377

(Oyo Toshitsu Kagaku (J. Appl. Glycosci.), Vol.43, No.3, p.377-384 (1996))

Review

Structure-Property Relationships of Water-Soluble Polysaccharides

James N. BEMILLER

Whistler Center for Carbohydrate Research, Purdue University

(West Lafayette, Indiana 47907-1160, USA)

The need for understanding structure-physi

cal and functional property relationships of

water-soluble polysaccharides is still strong. It is estimated that at least 1011 tons of polysac

charide are generated annually on planet earth

because the compositions of both land and marine plants is dominated by polysaccharides.

Greater use of biomass, a renewable, replace-

able resource, will be required as petroleum-

derived materials become scarcer and more expensive, should be done immediately as a

conservation measure, and depends in part on

determining polysaccharide structures and how they determine physicochemical properties.

For humankind to make greater practical use of

natural biomaterials, the ability to improve specific properties for specific applications is

necessary. Fortunately, polysaccharide struc

tures, diverse in nature, can be easily modified and their properties manipulated enzymically,

chemically, and genetically.

The origin of polysaccharide structure-property relationships is rooted in the concepts of WHISTLER.1)REEs pioneered the use of instrumental methods to relate structures and

physicochemical properties, gave us insight into the shapes of polysaccharides, appears to have first used the term "junction zones" with regards to polysaccharide gel structures, and coined the term "egg box" to describe the nature of the junction zones in calcium alginate

gels. The fundamental concepts he developed were summarized in two small books2,3) (1967 and 1977) and a review paper4) (1972). REES

(1972) also outlined the various mechanisms of gel formation involving polysaccharides, which are 1) double helix formation followed by

packing of the double helices, in some cases the packing of the double helices being mediated by cations, 2) formation of an "egg-box" junction zone in the presence of divalent cations, and 3) association of two different polymer molecules. It is now known that gels can also be formed by an entanglement of polysaccharide chains that results in network formation, that

junction zones can be formed between anionic polysaccharides and protein molecules (via ionic interactions in which the protein serves as a polycation), and that, as will be discussed below, at least in the "mixed aggregate" type of gel formation exhibited by xanthan plus locust bean gum (LBG), there is evidence that LBG serves as a crosslinker for xanthan super-strands. ARNOTT5), employing x-ray fiber diffraction analysis, provided an understanding of the basic helical nature of polysaccharides.

Research and hypothesis formation on relationships between chemical structures, molecular architectures, physical properties, and functional properties of water-soluble gums has been continued by a few groups in industrial,

governmental, private, and university research labs who have employed additional experimental techniques as they and the required instrumentation became available. Techniques now employed to examine polysaccharide structures and/or behaviors include pulsed electric birefringence; vacuum UV circular dichroism; electron microscopy; solid state and liquid NMR; dynamic rheology; small-angle light, neutron, and x-ray scattering; sedimentation velocity

* Presented at the Annual Meeting of the Japanese

Society of Applied Glycoscience, Osaka, September 1995.

378 Oyo Toshitsu Kagaku (J. Appl. Glycosci.), Vol. 43, No. 3 (1996)

determinations; FTIR and Raman spectroscopy; thermal analysis; and X-ray fiber diffraction. We can expect additional information in the future from atomic force microscopy. Coupling of experimental data with molecular modeling is often required to give the most probable analysis. The dynamics of systems may be the next frontier. This review covers a fraction of the available information, with a purpose of

presenting through two examples developments that have occurred in our ability to relate chemical structures, conformations, physical properties, and f unctionalities of water-soluble

polysaccharides (gums) and something of the current state of the science. Some general principles, that can be expressed as the following sequential tenets, have been developed.6-8) Polysaccharides with regular repeating-unit sequences have a natural tendency to adopt helical conformations. Determinations of the conformations of polysaccharides largely involves characterization of the helix. With polysaccharides, there is generally a preference for polymer-polymer contacts over poly-mer-water contacts.9) Behaviors of polysaccharide solutions and gels are related to inter-actions between helices. These interactions are responsible for the associative properties of polysaccharides. Formation of associated regions of polysaccharide chains is a crystallization process that follows the three-step mechanism for the crystallization of partially crystal-line polymers.10) Interactions between helical segments are effected by surrounding water molecules (and cations in the case of anionic

polymers) which, along with inter- and intramolecular hydrogen bonding, play a definite, specific role in intermolecular associations.

(For examples of the role played by water molecules, see References 11 and 12.) Interactions of the polymer molecules must be limited; otherwise insolubility results. Interactions restricted to junction zones are the ones that can affect properties of solutions and gels.

XANTHAN-LOCUST BEAN GUM GELATION

The first example will be that of the synergis

tic interaction between xanthan and galacto

mannans, especially locust bean gum, a syner

gistic interaction that has been described, studied, and used for at least 20 years.

Xanthan has been in commercial use for more

than 30 years. It is a non-gelling polysaccharide that imparts unique and useful

rheological properties to aqueous systems.

Prominent among these useful properties is

high viscosity at low concentration and low shear rates13) and a high degree of pseudoplas

ticity. It is known commercially as "xanthan

gum." Xanthan undergoes a conformational tran

sition as the properties of the solvent are changed. An ordered, helical conformation is stabilized by high ionic strength and/or low temperature, both of which increase inter-molecular associations, with the conformational transition midpoint temperature increasing with increasing ionic strength.l4-20) Whether the ordered conformation of xanthan in solution is that of a single17,21) or double helix22-25) or both and the conditions required for either remains an open question; but chain aggregation, whether aggregation of single or double helices, is accepted.25> Results from a variety of

physicochemical techniques suggest that, in solution, xanthan is a rather stiff molecule with a low degree of flexibility,17,25,27,28) which accounts for the very pseudoplastic nature of its solutions. Xanthan does not, by itself, form

gels; but elastic, thermoreversible gels are formed upon cooling hot solutions containing both xanthan and locust bean (carob) gum, a

galactomannan and another nongelling polysaccharide.

There are two principal commercial galacto

mannans, guar gum, the main component of

which is guaran, and locust bean gum.29)

Guaran has a Man: Gal ratio of 1: 0.65 and is the more soluble of the two polysaccharides.

Guaran derives its solubility from irregular substitution of the mannan backbone with a-D-

galactopyranosyl units, as determined by MCCLEARY.30) Locust bean gum has a Man : Gal ratio of 1: 0.28, contains backbone stretches

that are unsubstituted, and is less soluble than

guar gum, presumably because of inter-molecular associations between unsubstituted

379Structure-Property Relationships of Water-Soluble Polysaccharides

segments. Locust bean gum preparations can

be fractionated into a series of polysaccharide

fractions with Man: Gal ratios between approximately 1: 0.33 and 1: 0.20, the fraction with the

lowest content of a-D-galactopyranosyl side

units being the least soluble.31,32> Neither polysaccharide is represented by a single uniform

structure.

The molecular architecture of galactomannan molecules has not been described, but they are reported to be fluctuating, disordered, mobile coils in solution.33) Intermolecular associations between unsubstituted regions of chains occur. MORRIS et al.33,34) suggest that these associations are in addition to normal topological entanglements characteristic of

polysaccharides with 1, 4-diequatorial geometry and result in a deviation from the rheological

properties of typical random-coil polysaccharides. Konjac glucomannan35) and tamarind xyloglucan,36) both of which have backbone chains in which the monomer units are joined by diequatorial (1-4) glycosidic bonds, behave similarly.

In a binary solution of xanthan and locust bean gum, the two polymers interact and form a supermolecular network, i.e., a gel. Gelation is most pronounced with a locust bean gum fraction with a high Man: Gal ratio.31,32,37) (The structure of xanthan is constant.) It has been

proposed by LUNDIN and HERMANSSON37) from electron microscopic observations, that the elastic network is formed from supermolecular strands of xanthan and that strand formation is neither hindered nor altered, i, e., is unaffected, by the presence of locust bean gum, that locust bean gum polysaccharide molecules are able to bind both to single-stranded xanthan helices and to surfaces of xanthan superstrands, and that in a xanthan-locust bean gum gel, xanthan super-strands are connected by bridges of much smaller locust bean gum polysaccharide molecules (Fig. 1). (Gels consist of a supermolecular network of polymer molecules or a network of interacting particles. Polysaccharide gels are composed of a statistical ensemble of polymer molecules with various sizes, degrees of helical character, and interchain associations, i.e., they are not homogeneous at the molecular level.)

Fig. 1. Schematic depiction of how locust bean gum (LBG) (thin lines) might leash together supermolecular strands of xanthan (thick lines) after

LUNDIN and HERMANSSON.37)

According to the prevailing hypothesis, only smooth,

i, e., unbranched, regions of LBG bind to xanthan super-

strands. Only the LBG molecules that may be involved in crosslinking are depicted. It is reasonable to assume

that the intermolecular binding is dynamic.

Electrolytes influence the viscosity and rheology of xanthan solutions26) through an influence on molecular structure. Using a combination of low-amplitude oscillation rheometry and thermal analysis, MORRIS and FOSTER38) concluded that gelation onset begins at the same temperature (ca. 60t) whether salt is present or absent. However, at the xanthan concentration and under the conditions used, the disorder-order transition temperature in water was below the gelling temperature, so association of the two polysaccharides occurred with xanthan in its disordered form. This gelation mechanism was proposed earlier by CAIRNS et al.39>40) MORRIS and FOSTER38) further reported that addition of sodium chloride to a concentration of 30 mM raised the disorder-order transition temperature to a temperature above that at which gelation occurs, so in this case, xanthan is in an ordered conformation when network for-

380 Oyo Toshitsu Kagaku (J. Appl. Giycosci.), Vol. 43, No. 3 (1996)

mation begins. WILLIAMS et al.20) also reported that xanthan is in an ordered state as the net-work is formed. (LUNDIN and HERMANSSON37) used no salt, but the xanthan they used was completely in the sodium salt form.) Both MORRIS and FOSTER38) and ZHAN et al.41) found no dependence of the gel melting temperature on salt concentration. MORRIS and FOSTER38) concluded that xanthan-locust bean gum gels can be formed under conditions where xanthan is either ordered or disordered, i, e., that neither condition is required for gelation, that the same structure is formed in either case, and that the xanthan helix can undergo conformational rearrangement to accommodate binding inter-actions with locust bean gum. Both groups conclude that the interaction between xanthan and, at least a fraction of, locust bean gum occurs as xanthan helices are formed or that locust bean gum promotes helix formation, that the addition of salt leads to a decrease in net-work strength due to promotion of formation of aggregates from stabilized xanthan helices, and that the interaction between xanthan and, at least a fraction of, locust bean gum is competi-tive with the formation of aggregates of xanthan helices. Clearly, the complete mechanism is not known and there will be more work and results in this area.

THE GELLAN FAMILY

The second example will be that of the gellan family of polysaccharides. The principal member of this family is gellan gum, a commercial multi-functional, gelling, bacterial polysaccharide.26,42) The native polysaccharide is called gellan. It is one of seven known, naturally occurring polysaccharides of closely related chemical structures8) (Fig. 2) known as the gellan family. Five of the seven members of this family have a single mono- or disaccharide branch unit on the tetrasaccharide repeat unit of the main chain. The diversity in

properties exhibited by members of this family can be correlated to the location and type of these side units,8,42) and in several cases, chemical structures of the polymers can be related to their three-dimensional structures.8)

Fig. 2. Repeating unit structures of eight members of

the gellan family of polysaccharides.

Gellan contains an acetyl group and a glyceryl group on one of the a-D-glucopyranosyl units of the tetrasaccharide repeating unit (unit 1, Fig. 2) and is practically non-gelling.26a Gellan gum is the commercial name of deesterified (deacylated) gellan. In the presence of cations,

gellan gum forms firm, brittle, and both thermoreversible and irreversible gels. In aqueous solution, it behaves as a stiff coil43) that, like other gelling polymers and polymer systems, undergoes a disorder-order transition during

gelation and an order-disorder transition during melting.44-46) However, ordered structures remain in solution after melting.47) Pulsed electric-birefringence suggests that gellan solutions contain highly extended, rigid polymer chains or rod-like aggregates.48)

381Structure-Property Relationships of Water-Soluble Polysaccharides

Using combinations of x-ray fiber diffrac

tion and molecular modeling techniques,

CHANDRASEKARAN and coworkers have deter-mined the shapes of the polymer molecules of

two members of the gellan family of polysac

charides: gellan8,49-51) and welan,52,53) in regions where they interact with one another. A basic

question addressed by them is why do groups appended to the main chain inhibit inter-molecular associations in the case of some lin-

ear polysaccharides, while in other cases,

appended groups enhance associations. The CHANDRASEKARAN group has determined that the molecular structure of gellan gum is that of a double helix formed by the intertwining of two left-handed, threefold helical chains in parallel. A translation of one chain along the helix axis by half the pitch leads to its exact superposition on the other chain.49) Strong union is provided by monovalent cations octahedrally coordinated in the vicinity of the carboxylate group of each tetrasaccharide repeat unit, the ligands being both carboxylate oxygen atoms and the oxygen atoms of two hydroxyl

groups of one chain, the oxygen atom of one hydroxyl group of the other chain, and the oxygen atom of a crystalline water molecule.11) To form a junction zone of a supermolecular gel structure, gellan double helices become aligned in an antiparallel fashion and connected by strong carboxylate-cation-water moleculecation-carboxylate interactions.g) A finite concentration of each cation is required for

gelation.26,42) As the concentration is increased beyond the minimum required for gelation, gel hardness and modulus increase to a maxi-mum.26,42) Water-soluble substances lower the ionic requirement.26,42) Calorimetric and chiroptical data revealed a salt-induced, thermally reversible conformational transition of gellan

gum in aqueous solution.54) When divalent cations, notably calcium ions,

are used for gelation, each bridge is replaced by a single divalent cation.50) Only about 1/40th as much calcium ions, as compared to potassium ions, which are quite effective monovalent cat-ions, are required to give maximum gel hard-ness and modulus.26,42>

Native (acylated) gellan will form gels with

potassium and calcium ions, but the gels are weak and elastic, as compared to the firm, brittle gels formed by deacylated gellan.26,42) In acylated gellan, the two hydroxyl groups of the

glyceryl group occupy about the same sites as the monovalent cation and the water molecule, thereby shielding the carboxylate group, but also introducing a new interchain hydrogen bond that adds stability to the double helix. Due to the shielding, the occupancy of the cation is reduced to one half and the ion is displaced relative to its position in deacylated gellan. The presence of the glycerate ester group also increases the distance between chains by about 5%, further weakening ion-mediated, interchain bonding. The acetyl groups of native gellan do not interfere with chain packing because they

protrude from the helix. Welan is a non-gelling, high-viscosity thick

ener with exceptional thermal stability, which is

the basis for it use in oil fields. At shear rates

of <10 sec-1, its solutions are more viscous than

those of xanthan. For welan solutions, the

elastic modulus, G', predominates over the

viscous modulus, G", through an entire dynamic

rate sweep, while for xanthan solutions, the

viscous modulus predominates at frequencies

of < 1 rad/sec.42,55) (In reference 42, the legends

to Figs. 8 and 10 are reversed.) At frequencies

around 0.1 rad/sec, welan solutions have elastic

modulus values about five times larger than

those of xanthan solutions of the same concen

tration. Solutions of both gums are very

pseudoplastic. This information indicates that

welan solutions contain even more structure

than do xanthan gum solutions. Welan mole

cules also bind calcium ions, which is the basis

for welan's use in concrete, especially where it

is to be set under water. Welan is the simplest

branched member of the gellan family of

polysaccharides. It has the same backbone

structure of gellan, but contains either an a-L-

rhamnopyranosyl or an a-L-mannopyranosyl

unit (ratio about 2:1) on unit 3 of the main

chain (Fig. 1). Approximately 85% of the 0-3

substituted ƒÀ-D-glucopyranosyl units (Fig. 2,

unit 1) contain an acetyl group at 0-2,56)

Replacement of a monosaccharide branch unit

with a disaccharide (rhamnobiosyl) branch unit

382 Oyo Toshitsu Kagaku (J. Appl. Glycosci.), Vol. 43, No. 3 (1996)

(polysaccharide S-657) imparts even greater heat and shear stability to the molecule.42)

The CHANDRASEKARAN group has determined that welan forms a half-staggered, parallel,

gellan-like double helix in the presence of calcium ions57) that is more stable than the gellan double helix, but the double helices do not associate to form junction zones of a super-molecular network that would provide a gel structure. The side units fold back along the backbone and both coordinate chain association and stabilize welan's ordered helical structure,52,53) similar to the presumed function of the trisaccharide side chains of xanthan.17,13,21) Molecular stability is enhanced by side-unit shielding of the carboxylate group. And, because of the shielding, welan behaves more like a neutral polysaccharide than like an anionic polysaccharide.42) Calcium ions cross-link carboxylate groups in neighboring welan double helices, a crosslinking that is correlated with its rheological properties.42) In my opinion, this analysis of conformation

of these two polymers in areas in which like

polymer chains are associating is the most definitive example to date in relating molecular

architectures to physical properties imparted by

polysaccharides to aqueous systems. There have been other recent contributions to

structure-function relationships of gellan. Mentioned here is that of QUINN et al.47) who have reported the following hypothesis. Slow cooling of gellan gum solutions allows the water envelope to average and results in more homo

geneous supermolecular systems than are produced by rapid solution cooling. In gellan gels, initial junction zones contain an estimated four double helices with a length of five repeat units (20 monosaccharide units). The length of the junction is increased to seven repeat units by annealing. Junction zones are linked by extended, helical single chains. Melting of a

gellan gum gel upon heating involves the follow-ing transitions: junction zones-individual double helices-stiff, extended helical single chains. Some junction zones and double helices persist in the resulting solution, so the gel to solution transition is not an all-or-none transition, but a

gradual, partial destructuring and some reag

gregation. Aggregates that form in solution

are composed of double helices, extended single

helices, and perhaps other chains in a super-

molecular, liquid crystalline structure. Like-

wise, the gel•¨solution transition in xanthan

locust bean gum gels is a gradual one in which,

at any given temperature within the range,

various states of conformation and association

occur.17) (See also references 58 and 59.)

CONCLUSIONS AND THE FUTURE

Increased utilization of polysaccharides will occur because they are abundant; replenishable; inexpensive; functional; amenable to chemical, biochemical, and physical modification; safe/ nontoxic; and biodegradable. However,

greater practical use of polysaccharides in foods, in non-food industrial applications, and for their biological activities, depends, in some cases, on being able to modify their structures chemically, enzymically, and/or genetically to improve specific properties. In order to make use of the ability to modify their structures, we must understand better their structure-physical

property-functionality relationships. Much progress has been made in this area

over the past two decades as new, more power-

ful, more discerning experimental and molecu

lar modeling techniques have been applied; but

progress has been relatively slow because of a rather limited number of investigators. This

science relies upon a thorough understanding of

both the rheological properties of a particular system containing a gum and the molecular

architecture of the hydrated gum molecules or, much more often, hydrated gum molecules in

intermolecular associations with like molecules

or with other hydrophilic polymer molecules

and how they are related. Both aspects require contemporary physicochemical and modeling

techniques. There is a continuing need for the kind of information given by this science, both

from a standpoint of curiosity and from a prac

tical point of view. The curiosity aspect includes understandings of the biological func

tions of polysaccharides, but such understand

ings could also lead to practical extraction and recovery processes and applications.

383Structure-Property Relationships of Water-Soluble Polysaccharides

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(Received June 25, 1996)