation, sols, solubilization by NaOH, structures, sulfated, suprastructures, trans, triple helices in water, types, A, B, ultrastructure, water-soluble, WPI, temperature effects, xerogel, Polyuronates, Polyuronic acids, Potential electrochemical, energy, 25 zeta, Power-law equation, Power law model, Precipitation, Primary structure, Proteins, structural organization, Protective colloid, Protein-polysaccharide interactions, synergism, Protopectin, Pseudoplastic behavior, Pullulan, Pyranose ring forms,

Quasistatic tests, Quaternary structure(s),

Radius of gyration, Random coil polymer(s), Random coil(s), 9, 44 Recoverable shear,

335 Regime concentrated, 4 dilute, 4 semi-dilute, 10 Relaxation, 6, 91 temperature dependence, 93 times, 291 Repeptization, 276 Reptation, 10 Resistant starch, 78, 80 Retardation time, 301 Retrogradation, 70, 252 effect starch-lipid complexes on, 248 effect of sugars, 127, 128 prevention or retardation of, 131 Rhamnose kink, 30 Rheology, 290 Rods, 4 Root mean square end-to-end distance, 5, 24

Scatchard plot(s), 275, 278 Schardinger dextrins, 207 Schizophyllan, 45 Screloglucan, 38, 45 Semi-dilute regime, 10 Shear deformation, 300 rate, 294 stress, 294 -thickening, 293, 304 -thinning, 291, 293 Sol-gel transition, 38, 274, 303 Stability foam, 181 kinetic, 10 ring, 20 thermodynamic, 10 Starch, 57, 259, 293 A, B, C polymorphs, 69, 72, 105 acid-resistant, 59 aging, 78, 124, 313 alkylated, 3

[Starch] amorphous, 72, 73, 87, 91 amylose-amylopectin incompatibility, 74 annealing effects, 94, 95 crystalline, crystallization, 72, 125 water effects on, 125 DSC thermal curve, 98, 99 effects of annealing, 94 effects of heating, 94, 96, 103 effects of hydrocolloids, 107, 129 effects of milling, 105 effect of water, 87, 91, 92 enthalpies, 239 enzyme-resistant, 252 esterification, 3 etherification, 3 functionality, 62 gelatinization, 78, 82, 94, 96, 102, 105, 248, 303 effects of salt, 107 effect of sugars, 105, 106, 109 rheological changes, 302, 304 gelatinized suspensions, 304 gels, 57, 109, 110, 120, 310, 311, 313 effect of proteins, 316 influence of hydrocolloids, 315, 316 influence of lipids and emulsifiers, 315 glass transition, 83, 85, 87, 89, 90, 93 granule differentiation, 58, 59 granule organization, 68 hydroxylated, 3 instant, 108 -iodine reactions, 62 lintnerization, 103 -lipid complex formation, 80, 234 macromolecules, 60 Maltese 67 melting behavior, 100 molecular and solid-state structure 58, 71

336 [Starch] order-disorder, 82 pastes, 57, 78 phase separation, 117 resistant, 78, 81 retrogradation, 79, 82, 123 effect of lipids, surfactants/ emulsifiers, 248 effect of sugars, 118, 127, 128 prevention, 131 retrograded structures, 78, short-chain precipitation of, 63 small-angle X-ray scattering by, 62 sol-gel transition in, 303 structure-property relationships, 139 syneresis, 79, 123 temperatures, 239 three-microphase model, 86 transitions, 82, 87, 98, 102 of V-complexes, 131 V-polymorph, 74, 92, 102, 234 viscoelasticity, 122, 126, 304 X-ray diffraction patterns of, 70 Steric stabilization, Stopped-flow polarimetry, 30 Strain, 6 Stress relaxation, 298 Stress tensor, 290 Structure(s), branched, 27 double-helix, 15 levels of organization, 16 molecular, definition, 15 monosaccharide, 17 ordered crystalline, 27 polysaccharide, 15 primary, 2, 16 quaternary, 4, 16 retrograded starch, 78, 116 ring, 20 secondary, 2, 16 supramolecular, 7 tertiary, 4, 16 Succinoglycan, 38, 45, 46, 279 Sucrose, 22, 127

Index Sugars, 89, 105, 106, 109, 116, 118, 127, 172, plasticizing effect of, 89, 172 Surface area, 9, 10 Surface tension, 7 Surfactancy, 1, 127 Surfactant clusters, 5 Syneresis, 42, 52, 70, 79, 123, 279 control, 181 Synergism, 6, 52, 266 whey protein/polysaccharide, 265

Temperature@) consolute, 11 effect on metal binding, 279 effects on polysaccharide/WPI mixtures, 266 endpoint, 309 glass transition, 4, 233 dependence on, 124 Tertiary structure, 4 Thermodynamic equilibrium, 11 incompatibility, 313 stability, 9 Thixotropy, 4, 304, 310 Time-dependent retarded elastic region, 301 Topology, 3 Transition@), coil-helix, 115, 228, 277, 282 disorder-order, 2 effect of water on melting, 132 helix-to-coil, 30 order-disorder, 2 in alkali, 44 phase, 279 sol-gel, 38 V-complexes, 131 Transport parameter, 276

Ultrastructures in sol-gel transition, 38 Uniaxial deformation, 300

Urea, Uronans,

V-complexes, Vesicles, V i a l coefficients, Viscoelastic fluids, material, Viscoelasticity, effect of emulsifiers on, of WPIIK-carrageenan, Viscosity, apparent, biaxial extensional, complex, -concentration relationship, electroviscous region, infinite shear, intrinsic, number, shear, Volume excluded, free, of a dispersion,

Water, compressibility, effect of, on DSC curves,

[Water] effect on starch of restricted, free-draining, non-free-draining, of hydration, 5 plasticizing effect of, stabilizing effect of, Weissenberg effect, Whey protein isolate (WPI) gels, gelation, WPI/K-carrageenan, effect of NaCl on, WPIImaltodextrin, WPIIstarch, WPIIxanthan, Williams-Landel-Ferry equation, Work, WPIIprotein synergism, Xanthan, single- and double-stranded fibrils in, Xerogel, dissolution, Yield point, Yield stress, dynamic, Zeta potential, 8