Gelation of Gellan a Review

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Food Hydrocolloids 28 (2012) 373e411

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Food Hydrocolloids

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Review

Gelation of gellan e A review

Edwin R. Morris a, Katsuyoshi Nishinari b,*, Marguerite Rinaudo c

aDepartment of Food and Nutritional Sciences, University College Cork, Cork, IrelandbDepartment of Food and Nutrition, Graduate School of Human Life Science, Osaka City University, Sumiyoshi, Osaka 558-8585, JapancCentre de Recherches sur les Macromolecules Végétales (affiliated with the Joseph Fourier University of Grenoble), CERMAV-CNRS, BP 53, 38041 Grenoble,Cedex 9, France; Present address: ESRF, BP 220, 38043 Grenoble Cedex 9, France

a r t i c l e i n f o

Article history:Received 12 August 2011Accepted 3 January 2012

Keywords:GellanGelationHigh acylAggregationDouble helixPolyelectrolyte

* Corresponding author. Tel.: þ81 6 6605 2818; faxE-mail address: [email protected] (K

0268-005X/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.foodhyd.2012.01.004

a b s t r a c t

Gellan is an anionic extracellular bacterial polysaccharide discovered in 1978. Acyl groups present in thenative polymer are removed by alkaline hydrolysis in normal commercial production, giving thecharged tetrasaccharide repeating sequence: / 3)-b-D-Glcp-(1 / 4)-b-D-GlcpA-(1 / 4)-b-D-Glcp-(1 / 4)-a-L-Rhap-(1 /. Deacylated gellan converts on cooling from disordered coils to 3-fold doublehelices. The coilehelix transition temperature (Tm) is raised by salt in the way expected from poly-electrolyte theory: equivalent molar concentrations of different monovalent cations (Group I andMe4Nþ) cause the same increase in Tm; there is also no selectivity between different divalent (Group II)cations, but divalent cations cause greater elevation of Tm than monovalent. Cations present as coun-terions to the charged groups of the polymer have the same effect as those introduced by addition ofsalt. Increasing polymer concentration raises Tm because of the consequent increase in concentration ofthe counterions, but the concentration of polymer chains themselves does not affect Tm. Gelation occursby aggregation of double helices. Aggregation stabilises the helices to temperatures higher than those atwhich they form on cooling, giving thermal hysteresis between gelation and melting. Melting ofaggregated and non-aggregated helices can be seen as separate thermal and rheological processes.Reduction in pH promotes aggregation and gelation by decreasing the negative charge on the polymerand thus decreasing electrostatic repulsion between the helices. Group I cations decrease repulsion bybinding to the helices in specific coordination sites around the carboxylate groups of the polymer.Strength of binding increases with increasing ionic size (Liþ < Naþ < Kþ < Rbþ < Csþ); the extent ofaggregation and effectiveness in promoting gel formation increase in the same order. Me4Nþ cations,which cannot form coordination complexes, act solely by non-specific screening of electrostaticrepulsion, and give gels only at very high concentration (above w0.6 M). At low concentrations ofmonovalent cations, ordered gellan behaves like a normal polymer solution; as salt concentration isincreased there is then a region where fluid “weak gels” are formed, before the cation concentrationbecomes sufficient to give true, self-supporting gels. Aggregation and consequent gelation with Group IIcations occurs by direct site-binding of the divalent ions between gellan double helices. Highconcentrations of salt or acid cause excessive aggregation, with consequent reduction in gel strength.Maximum strength with divalent cations comes at about stoichiometric equivalence to the gellancarboxylate groups. Much higher concentrations of monovalent cations are required to attain maximumgel strength. The content of divalent cations in commercial gellan is normally sufficient to give cohesivegels at polymer concentrations down to w0.15 wt %. Gellan gels are very brittle, and have excellentflavour release. The networks are dynamic: gellan gels release polymer chains when immersed in waterand show substantial recovery from mechanical disruption or expulsion of water by slow compression.High concentrations of sugar (w70 wt % and above) inhibit aggregation and give sparingly-crosslinkednetworks which vitrify on cooling. Gellan forms coupled networks with konjac glucomannan andtamarind xyloglucan, phase-separated networks with kappa carrageenan and calcium alginate, inter-penetrating networks with agarose and gelling maltodextrin, and complex coacervates with gelatinunder acidic conditions. Native gellan carries acetyl and L-glyceryl groups at, respectively, O(6) and O(2)of the 3-linked glucose residue in the tetrasaccharide repeat unit. The presence of these substituentsdoes not change the overall double helix structure, but has profound effects on gelation. L-Glyceryl

: þ81 6 6605 3086.. Nishinari).

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E.R. Morris et al. / Food Hydrocolloids 28 (2012) 373e411374

groups stabilise the double helix by forming additional hydrogen bonds within and between the twostrands, giving higher gelation temperatures, but abolish the binding site for metal ions by changing theorientation of the adjacent glucuronate residue and its carboxyl group. The consequent loss of cation-mediated aggregation reduces gel strength and brittleness, and eliminates thermal hysteresis. Aggre-gation is further inhibited by acetyl groups located on the periphery of the double helix. Gellan witha high content of residual acyl groups is available commercially as “high acyl gellan”. Mixtures of highacyl and deacylated gellan form interpenetrating networks, with no double helices incorporatingstrands of both types. Gellan has numerous existing and potential practical applications in food,cosmetics, toiletries, pharmaceuticals and microbiology.

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Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3742. Conformation in the solid state and in solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .376

2.1. Structure of gellan in the solid state . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3762.2. Interactions of cations with anionic polyelectrolytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3762.3. Conformational transitions of gellan in solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3772.4. Light scattering, osmometry and small-angle X-ray scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379

3. Cation-induced gelation of gellan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3803.1. Rheology of solutions and gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3803.2. Critical gel point . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3813.3. “Weak gels” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3823.4. Gelation of gellan with Group I (alkali metal) cations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3833.5. Gelation of gellan with Me4Nþ cations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3863.6. Gelation of gellan with Group II (alkaline earth) cations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3863.7. Effect of excess salt or low pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3883.8. Summary and interpretation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388

4. Gelation of gellan in water, with no added salt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3895. Topology and properties of gellan networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393

5.1. Internal structure of gellan gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3935.2. Dimensions of strands in gellan networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3945.3. Gelation by cations at ambient temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3955.4. Conformational freedom and release of polymer chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3955.5. Texture of gellan gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3965.6. Mobility of water in gellan networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3975.7. Syneresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3975.8. Flavour release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3985.9. Gellan liquid crystals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399

6. Effect of sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3997. Effect of acyl substituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402

7.1. Acyl groups in native gellan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4027.2. High acyl gellan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4027.3. Blends of high acyl and deacylated gellan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4027.4. Partially deacylated gellan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4037.5. Individual roles of glyceryl and acetyl groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403

8. Mixtures and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408

1. Introduction

Gellan is the most recent addition to the range of gellingagents available commercially for use in food (Gibson &Sanderson, 1997; Sanderson, 1990). It is an extracellular bacte-rial polysaccharide synthesised (Pollock, 1993) by Sphingomonaselodea (ATCC31461), formerly known as Auromonas elodea orPseudomonas elodea, and was identified as having commercialpotential (Sanderson, 1990) in 1978, during an extensivescreening programme of soil and water bacteria by Kelco (SanDiego, USA), the company that was also the first to producexanthan as an industrial polysaccharide.

Gellan, which was known initially as polysaccharide S-60, isa linear anionic polymer with a tetrasaccharide repeating sequence

(Jansson, Lindberg, & Sandford, 1983; O’Neill, Selvendran, & Morris,1983) which consists of two residues of b-D-glucose, one of b-D-glucuronate and one of a-L-rhamnose (Fig. 1). The native poly-saccharide, as biosynthesised, has an L-glyceryl substituent on O(2)of the 3-linked glucose residue of the tetrasaccharide sequence(Fig. 1) and, in at least some of the repeat units, an acetyl group atO(6) of the same residue (Kuo, Mort, & Dell, 1986). In normalcommercial production, however, both types of substituent areremoved by treatment of the fermentation broth with hot alkali.The resulting deacylated polymer is known generically as “gellangum” or by the proprietary names of Kelcogel (food-grade) orGelrite (for non-food applications). Most (or perhaps all) of the acylgroups can be preserved by use of milder extraction procedures,giving “high acyl” gellan, which is now also available commercially.

Table 1Cation content (wt %) of the common samples used in Japanese collaborativeresearch.

Sample Naþ Kþ Ca2þ Mg2þ

KGG-1 (1993) 0.19 2.08 0.512 0.146NaGG-2 (1996) 3.03 0.19 0.11 0.02NaGG-3 (1999) 2.59 0.009 0.02 0.001

Fig. 1. Tetrasaccharide repeating unit of deacylated gellan. The sites of attachment of glyceryl and acetyl substituents in high acyl (“native”) gellan are indicated.

E.R. Morris et al. / Food Hydrocolloids 28 (2012) 373e411 375

Throughout this review, however, we will use the term “gellan” torefer to the deacylated polysaccharide, unless otherwise specified.

Gellan was granted approval for food use in the USA inNovember 1992, followed by EU approval as E418, and is nowallowed as a food additive in many other countries worldwide(including Canada, Australia, South Africa, and most countries ofSouth America and Southeast Asia). Approval for food use in Japancame much earlier, in 1988, when, as a product of fermentation,gellan was accepted as a “natural food additive” (Gibson &Sanderson, 1997). This prompted an upsurge of interest by Japa-nese companies and researchers, and formation of a group focus-sing specifically on the conformation, gelation and industrialapplications of gellan within the Research Group on Polymer Gelsaffiliated to the Society of Polymer Science, Japan.

It is well known in the history of the development of rheologythat collaborative research on NBS (National Bureau of Standards,USA) polyisobutylene played an important role. Using the samesample, various groups participating in the collaboration comparedtheir results, and thus made a great contribution to the establish-ment of a timeetemperature superposition principle and a reducedvariable method. Following this approach, the research initiative inJapan used different, complementary, experimental techniques tostudy a single batch of gellan, thus avoiding complications fromvariations between different samples.

One possible source of variation is molecular weight. Plant andalgal polysaccharides show very large differences in molecularweight from sample to sample, reflecting differences in, forexample, botanic source, growth conditions, maturity at harvest,andmethods of extraction of the polymer from the tissuematrix. Asa product from fermentation of a well-defined medium by a purebacterial culture, with strict control of process parameters (Kang,Veeder, Mirrasoul, Kaneko, & Cottrell, 1981) such as pH, tempera-ture, aeration and agitation, and release from a fluid broth ratherthan from cohesive tissue, gellan would be expected to show farless variability. Nonetheless, various groups have reported widelydifferent molecular weights, although, as discussed in Section 2.4,this may reflect differences in experimental procedure, rather thangenuine differences in molecular weight between gellan samples.

A more significant factor is the nature of the counterions thatbalance the charge fromthecarboxylate groupsof thepolymerchains(Fig.1). In food-gradegellan, theseare confined to sodium, potassium,magnesium and calcium cations, present in the nutrient saltsrequired for growth of the synthesising bacteria and/or introducedduring post-fermentation processing. It was established by earlystudies within Kelco (Sanderson, Bell, Clark, & Ortega, 1988) andexternally (Grasdalen & Smidsrød, 1987) that divalent cations (Ca2þ

and Mg2þ) are much more effective in promoting gelation of gellanthan monovalent cations (Naþ and Kþ) and that Kþ is more effectivethan Naþ. The cation content of gellan samples is therefore crucial.

In the first phase of the Japanese collaborative research initiative,the principal cation in the common sample used (KGG-1) was Kþ

(Table 1), but with an appreciable content of other cations, particu-larly Ca2þ. This material was distributed to the collaborating labo-ratories, and the results obtained were published in a special issue ofthis Journal (Food Hydrocolloids, Volume 7, number 5, December1993). The experimental approaches used were light scattering(Okamoto, Kubota, & Kuwahara, 1993), ESR (Tsutsumi et al., 1993),small-angle X-ray scattering (Yoshida & Takahashi, 1993; Yuguchi,Mimura, Kitamura, Urakawa, & Kajiwara, 1993), osmotic pressure(Ogawa, 1993), ultrasonic velocity (Tanaka, Sakurai, & Nakamura,1993) and viscoelastic measurements (Nakamura, Harada, &Tanaka, 1993; Shimazaki & Ogino, 1993; Watase & Nishinari, 1993).

The second common sample (NaGG-2) was predominantly inthe Naþ salt form, although the content of other cations, particu-larly Kþ and Ca2þ, could still not be regarded as negligible (Table 1).This sample was studied by 17 collaborating laboratories and theresults were published in a special issue of the international journalCarbohydrate Polymers (Volume 30, No. 2/3, June/July 1996), whichalso included two papers on gellan from groups outside Japan(Milas & Rinaudo, 1996; Morris, E.R., Gothard, Hember, Manning, &Robinson, 1996).

The third common sample (NaGG-3), which, like NaGG-2, wasspecially prepared by San-Ei-Gen FFI (based on advice from Dr. G.R.Sanderson of Kelco) for use in the Japanese collaborative researchprogramme, can be regarded as essentially pure Naþ gellan, havingonly a negligible content of other cations (Table 1). The results fromwork on this sample were again presented in a special issue of aninternational journal (Progress in Colloid and Polymer Science,Volume 114, 1999), along with four papers from outside Japan(Morris, V.J., Kirby, & Gunning, 1999; Morris, E.R., Richardson, &Whittaker, 1999; Morrison, Sworn, Clark, Chen, & Talashek, 1999and Sworn & Kasapis, 1999).

During the course of the collaborative research initiative, twointernational meetings were held in Osaka, Japan, to promote anactive exchange of information and ideas between scientists,industrialists and food technologists. The first was the InternationalWorkshoponGellan andRelatedPolysaccharides (14e15November,1994), preceding publication of the special issue of CarbohydratePolymers in 1996. The second, four years later, was the 4th

Fig. 2. Double helix structure of deacylated gellan (Chandrasekaran, Millane et al.,1988) viewed (a) perpendicular to the helix axis and (b) along the helix axis. Thesites of attachment of glyceryl and acetyl substituents in high acyl gellan, relative tothe position of the carboxyl group on the neighbouring glucuronate residue, areindicated in (b).


International Hydrocolloids Conference. This included an informalworkshop attended by many of the participants in the Japanesecollaborativeprogrammeandbyscientistworkingongellan in othercountries (M. Dentini, E.R. Morris, V.J. Morris and M. Rinaudo),where therewas a frank (and sometimes heated) exchange of viewsewhich has continued during the preparation of this review!

Previous reviews of gellan include articles by Morris, V.J. (1995),Nishinari (1996), Gibson and Sanderson (1997), Rinaudo and Milas(2000), Sanderson (1990), Sworn (2009) and Valli and Clark (2010).Since the discovery of gellan, Kelco has gone through severalchanges of ownership, and eventually the part of the companydealing with “biogums” such as xanthan and gellan was split fromthe original alginate business and merged with Copenhagen Pectinas CPKelco. An overview of the properties and applications ofgellan by this company is available on-line at: http://www.appliedbioscience.com/docs/Gellan_Book_5th_Edition.pdf.

2. Conformation in the solid state and in solution

2.1. Structure of gellan in the solid state

Gellan fibres obtained by slow stretching of gels to promotealignment and lateral packing before drying (Upstill, Atkins, &Atwool, 1986) gave X-ray fibre diffraction patterns of very highquality, the pattern for the Liþ salt form being arguably the best thathad ever been obtained for any polysaccharide. Models proposedfrom initial analysis of the diffraction data (Upstill et al., 1986) wereunconvincing, but subsequent re-examination (Chandrasekaran,Millane, Arnott, & Atkins, 1988) showed conclusively that theordered structure of gellan in the solid state is a coaxial doublehelix (Fig. 2). In this structure, each strand is a 3-fold, left-handedhelix with a pitch of 5.64 nm. The two chains run parallel to oneanother and are exactly half-staggered (i.e. with each chain rotatedby 180� and translated by half a pitch relative to the other), so thatthe repeat distance (pitch) of the double helix is half that of theindividual strands (2.82 nm), an arrangement similar to thatobserved (Arnott, Scott, Rees, & McNab, 1974) for iota carrageenan.

Three of the four glycosidic linkages in the tetrasacchariderepeating sequence of gellan (Fig.1) involve equatorial bonds at C(1)and C(4) of the participating residues. Polysaccharide chains inwhich all the linkages are (1/4)-diequatorial (such as cellulose, orthe mannan backbone of galactomannans) adopt flat, ribbon-likestructures in the solid state (Rees, Morris, Thom, & Madden, 1982).The remaining linkage in the gellan repeat, however, is (1 / 3),which introduces a systematic “twist” in direction of the chain andpromotes helical geometry, in the sameway as the (1/3) linkagesin the alternating (1 / 3), (1 / 4)-linked disaccharide repeatingsequences of polysaccharides in the agar/carrageenan series.

2.2. Interactions of cations with anionic polyelectrolytes

When anionic polysaccharides such as gellan are dissolved inwater, the only cations present in the resulting solution are, ofcourse, those present as counterions to the charged groups of thepolymer chains. The effective concentration (activity) of thesepositively-charged ions in the bulk of the solution is reduced(Katchalsky, 1971; Manning, 1969a,b) by electrostatic attraction tothe negatively-charged polymer chains. The strength of attractionis determined by the linear charge density (charge per unit length)of the polyanion and the charge of the individual cations, withdivalent cations therefore being attracted twice as strongly asmonovalent. Cation activity can be quantified experimentally bymeasurement of the activity coefficient (g) by potentiometry, theosmotic coefficient (f) from measurements of osmotic pressure, orthe transport coefficient (f) from conductivity or free diffusion of

Fig. 3. Temperature-dependence of optical rotation (302 nm) for 1.2 mM (0.086 wt %)Me4Nþ gellan on heating (B) and cooling (C) in the presence of Me4NCl at concen-trations (mM) of (a) 0; (b) 30; (c) 75; (d) 92; (e) 120; (f) 150 and (g) 250 (Crescenziet al., 1987).


counterions. These three parameters are not normally identical toone another, but there are known quantitative relationshipsbetween them (Rinaudo, 2009) which allow the activity of thecations to be derived by any of the investigative techniquesmentioned.

When extraneous salt is added, the total concentration ofcations (CT) becomes:

CT ¼ Cp þ CS (1)

and the total cation activity (aT) in dilute solution (assuming theactivity coefficient of small ions is equal to 1) is given by:

aT ¼ gCp þ CS (2)

where CP is the concentration of charged groups on the polymer,CS is the concentration of added salt, and g is the fraction ofthermodynamically-free counterions.

For anionic polysaccharides such as carrageenans that convertfrom a disordered coil to an ordered (double helix) conformation oncooling (Rees et al., 1982) the inverse of the midpoint temperature(Tm) of the disordereorder transition varies linearly (Rochas &Rinaudo, 1980) with the logarithm of cation activity:

dð1=TmÞ=dlog aT ¼ �Rðfc � fhÞ=DH (3)

The slope of 1/Tm versus log aT is determined by two factors: thetransition enthalpy (DH) and the decrease in osmotic coefficientfrom fc when the polymer is in the coil form to fh for the orderedform (Milas, Shi, & Rinaudo, 1990). This decrease arises from thehigher linear charge density of the double-stranded helix,promoting greater reduction in cation activity than the singlechains in the coil form.

In solutions where the concentration of added salt (CS) is at leastcomparable to the polymer concentration (CP) and the activitycoefficient is high or moderate (g > w0.5), aT in Eq. (3) can bereplaced by the total cation concentration (CT) without any seriousloss of linearity, although the resulting plots have somewhatsteeper slope than those obtained using aT.

2.3. Conformational transitions of gellan in solution

The procedure adopted in many investigations of gellan (andother anionic polysaccharides) to avoid complications from thepresence of different cations is to convert the polymer to a singlesalt form and to use a salt of the same cation to vary the total cationconcentration. For studies of gellan in solution, use of the tetra-methylammonium (M4Nþ) salt form avoids further complicationsfrom gel formation, since, as reported by Grasdalen and Smidsrød(1987) and described further in Section 3.5, gelation of gellanwith M4Nþ cations occurs only at very high values of CS.

The formula weight of the gellan tetrasaccharide repeat unit(Fig. 1), with the glucuronate carboxyl group in the charged (COO�)form, is 645. In the sodium salt, this is increased by 23, to 668. Thusa 1 M (1000 mM) solution of Naþ gellan has a concentration of668 g/L ¼ 66.8 wt %; a 1 wt % solution therefore corresponds to1000/66.8z 15mM. The higher mass of the M4Nþ cation raises theformula weight per repeat unit to 719 (645 þ 74), so that a 1 wt %solution now corresponds to 1000/71.9 z 13.9 mM.

One of the techniques most commonly used to monitorconformational transitions of polysaccharides is optical rotation(OR). Fig. 3 shows plots of OR (at 302 nm) versus temperatureobtained by Crescenzi, Dentini, and Dea (1987) for dilute solutionsof M4Nþ gellan (1.2 mMz 0.086 wt %) inwater and in the presenceof M4NCl at concentrations ranging from 30 to 250mM. The plot forthe solution in water is featureless, showing only a slight,

approximately linear, increase in (negative) optical rotation withdecreasing temperature. For the solutions with added M4NCl, bycontrast, there is a well-defined sigmoidal change, which moves toprogressively higher temperature with increasing concentration ofsalt, and which shows no thermal hysteresis between valuesrecorded on cooling and on heating. Similar sigmoidal changes inOR have been observed for other polysaccharides and shown toarise from a conformational transition between an ordered struc-ture at low temperature and a disordered coil state at hightemperature (Rees et al., 1982).

The thermally-reversible changes in optical rotation of M4Nþ

gellan are accompanied by large changes in spectra (Fig. 4) obtainedby the related chiroptical technique of circular dichroism (CD).Maximum change in CD between the disordered state at hightemperature and the ordered state at low temperature comes atw202 nm, and measurements of CD ellipticity at or near thiswavelength can also be used to follow the temperature-course ofconformational change (Matsukawa, Tang, & Watanabe, 1999;Matsukawa & Watanabe, 2007; Nitta, Ikeda, Takaya, & Nishinari,2001; Nitta et al., 2003; Ogawa, Takahashi, Yajima, & Nishinari,2006; Tanaka, Sakurai, & Nakamura, 1996).

A more versatile and convenient technique for monitoringthermally-induced conformational transitions is differential scan-ning calorimetry (DSC). Unlike OR and CD, DSC does not requireoptically-clear samples, and can therefore be used at much higherconcentrations of polymer. Manning (1992) observed well-defined

Fig. 5. Salt (Me4NCl) dependence of transition midpoint temperature (Tm) for Me4Nþ

gellan. Results obtained by Crescenzi et al. (1987) using optical rotation (Fig. 3) at302 nm with a polymer concentration of Cp ¼ 0.087 wt % (B) are compared withvalues obtained by Manning (1992) using optical rotation at 436 nm (C), circulardichroism (6) and DSC (-) with values of Cp in the range 1e2 wt %.

Fig. 6. Temperature-dependence of reduced specific viscosity on heating (:) andcooling (B) for 0.05 wt % Me4Nþ gellan in 30 mM Me4NCl (Crescenzi et al., 1987).

Fig. 4. Circular dichroism spectra recorded (Manning, 1992) for 1.0 wt % (13.9 mM)Me4Nþ gellan in water, in the disordered state at 40 �C (B), the ordered state at 11 �C(6) and part way through the disordereorder transition (at 20 �C; C).


exotherms on cooling and corresponding endotherms on heatingfor solutions of M4Nþ gellan (1.0e2.0 wt %) in water and in thepresence of various concentrations of M4NCl. For each solution,the transition midpoint temperatures from cooling and heatingscans extrapolated to the same value at zero scan rate, consistentwith the absence of detectable thermal hysteresis in OR (Fig. 3).

In the same investigation (Manning, 1992) the conformationaltransition of M4Nþ gellan in water and with added M4NCl wasmonitored by measurements of optical rotation at higher wave-length (436 nm) and much higher concentration of polymer (1.0 wt%) than in the study by Crescenzi et al. (1987), and by circulardichroism (Fig. 4). Despite the differences in experimentalapproaches and polymer concentration, the values of Tm from thetwo investigations agree well (Fig. 5) and their variation with totalconcentration of M4Nþ (counterions to the polymer plus addedM4NCl) gives good linearity when plotted according to Eq. (3).

As shown in Fig. 6, Crescenzi et al. (1987) found that theconformational changes described above were accompanied bya massive increase in reduced specific viscosity (hsp/C, where C ispolymer concentration) on cooling and a corresponding reductionon heating, with no thermal hysteresis. Since intrinsic viscosity, [h],which is directly related to hydrodynamic volume in solution(Bohdanecký & Kovár, 1982), is defined as reduced specific viscosityextrapolated to C ¼ 0, the values of hsp/C in Fig. 6, which wereobtained at very low polymer concentration (0.05 wt % in 30 mMM4NCl), will approximate closely to [h], and themuch higher valuesat low temperature therefore demonstrate that the ordered struc-ture of M4Nþ gellan is stiffer, and has a much greater hydrodynamicvolume, than the disordered state.

In a more recent study, Ogawa et al. (2006) measured thetemperature-dependence of intrinsic viscosity (in 25 mM NaCl) forsix samples of Naþ gellan ranging in molecular weight from 120 to17 kD. For the sample of highest molecular weight there wasa sharp, sigmoidal increase in [h] between the disordered state athigh temperature and the ordered state at low temperature (Fig. 7),closely similar to the increase shown in Fig. 6. For the samples ofprogressively lower molecular weight the increase in [h] on coolingbecame progressively smaller (Fig. 7), and the sample of lowestmolecular weight (17 kD) showed no sigmoidal change, suggestingthat the chains were too short to adopt the ordered conformation,

even at the lowest temperature studied (10 �C). DSC exothermsobserved (Ogawa et al., 2006) for the same gellan samples oncooling (at 1 wt % in 25 mM NaCl) also showed progressivereduction in intensity with decreasing molecular weight. Decreasein the extent of conformational ordering with decreasing chain-length has also been observed (Rochas, Rinaudo, & Vincedon, 1983)for kappa carrageenan.

Solutions of small molecules, such as sugars, give sharp lines inhigh-resolution nuclear magnetic resonance (NMR) spectra. Forsolutions of disordered polysaccharide coils the lines are broader, butstill discernable. On conversion of the polymer to a rigid, orderedstructure, however, the lines become so broad that they are effec-tively flattened into the baseline, and can no longer be detected. Loss

Fig. 7. Temperature-dependence of intrinsic viscosity, [h], for Naþ gellan in 25 mMNaCl (Ogawa et al., 2006). The molecular weights (kD) of the samples studied(measured in the disordered state at 40 �C) were: G1: 120; G2: 71; G3: 62; G4: 57;G5: 32 and G6: 17.


of detectable high-resolution NMR signal can therefore be used tomonitor conformational ordering (Rees et al., 1982).

As shown in Fig. 8, Milas and Rinaudo (1996) observeda sigmoidal reduction in intensity of 1H high-resolution NMRspectra with decreasing temperature for solutions of Naþ gellan(0.4 wt % in D2O). The reduction was quantified by measurement ofthe intensity of the resonance from the methyl group of rhamnose(Fig. 1), which is well resolved from resonances of other hydrogenatoms in gellan, relative to the NMR intensity of an external stan-dard (sodium succinate) of known concentration. Loss of detectablesignal (Fig. 8) confirms that the conformational transition observedby other techniques does indeed correspond to conversion froma disordered state at high temperature to a rigid, ordered structureat low temperature. It was also found (Milas & Rinaudo, 1996) thatthe transition midpoint temperature for solutions of gellan in D2Owas w6 �C higher than in water, indicating stabilisation of doublehelices by hydrogen bonding within/between the constituentstrands and to surrounding water molecules.

The effectiveness of different cations in promoting conforma-tional ordering of gellan in solutionwas explored (Milas & Rinaudo,1996) by converting the polymer to specific salt forms by cationexchange, adjusting total cation content by addition of chloride

0

20

40

60

80

100

0 20 40 60 80

Temperature (°C)

Vis

ible

sig

nal (

%)

Fig. 8. Loss of detectable high-resolution 1H signal on conformational ordering of Naþ

gellan (0.4 wt % in D2O); changes were quantified by the intensity of the well-resolvedresonance of the rhamnosyl methyl group (Fig. 1) relative to an external standard(sodium succinate) of known concentration (Milas & Rinaudo, 1996).

salts of the same cations, and measuring midpoint temperature ofthe disordereorder transition by optical rotation. As shown inFig. 9, the variation of Tm with total cation activity (aT) gave goodlinearity when plotted according to Eq. (3). Divalent cationsinduced conformational ordering at higher temperature thanmonovalent, but with no evident difference in effectivenessbetween Ca2þ and Mg2þ. There was also no evidence of selectivitybetween the Group I metal ions studied (Kþ, Naþ and Liþ) orbetween them and M4Nþ, in contrast to the pronounced selectivityobserved (Grasdalen & Smidsrød, 1987) in the ability of differentmonovalent cations to induce formation of gellan gels. Conductivitymeasurements of the decrease in concentration of free Naþ or Kþ

cations on conversion of the polymer from the disordered to theordered form gave values of Tm in close agreement with thoseobtained by OR (Milas et al., 1990).

These observations are fully consistent with polyelectrolytetheory (Katchalsky, 1971; Manning, 1969a,b) in which attraction ofcations to anionic polyelectrolytes depends solely on the charge perunit length of the polyanion and the concentration and charge ofthe cations, and not on the chemical nature of either.

2.4. Light scattering, osmometry and small-angle X-ray scattering

In an early investigation, Brownsey, Chilvers, I’Anson, andMorris (1984) used static light scattering to determine theweight-average molecular weight (Mw) of gellan. The samplestudied was a mixed salt form, containing w2% Kþ, w0.5% Mg2þ,and smaller amounts (w0.1%) of Naþ and Ca2þ. Solutions wereprepared in a mixed solvent of 90% DMSO and 10% water, andpassed through a 0.5 mm filter. Light scattering measurements weremade at 25 �C, yielding values of Mw ¼ 880 and 960 kD fromreplicate determinations.

In subsequent studies by light scattering, Dentini, Coviello,Burchard, and Crescenzi (1988) obtained Mw ¼ 434 kD for M4Nþ

gellan in the ordered state (in 75 mM M4NCl at 25 �C) for solutions

Fig. 9. Inverse of transition midpoint temperature (Tm) on cooling as a function of theactivity of Kþ (B), Naþ (6), Liþ (C), Me4Nþ (:), Ca2þ (,) and Mg2þ (-) counterions insolutions of deacylated gellan. Gellan samples were converted to these specific salt formsby ion exchange, and the total activity (aT) of each cation was adjusted by addition of thecorresponding chloride salt (Milas & Rinaudo, 1996). Corresponding values for thesolegel transition temperature (Tsg) of high acyl gellan with monovalent (Naþ, Kþ) anddivalent (Ca2þ, Mg2þ) cations (Huang et al., 2004) are shown by dashed lines.


clarified using a 0.45 mm filter, but Okamoto et al. (1993), usinga different sample of M4Nþ gellan but essentially identical experi-mental conditions, reported a substantially lower value of Mw(238 kD). The number-average molecular weight (Mn) of the M4Nþ

gellan used by Okamoto et al. (1993) was also characterisedby osmotic pressure measurements (Ogawa, 1993), yieldingMn ¼ 55 kD. This value cannot, however, be compared directly withthe value of Mw ¼ 238 kD from light scattering, since themeasurements of osmotic pressure were for the disordered form at40 �C, rather than the ordered form characterised in the lightscattering experiments (Okamoto et al., 1993) at 25 �C. Somewhatlower values of Mn from osmometry were reported by Ogawa,Matsuzawa, and Iwahashi (2002) for a single batch of commercialgellan converted by cation exchange to the Liþ, Naþ and Kþ saltforms (48, 43 and 49 kD, respectively, in the disordered state at45 �C).

In a further light scattering study of (ordered) M4Nþ gellan in75mMM4NCl at 25 �C, Gunning andMorris (1990) compared valuesofMw and Rg (radius of gyration) for solutions that had been passedthrough filters with a pore size of either 0.45 mm (as used by Dentiniet al., 1988) or 3 mm. Filtration at 3 mmyieldedMw ¼ 4500� 100 kDand Rg ¼ 159 � 10 nm; on reduction in pore size to 0.45 mm, bothvalues decreased dramatically, to Mw ¼ 106 � 6 kD andRg¼ 72� 4 nm. It seems likely, therefore, that much of the variationbetween reported values of themolecularweight of gellanmayhavearisen from differences in the experimental procedure used inpreparation of solutions, with the presence of residual intermolec-ular aggregates giving spuriously high values.

Differences in sample preparation do not, of course, arise whenthe same solutions are measured at different temperatures. Ina study of M4Nþ gellan (in 25 mM M4NCl) by light scattering, Milaset al. (1990) observed an approximate doubling in molecularweight (from 250 to 490 kD) on going from the disordered form (at36 �C) to the ordered form (at 24 �C), with an accompanyingincrease in Rg from 69.5 to 127 nm and in persistence length from5.9 to 71.2 nm. In a more recent investigation, Takahashi et al.(2004) studied nine samples of Naþ gellan (in 25 mM NaCl) bystatic and dynamic light scattering. In the disordered form (at40 �C) the molecular weights (Mw) of these samples ranged from34.7 to 115 kD; there was again an approximate doubling in Mw ongoing to the ordered state (at 25 �C), accompanied by an increase inpersistence length (determined by application of unperturbedwormlike chain models to data from hydrodynamic measure-ments) from 9.4 nm at 40 �C to 98 nm at 25 �C. For all nine samples,the ratio ofMw for the ordered state to that for the disordered statewas within the range 1.99e2.07. Furthermore, doubling of mass perunit length and cross-sectional radius of gyration on going from thedisordered form to the ordered form was observed by small-angleX-ray scattering in a study of Naþ gellan (NaGG-2, Table 1) byYuguchi et al. (1996).

Taken together, these investigations give compelling evidencethat the ordered form adopted by gellan on cooling in the solutionstate is the double-stranded helical structure (Fig. 2) characterisedin the solid state by X-ray fibre diffraction, which then reverts to thesingle-stranded disordered coil state on heating, with no thermalhysteresis between the coilehelix and helixecoil transitions.

3. Cation-induced gelation of gellan

3.1. Rheology of solutions and gels

The most common way of characterising the mechanical(rheological) properties of gels is compression testing, usuallycarried out on cylindrical samples. The three main parameters thatcan be derived from the variation of resistance to deformation

(stress, s) as the extent of compression (strain, 3) is increased arethe initial slope of the compression curve, which gives Young’smodulus (E ¼ s/ 3), and the values of stress and strain at the pointwhere the gel breaks (sb and 3b). For materials whose volumeremains constant during compression, reduction in height isaccompanied by lateral expansion. The change in diameter dividedby the change in height is known as Poisson’s ratio. Lateralexpansion can also be characterised by the “stretch ratio”, definedas the diameter at any particular degree of compression divided bythe original diameter of the sample.

A more versatile and informative procedure, which can beapplied to both solutions and gels and can be used to follow theformation and melting of gel networks, is measurement of resis-tance to low-amplitude oscillatory deformation (Morris, 1985;Ross-Murphy, 1984; Te Nijenhuis, 1997). Elastic (solid-like) resis-tance is greatest at the extremes of the oscillatory cycle, where thedisplacement (strain) is greatest, and drops to zero in the middle ofthe cycle. The rate of deformation, which determines the resistanceof ideal liquids, is greatest in the middle of the oscillatory cycle anddrops to zero at the extremes (where the direction of movement isreversed). Thus the stress generated by perfect solids is exactly inphase with the oscillatory strain, whereas for perfect liquids it isexactly (90�) out of phase.

For “viscoelastic” materials, such as polysaccharide solutionsand gels, the total stress can be resolved into an in-phase compo-nent and an out-of-phase component; dividing these by the appliedstrain gives, respectively, the “storage modulus”, which character-ises the solid-like (elastic) response of the sample, and the “lossmodulus” which characterises the liquid-like response. For longi-tudinal oscillation (i.e. alternate compression and extension of thesample), the storage and loss moduli are termed E0 and E00

(following Young’s modulus, E, for unidirectional extension orcompression). It is more common, however, to use shear strain, andthe moduli are then denoted by G0 (storage modulus) and G00 (lossmodulus). The ratio of the total, unresolved stress to the appliedstrain is known as the complex modulus, jG�j, which is related to G0

and G00 by:

jG�j ¼�G02 þ G002

�1=2(4)

Dividing jG�j by the frequency of oscillation (u) gives thecomplex dynamic viscosity, jh�j, which can be regarded as theoscillatory analogue of steady-shear viscosity (h) from rotationalmeasurements or capillary viscometry.

jh�j ¼ jG�j=u ¼�G02 þ G002

�1=2�u (5)

jG�j and jh�j are often written simply as G* and h*, with nochange in meaning. Another informative parameter is the losstangent, tan d, which is given by:

tan d ¼ G00=G0 (6)

The variation of G0 and G00 with frequency (normally plotted onlogarithmic axes) is known as the “mechanical spectrum” of thematerial; jh�j is often also included in the spectrum. Typicalmechanical spectra for polysaccharide solutions and gels are shownin Fig. 10.

For gels (Fig. 10a), solid-like character (G0) predominates overliquid-like, viscous response (G00), usually by at least an order ofmagnitude. There is little change in either modulus on varyingfrequency (u), fromwhich it follows (Eq. (5)) that log jh�j decreaseslinearly as log u is increased, with a slope close to �1. Formation ofa continuous gel network occurs only when the polymer concen-tration (C) reaches a minimum critical value, Co. At concentrations

Fig. 10. Typical mechanical spectra of (a) a true gel, (b) a semi-dilute solution ofentangled polymer coils, and (c) a dilute polymer solution.


well above Co, plots of log G0 versus log C for typical gellingbiopolymers have a constant slope of w2 (i.e. G0 w C2); C2-dependence is also commonly observed for E and E0. There is then,however, a progressive increase in slope (i.e. progressively steeperconcentration-dependence of modulus) as C is decreased towardsCo (Clark & Ross-Murphy, 1985).

For dilute solutions of disordered coils free to move indepen-dently (Fig. 10c) G00 predominates over G0, since resistance todeformation arises mainly from movement (flow) of polymermolecules through the solvent; log G00 increases linearly onincreasing log u, with a slope ofþ1 (i.e. G00 w u). The variation of logG0 with log u is also linear, but with the steeper slope of þ2 (i.e.G0 w u2), which reflects progressively greater storage of energy bycontortion of individual polymer coils as the frequency of

oscillation is increased. As polymer concentration is raised, a pointis reached at which the individual coils are forced to interpenetrateone another and form an entangled network (Graessley, 1974). Theconcentration at which this occurs is known as C*, and solutions ofhigher concentration are termed “semi-dilute”.

At low frequencies, where there is sufficient time for entangle-ments to come apart within the period of oscillation, semi-dilutesolutions respond predominantly by flow, and their mechanicalspectra (Fig. 10b) are similar to those of dilute solutions (Fig. 10c).However, at higher frequencies, where there is less time fordisentanglement, the predominant response to oscillatory strainbecomes elastic distortion of the entangled network, and themechanical spectra (Fig. 10b) become similar to those observed forgels (Fig. 10a). The frequency-dependence of jh�j superimposesclosely on the shear-rate dependence of steady-shear viscosity(h ¼ s= _g, where s ¼ shear stress and _g ¼ shear rate) at equivalentvalues of _g=s�1 and u/rad s�1, reflecting the same dependence ofrheology on the timescale of molecular rearrangement (entangle-ment/disentanglement) in response to both small-deformation(oscillatory) and large-deformation (rotational) perturbation.

Systems that display superposition of h and jh�j are said to obeythe “CoxeMerz rule” (Cox & Merz, 1958) and include simple liquidsand dilute polymer solutions, as well as entangled networks.

3.2. Critical gel point

Intermolecular association of polymer molecules in solutionresults initially in formation of small, soluble clusters of chains. Asthe extent of association increases these clusters grow, until ulti-mately one becomes large enough to span the entire volume of thesolution and form a continuous crosslinked network: this is thecritical gel point.

Mechanical spectra at the gel point have a characteristic form(Durand, Delsanti, Adam, & Luck, 1987; Te Nijenhuis & Winter,1989) in which log G0 and log G00 vary linearly with log u overmany decades of frequency, and have the same slope, n (i.e. G0 w un

and G00 w un), so that tan d (Eq. (6)) is independent of frequency. Insome, but by no means all, gelling systems, the values of G0 and G00

at the critical gel point are close to one another, giving tan dz 1. Formaterials that gel on cooling (such as carrageenans and gellan) thetemperature at the critical gel point (Tc) can be obtained by plottingcurves of tan d versus temperature (T) for a range of differentfrequencies of oscillation. The curves cross one another at a singlepoint of intersection where T ¼ Tc.

Theoretical values of the common slope (n) of log G0 and log G00

versus log u lie mainly within the range 0.50e0.75 (Picout & Ross-Murphy, 2003) and include n ¼ 0.5 from the WintereChambonmodel (Chambon & Winter, 1985; Winter & Chambon, 1986;Winter & Mours, 1997) and n ¼ 0.67 (2/3) from percolation theory,assuming Rouse-like dynamics (Martin, Adolf, & Wilcoxon, 1989).Experimental values for gelling polysaccharides include n ¼ 0.7 forcalcium-induced gelation of pectin (Lopes da Silva, Gonçalves,Doublier, & Axelos, 1996) and n ¼ 0.42 for gelation of 1 wt % iotacarrageenan on cooling, decreasing monotonically as the carra-geenan concentrationwas increased (Hossain, Nemoto, & Nishinari,1997). In a study of commercial gellan (Gelrite) in water, Dai, Liu,Liu, and Tong (2008) obtained values of n ¼ 0.77, 0.53, 0.43 and0.38 at polymer concentrations of 1.0, 1.5, 2.0 and 2.5 wt %,respectively. Variation of n with concentration appears to beconfined to polymers that form “physical gels” by co-operative non-covalent association, and may reflect departure from the randomgrowth of network structure that occurs on chemical (covalent)crosslinking and is assumed in theoretical models that predict“universal” values of n.


The onset of intermolecular association into soluble clusters isaccompanied by a sharp increase in G00, reflecting increased resis-tance to movement through the solvent. The temperature at whichthis occurs can be taken as an index of the coilehelix transitiontemperature (Tch). After initial formation of a continuous networkat the critical gel point, G0 increases steeply with increasing inter-molecular association on further cooling, until stabilising when thenetwork is fully formed. The point at which the curves of G0 and G00

versus temperature cross one another (i.e. going from G0 < G00 toG0 > G00) is often taken as the solegel transition temperature (Tsg).Although less rigorous than finding the temperature at which tan d

becomes independent of frequency, use of the G0, G00 crossovercriterion is far simpler and less time consuming. Its validitydepends on the frequency at which the measurements are made.

As discussed above, the response of semi-dilute solutions tohigh-frequency oscillatory deformation (Fig. 10b) may be domi-nated by elastic distortion of the entangled network, and furtherincrease in G0 during cooling may simply reflect greater entangle-ment between individual growing clusters of crosslinked chains,rather than formation of a continuous network. However, at lowfrequencies, where entanglement makes little contribution tosolid-like (elastic) response, the crossover of G0 and G00 givesa reasonably valid index of Tsg.

3.3. “Weak gels”

Passing through the critical gel point does not necessarily implyformation of a cohesive gel. A number of polysaccharides givesolutions that flow freely but have mechanical spectra qualitativelysimilar to that shown in Fig. 10a for a typical gel network. Systemsof this type, which show predominantly elastic (gel-like) responseto small perturbations but which cannot support their own weightand can be stirred and poured like normal solutions, are commonlyknown as “weak gels” (Kavanagh & Ross-Murphy, 1998; Morris,1985; Picout & Ross-Murphy, 2003; Ross-Murphy, 1984).

“Weak gels” should not be confused with conventional gels thatare “weak” only in the sense of having low moduli. Conventionalgels, which are often described as “self-supporting” or “demould-able”, or as “true gels” (the term used in this review), respond tohigh stress by fracturing, whereas “weak gels” flow. To avoid suchconfusion, other descriptions such as “pourable gels” (Morris, 1991)or “fluid gels” (Sworn, Sanderson, & Gibson, 1995) have been used,and indeed Professor S.B. Ross-Murphy, who was one of the first toadopt the term “weak gel”, has suggested more recently that“structured liquid” might be better (Ross-Murphy, 2008). However,the term “weak gel” seems firmly established in the literature, andwe will continue to use it in this review. Mechanical spectra of“weak gels” normally differ from those of true gels in having greaterfrequency-dependence of G0 and G00 and smaller separationbetween the two moduli, but the main distinction is the differencein response to unidirectional stress.

The most extensively-studied polysaccharide with “weak gel”properties is xanthan. At high temperature and low ionic strengthxanthan is disordered in solution and shows normal solutionproperties. On cooling and/or addition of salt, however, itundergoes a (thermally-reversible) transition to a stiff, orderedstructure (Norton, Goodall, Frangou, Morris, & Rees, 1984). Atconcentrations abovew0.3 wt %, solutions of ordered xanthan givegel-like mechanical spectra, and are capable of holding smallparticles in suspension over long periods of time (which is centralto many of the industrial and food applications of xanthan).

“Weak gel” properties have also been reported for solutions ofother ordered polysaccharides, including welan and rhamsan(Morris et al., 1996) which are branched variants of gellan, and fordispersions (Haque, Richardson, Morris, & Dea, 1993) of ispaghula

(milled seed husk of Platago ovata Forsk). The common feature ofthese systems is that the polysaccharide is in a rigid form, either atthe molecular level or in large, supramolecular assemblies.Formation of true gels by association of disordered coils intoordered junctions involves substantial loss of conformationalentropy, and will therefore occur only if the enthalpic advantage ofassociation is correspondingly large (i.e. if the non-covalentbonding within the intermolecular junctions is strong). Ruptureof the resulting networks therefore requires considerable force.Association of rigid structures, by contrast, involves little loss ofentropy, and can therefore occur even if the bonding is weak, givingrise to tenuous “weak gel” networks that come apart at low stress.

Polysaccharides that form true gels on cooling under quiescentconditions can also give dispersions of microscopic gel particleswith “weak gel” properties if they are subjected to shear on coolingthrough the temperature-range of the solegel transition (Harris &Pointer, 1986; Norton, Jarvis, & Foster, 1999). By applying thisapproach to gellan, Sworn et al. (1995) obtained “weak gel”networks capable of suspending small particles at very low poly-mer concentration (0.125 wt %) where ordered xanthan shows onlythe predominantly liquid-like properties of a normal poly-saccharide solution.

The internal rheology of the gellan microgel particles in suchpreparations was compared with the macroscopic “weak gel”properties in an investigation by Caggioni, Spicer, Blair, Lindberg,and Weitz (2007). Mixed solutions of commercial gellan (Kelco-gel) and NaCl were prepared at 80 �C and cooled (at 0.5 �C/min) to25 �C, either quiescently or under shear (at a constant shear rate of100 s�1). As would be expected, the true gels formed on quiescentcooling had higher moduli (G0 and G00) than the “weak gels” formedunder shear. The “microrheology” of both was probed on a micro-metre length-scale by video imaging of the Brownian motion oftracer particles (1 mm diameter polystyrene beads) incorporated inthe gellan solutions prior to cooling. The results obtained for thesheared and non-sheared preparations were indistinguishable,demonstrating that the microgel particles formed by cooling undershear have the same internal structure as the continuous gelnetworks obtained on quiescent cooling. The “weak gel” networkswere capable of holding the polystyrene tracer particles insuspension over long periods of time: at a gellan concentration of0.05wt % (in 100mMNaCl) only slight sedimentationwas observedafter storage for 2 months.

On application of small stresses (steady or oscillatory) theresulting deformation of the “weak gels” increased in directproportion to the stress (giving constant elastic modulus, as wouldbe observed for a true gel). Above a critical stress, however, thestrain increased steeply, showing failure of the “weak gel” network,with elastic deformation being replaced by viscous flow. The stressat the point of failure increased in direct proportion to gellanconcentration (over the range 0.05e0.40 wt % in 100, 200 or300 mM NaCl), and the strain at failure decreased inversely (i.e.with the onset of flow occurring at progressively smallerdeformation as the concentration of gellan was raised). Yieldingof the “weak gel” network of microgel particles was tentativelyattributed to a shear-induced transition from a “jammed” to an“un-jammed” state.

Failure of “weak gel” networks formed by gelation of gellanunder shear was investigated further by Garcia, Alfaro, Calero, andMuñoz (2011). A fixed concentration of NaCl (0.22 M) was incor-porated in solutions of Kelcogel (0.025e0.25 wt %) at 80 �C. Theonset temperature for gelation on cooling, as determined by low-amplitude oscillatory measurements of G0 and G00, was w41 �C.“Weak gels” were prepared by shearing at this temperature for1 min at 700 rpm, and were then stored for at least 2 days at 4.5 �Cbefore characterisation at 20 �C.

Fig. 11. Representative DSC cooling and heating traces for 1 wt % (15 mN) Naþ gellan attotal concentrations (mM) of Naþ (counterions to the polymer þ added NaCl) of: A: 25;B: 67; C: 115 and D: 145 (Manning, 1992; Robinson et al., 1991). Baselines have beensubtracted from the cooling curves; the heating curves are displaced vertically byarbitrary amounts to avoid overlap.


The sheared preparations were visually cloudy, and confocallaser scanning microscopy (CSLM), with fluorescent labelling ofgellan by fluoresceinamine, showed irregularly-shaped particleswith dimensions in the range 0.1e1 mm (for 0.25 wt % gellan),about 10-times larger than in the investigation by Caggioni et al.(2007) where shearing was more vigorous and prolonged.

The mechanical spectra recorded by Garcia et al. (2011) weresimilar to the typical true gel spectrum shown in Fig. 10a, with G0

about an order of magnitude greater than G00, and with bothmodulishowing only a slight decrease with decreasing frequency (u).“Equilibrium” values of G0 (Ge), obtained by extrapolation to u ¼ 0,varied with gellan concentration in the same way as the elasticmoduli of conventional (un-sheared) biopolymer gels (Section 3.1),with Ge w C2 for C ¼ 0.25e0.05 wt %, and a steeper decrease onfurther reduction to C ¼ 0.025 wt %, the lowest concentrationstudied.

Failure of the “weak gel” networks of microgel particles wascharacterised by measurement of transient changes in rheology inresponse to shear. After loading onto the rheometer, the sampleswere left to equilibrate (at 20 �C) until they had reached constantvalues of G0, which occurred within 10 min. They were then sub-jected to a constant shear rate ð _gÞ of 10 s�1 for 5 min. When theshear was applied, resistance (stress, s) rose almost instantaneouslyto an abruptmaximum, and then decreased progressively over timetowards a constant value (i.e. towards constant steady-shearviscosity, h ¼ s= _g). Recovery of “weak gel” rheology on cessationof shear became progressively slower with increasing concentra-tion of gellan.

The values of complex dynamic viscosity ðjh�jÞ for the initial“weak gel” networks were much (w20 times) higher than thevalues of h observed at the end of the 5 min period of steady shearat 10 s�1. Thus the gellan “weak gels” violate the CoxeMerz rule(Section 3.1): jh�j of the intact “weak gel” network is substantiallygreater than the viscosity (h) characterising flow after networkfailure. Similar violation of the CoxeMerz rule, and “stress over-shoot” in start-shear experiments, have been observed (Richardson& Ross-Murphy, 1987) for “weak gels” of ordered xanthan.

As pointed out by Garcia et al. (2011), the peak viscosity corre-sponding to the stress needed to initiate flow of “weak gels” is animportant consideration in engineering operations such as pump-ing the sheared networks from the tanks in which they wereprepared, with the power required being substantially greater thanwould be anticipated from steady-shear viscosity.

3.4. Gelation of gellan with Group I (alkali metal) cations

Fig. 11 shows representative DSC cooling and heating curvesfrom investigation (Manning, 1992; Robinson, Manning, & Morris,1991; Robinson, Manning, Morris, & Dea, 1988) of 1 wt % Naþ gel-lan (CP ¼ 15 mM) in the presence of NaCl at concentrations rangingfrom 0 to 130 mM (i.e. CT ¼ 15e145 mM; Eq. (1)). Throughout thisrange, the DSC cooling scans showed only a single exothermwhich,as expected from previous studies by other techniques (Section2.3), moved to progressively higher temperature as the concen-tration of Naþwas raised. Heating at low CT gave single endotherms(e.g. trace A in Fig. 11) which were essentially equal and opposite tothe exotherms observed for the same samples on cooling. At a saltconcentration of w50 mM NaCl (CT z 65 mM), however, a secondthermal process became evident in the DSC heating traces. Onfurther increase in salt concentration, the first endotherm in theheating curves became progressively smaller, but remained inapproximately the same position as the exotherm in the corre-sponding cooling curves. The second endotherm, by contrast,moved to much higher temperature and increased in size, until atthe highest concentration of NaCl studied (130 mM; CT ¼ 145 mM)

it became the only detectable process. Closely similar behaviour forthe same concentration of Naþ gellan was observed by Mazen,Milas, and Rinaudo (1999).

The proposed interpretation (Robinson et al., 1991), which nowseems to be widely accepted (e.g. Gunning, Kirby, Ridout,Brownsey, & Morris, 1996; Mazen et al., 1999; Miyoshi &Nishinari, 1999a; Nakajima, Ikehara, & Nishi, 1996) is that thesecond thermal process on heating comes from melting of doublehelices stabilised by aggregation, and the first from residualunaggregated helices which melt at the same temperature as theyformed on cooling, with the proportion and stability of aggregatedhelices increasing with increase in CT above a critical thresholdvalue (CT*). An alternative interpretation (Miyoshi, Takaya, &Nishinari, 1996; Robinson et al., 1988) in which the first DSCendotherm on heating was attributed to dissociation of aggregatesand the second to melting of unaggregated helices has now beenretracted by the senior coauthors of the papers in which it wasproposed (E.R. Morris and K. Nishinari).

The progressive increase in thermal hysteresis between forma-tion of individual helices on cooling and melting of helixehelixaggregates on heating as CT is raised above CT* is shown directlyin Fig. 12. At low concentrations of Naþ, up to CT z 25 mM, thesolutions of ordered gellan gave mechanical spectra (Morris, E.R.et al., 1999) similar to those of entangled polysaccharide coils(Fig. 10b). On further increase in CT to w40 mM the samples gavemechanical spectra similar to those of gels (Fig. 10a) but remainedfluid (i.e. showing the “weak gel” properties described in Section3.3). True gels were formed only when CT became greater than CT*(Fig. 12), with moduli then increasing steeply as the concentrationof Naþ was raised. The accompanying increase in extent of

Fig. 12. Effect of total concentration of Naþ on peak-maximum temperatures (Tmax) forthe single DSC exotherm observed on cooling (B) and the first (C) and second (-)endotherms on heating (see Fig. 11) for 1 wt % (15 mN) Naþ gellan with varyingconcentrations of NaCl (Manning, 1992). The three types of rheological responseobserved (Morris, E.R. et al., 1999) for the ordered form at low temperature overdifferent ranges of Naþ concentration are indicated at the foot of the figure (solution,“weak gel” and true gel).

Fig. 13. Variation of transition temperature (Tm) from optical rotation on cooling (opensymbols) and heating (filled symbols) with activity of Naþ (circles) and Kþ (squares) insolutions of gellan (Milas & Rinaudo, 1996). The cooling curve is identical to the lineshown for monovalent cations in Fig. 9, but some points have been omitted for clarityof presentation.

Fig. 14. Variation of Young’s modulus (E) with polymer concentration (C) for Kþ, Naþ

and Liþ gellan in 0.1 M KCl, NaCl or LiCl, respectively (Milas & Rinaudo, 1996).


aggregation, characterised by the magnitude of the second endo-therm in DSC (Fig. 11), and stability of the aggregates, characterisedby the degree of thermal hysteresis between formation of doublehelices on cooling and melting of helixehelix aggregates on heat-ing, indicates strongly that true gels are formed by association ofgellan double helices into stable aggregates.

Measurements of optical rotation (Milas & Rinaudo, 1996) alsoshowed hysteresis between values of transition temperature (Tm)obtained on cooling and on heating at cation concentrations abovea minimum threshold (Fig. 13). For Naþ ions, the value of CT*derived by OR was w45 mM, which is somewhat lower than thecorresponding value ofw65mM from DSC (Fig. 12). In both studies,however, the values of CT* were estimated by extrapolation fromhigher cation concentrations, and the comparatively smalldiscrepancy between them probably reflects the experimentalerror inherent in the extrapolation, rather than any genuinedifference between the processes characterised by the twodifferent techniques.

Despite this experimental imprecision, it is clear that the valueCT* z 20 mM (Fig. 13) derived by Milas and Rinaudo (1996) fromoptical rotation of Kþ gellan with added KCl is appreciably lowerthan the corresponding value for the Naþ salt form. Thus, incontrast to the lack of selectivity between different monovalentcations in promoting formation of gellan double helices (Section2.3), association of the helices into aggregates does appear todepend on which cation is used.

As shown in Fig. 14, the concentration-dependence of modulus(Milas & Rinaudo, 1996) for Naþ and Kþ gellan (in the presence ofthe corresponding chloride salt at a fixed concentration of 100mM)is close to the C2 relationship commonly found (Section 3.1) forgelling biopolymers at concentrations well above the minimumvalue (Co) required to form a continuous network, giving a slope ofw2 on a double logarithmic plot. At each concentration of gellan,the moduli for the Kþ salt form are about twice those observed forthe Naþ form, and the single value obtained for the Liþ salt form at

the highest gellan concentration studied (1.3 wt %) is about 30times lower, which is consistent with the order of effectivenessreported by Grasdalen and Smidsrød (1987) for gelation of gellanwith Group I cations: Liþ < Naþ < Kþ < Csþ.

Further evidence of differences in the effectiveness of differentGroup I cations in promoting aggregation of gellan double heliceshas come from investigation (Yuguchi, Urakawa, Kitamura,Wataoka, & Kajiwara, 1999) of 1.5 wt % Naþ gellan (NaGG-3,Table 1) in the presence and absence of 50 mM LiCl, NaCl, KCl orCsCl by small-angle X-ray scattering. Measured values of cross-


sectional radius of gyration for the ordered form (at 10 �C) were0.30 nmwith no added salt; 0.44 nmwith LiCl; 0.50 nmwith NaCl;1.01 nmwith KCl; and 1.37 nmwith CsCl. The increase in aggregatesize with increasing size of the cations from the added salt parallelsthe order of effectiveness in promoting gelation of gellan and isconsistent with the proposal (Robinson et al., 1991) that associationof gellan double helices into stable aggregates is responsible forformation of true gels.

In the disordered state, the coil dimensions of polyelectrolytessuch as gellan are expanded by intramolecular electrostatic repul-sion. On addition of increasing concentrations of salt the repulsionsare progressively screened by the simple anions and cations, withconsequent decrease in the dimensions of the polyelectrolyte coils,which in turn leads to reduction in viscosity and in dynamicmoduli, G0 and G00. Indeed, the extent to which intrinsic viscositydecreases with increasing concentration of salt has been used tocharacterise the stiffness of the polyelectrolyte (Smidsrød & Haug,1971). However, when the polymer is in an ordered conforma-tion, resistant to contraction, charge screening by added salt has theopposite effect of enhancing rheology, by suppressing repulsionbetween the ordered structures and thus facilitating aggregation.Such behaviour has been observed (Nishinari, 1996; Nishinari,Miyoshi, & Takaya, 1998) on addition of KCl to 1 wt % Naþ gellan(NaGG-2, Table 1) in the disordered and ordered states.

Non-specific charge screening, however, is determined simplyby ionic strength (Smidsrød & Haug, 1971), and cannot thereforeaccount for the observed selectivity between different monovalentcations in promoting aggregation and gelation of gellan. The mostlikely interpretation (Morris et al., 1996) is that Group I cationsattach directly to the gellan double helices by site binding (whichcan alternatively be described as formation of stable ion pairsbetween the metal cations and the carboxylate groups of gellan),with consequent reduction (or elimination) of repulsion betweenthe helices. In addition to electrostatic attraction, site binding ofcations to conformationally-ordered anionic polysaccharidesinvolves coordination (chelation) of the cation by appropriately-spaced oxygen atoms around the charged groups of the poly-saccharide, as postulated in the widely-accepted “egg box” model(Grant, Morris, Rees, Smith, & Thom, 1973) for cation-inducedgelation of alginate (Morris, Rees, Thom, & Boyd, 1978) or pectin(Morris, Powell, Gidley, & Rees, 1982). The order of effectiveness ofGroup I cations in promoting aggregation of gellan double helices

Fig. 15. Effect of temperature on 23Na, 39K and 87Rb NMR spectra for 20 mM NaCl, KCl or RbCwas observed between the changes seen as temperature was raised or lowered (Annaka et

would imply that Csþ ions give the best geometric fit to the bindingsite, with progressively less efficient coordination as the size of thecation decreases.

Site binding of Kþ ions to the gellan double helix in the solid statehas been demonstrated by X-ray fibre diffraction (Chandrasekaran,Puigjaner, Joyce, & Arnott, 1988). Each cation is coordinated(bound) to five oxygen atoms of the double helix: the two atomsfrom the carboxylate group of the glucuronate residue (Fig. 1) andO(2) of the glucose residue adjacent to it in the non-reducingdirection of one strand in the helix, and, from the second strand,O(2) of glucuronate and O(6) of the adjacent glucose in the reducingdirection. The separation of the Kþ ion from one of the oxygen atomsof the carboxylate group is greater than the distance required foroptimum coordination, suggesting that, as postulated above, largerGroup I cations (Rbþ and Csþ) would be bound more efficiently. Itwas proposed from the X-ray analysis that helixehelix aggregatesare formed by carboxylateeKþewatereKþecarboxylate interac-tions. However, although present in the solid state, it seems unlikelythat such “water bridges”would survive in the presence of the largeexcess of water in gellan gels.

Binding of Group I cations in gellan gels has been demonstratedby Annaka, Honda, Nakahira, Seki, and Tokita (1999) using multi-nuclear NMR. The samples studied contained 1.5 wt % Naþ gellan(NaGG-3, Table 1) with 20 mM NaCl, KCl or RbCl, and spectra for the23Na, 39K and 87Rb isotopes were recorded at temperatures spanningthe range of the coilehelix and helixecoil transitions of gellan. Asshown in Fig. 15, conformational ordering of gellan on cooling wasaccompanied by loss of detectable 39K and 87Rb resonances, whichimplies loss of mobility by attachment of the cations to the (rigid)gellan double helix. The changes were fully reversible on heating,and parallel the changes in intensity (Fig. 8) of detectable 1H high-resolution NMR signal from gellan itself. No such changes wereseen (Fig. 15) for the 23Na signal from solutions of 1.5 wt % (22.5 mM)Naþ gellan with 20 mM NaCl. However, the total concentration ofcations present (CT ¼ CP þ CS ¼ 22.5 þ 20 ¼ 42.5 mM) is below thecritical concentration (CT*) of Naþ at which cation-mediated aggre-gation can be detected (Figs.12 and 13), but above the correspondingcritical value for Kþ (Fig. 13), which could explain the difference inmobility (Fig. 15) of these two cations in the presence of orderedgellan. Immobilisation of Rbþ is then consistent with the evidencefrom other techniques, discussed above, that binding affinity ofGroup I cations to ordered gellan increases with increasing size.

l in the presence of 1.5 wt % Naþ gellan (NaGG-3; Table 1). Almost no thermal hysteresisal., 1999).


3.5. Gelation of gellan with Me4Nþ cations

Fig. 16 shows mechanical spectra recorded (L.E. Whittaker, R.K.Richardson & E.R. Morris, unpublished) for 1.0 wt % (13.9 mM)Me4Nþ gellan in water, and in 0.25, 0.4, and 0.7 M Me4NCl, aftercooling to the ordered state at 10 �C. The spectrum for orderedMe4Nþ gellan inwater (Fig.16a) is broadly similar to those observedfor semi-dilute solutions of disordered polysaccharide coils(Fig. 10b). With 0.7 M Me4NCl present in the sample, themechanical spectrum (Fig. 16d) has the form typical of a normalpolysaccharide gel (Fig. 10a). Formation of true gels by Me4Nþ

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Fig. 16. Mechanical spectra (10 �C; 1% strain) showing the frequency-dependence of G0

(-), G00 (B) and jh�j (:) for 1.0 wt % (13.9 mM) Me4Nþ gellan in (a) water, (b) 0.25 MMe4NCl, (c) 0.4 M Me4NCl and (d) 0.7 M Me4NCl (L.E. Whittaker, R.K. Richardson & E.R.Morris, unpublished).

gellan at high concentrations of Me4NCl was reported almost 25years ago by Grasdalen and Smidsrød (1987), although Me4Nþ

cations have often been described subsequently as “non-gelling”. Atlower concentration of Me4NCl (0.4 M), the rheological response(Fig. 16c) is still predominantly gel-like, but the moduli now showappreciable frequency-dependence and the separation between G0

and G00 is smaller. At 0.25 M Me4NCl (CT z 264 mM), log G0 and logG00 vary linearly with log u over the entire range of frequency (u) atwhich measurements could be made, with the same slope (w0.54)for both. As described in Section 3.2, such behaviour is typical ofa gelling system where the degree of crosslinking is just sufficientto give a continuous network, and pinpoints the onset of gelation.

Conformational ordering of Me4Nþ gellan on cooling under bothgelling and non-gelling conditions could be seen as a sharp increasein G0, with an accompanying sharp increase in G00. The effect ofincreasing concentration of Me4NCl on the temperature (To) at theonset of this steep increase on cooling and on completion of thecorresponding decrease on heating is shown in Fig. 17a. The twovalues are coincident up to the critical gel point (Fig. 16b) at 0.25 MMe4NCl, but then diverge progressively, with the extent of thermalhysteresis (i.e. the difference in To between heating and coolingscans) showing an approximately linear increase with increasingconcentration of Me4NCl.

At Me4NCl concentrations of 0.7 M and above, the gels becamestrong enough to be removed from cylindrical moulds and char-acterised by compression testing (Section 3.1). Fig. 17b shows thevariation of break stress (sb) for 1.0 wt %Me4Nþ gellanwith varyingconcentrations of Me4NCl, in direct comparison with the corre-sponding values of log G0 from small-deformation oscillatorymeasurements. At Me4NCl concentrations below w0.9 M, sbdecreased linearly with decreasing salt, extrapolating to zero atw0.6 M Me4NCl. The mechanical spectra, however, remained gel-like at salt concentrations down to the critical gel point at 0.25 MMe4NCl. There is thus a “window” of salt concentrations, extendingfromw0.25 tow0.6MMe4NCl, where 1.0 wt %Me4Nþ gellan shows“weak gel” properties (Section 3.3).

3.6. Gelation of gellan with Group II (alkaline earth) cations

Ina continuationof theworkdescribed in theprevious section, theeffect of divalent cations was explored by incorporation of CaCl2 innon-gelling solutionsof 2.0wt% (27.8mN)Me4Nþgellan.As showninFig. 18a, low concentrations of CaCl2 caused massive increases in G0,for example by about a factor of 10,000 at 5 mM (10 mN) Ca2þ. Theaccompanying increases in G00, though still massive, were smallerthanthoseofG0, and there is a sharpminimumintan datw9mNCa2þ,which is about 1/3 of the stoichiometric requirement of the carbox-ylate groups of the polymer (Fig. 1).

The effect of polymer concentration at fixed concentration ofCaCl2 (10 mM) was investigated by Rodriguez-Hernández, Durand,Garnier, Tecante, and Doublier (2003) using Naþ gellan preparedfrom Kelcogel by cation exchange. The variation of log G0 withconcentration (C) had the form typical (Section 3.1) of a gellingbiopolymer, with a progressive increase in slope as C was reducedtowards the minimum critical gelling concentration (Co), whichwas extremely low (slightly below 0.005 wt %), and G0 w C2 atC >> Co. Confocal laser scanning microscopy (CSLM) with fluores-cent labelling of gellan by fluoresceinamine showed the expectedprogressive increase in network connectivity as the polymerconcentration was raised.

In contrast to the behaviour observed with monovalent cations(Sections 3.4 and 3.5), where thermal hysteresis (Figs. 12, 13 and17a) occurs only when the cation concentration exceedsa minimum critical value (CT*), there was immediate thermalhysteresis on addition of CaCl2 to Me4Nþ gellan, with melting

Fig. 18. (a) G0 (-), G00 (C) and tan d (:) at 7 �C and (b) the temperature (To) at theonset of gelation on cooling (C) and completion of gel melting on heating (-) for2.0 wt % (27.8 mM) Me4Nþ gellan on progressive addition of CaCl2. Measurements weremade at 1 rad/s and 1% strain. (L.E. Whittaker, M.W.N. Hember & E.R. Morris,unpublished).

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Fig. 17. (a) Thermal hysteresis (:) between the temperature (To) at the onset ofgelation on cooling (C) and completion of gel melting on heating (-) and (b) breakingstress, sb (C) and G0 (-; 1 rad/s, 1% strain), measured at 5 �C, for 1.0 wt % (13.9 mM)Me4Nþ gellanwith varying concentrations of Me4NCl (L.E. Whittaker, R.K. Richardson &E.R. Morris, unpublished).


temperature (Fig. 18b) increasing linearly with Ca2þ concentration,and reaching 100 �C atw10 mN (5 mM). Formation of gels stable toabove 100 �C has been observed previously (Gibson & Sanderson,1997; Sanderson, 1990) on addition of low concentrations of Ca2þ

to commercial gellan. Comparative studies with MgCl2 (L.E. Whit-taker, R.K. Richardson & E.R. Morris, unpublished) showed nosignificant or systematic difference between gellan gels formedwith Ca2þ or Mg2þ, which is consistent with the study by Grasdalenand Smidsrød (1987) which showed no difference in gel strengthfor Group II cations (Mg2þ, Ca2þ, Sr2þ and Ba2þ), although divalentcations of transition metals (Zn2þ, Cu2þ and Pb2þ) were found togive stronger gels. Tang, Tung, and Zeng (1996), however, havereported that Ca2þ gives gels that are 1.1e1.4 times stronger thanthose formed with Mg2þ.

It is well established (e.g. Gibson & Sanderson, 1997; Grasdalen& Smidsrød, 1987; Milas & Rinaudo, 1996; Miyoshi, Takaya, &

Nishinari, 1994a, 1995, 1996; Sanderson, 1990) that the concentra-tion of divalent cations required to induce gelation of gellan is farlower than for monovalent cations, and that the resulting gels havegreater thermal stability. Indeed the content of divalent cations incommercial gellan is usually sufficient to give strong, stable gelswithout addition of extraneous salt. However, when divalent andmonovalent cations are present together, as would occur if saltssuch as CaCl2 or MgCl2 are added to solutions of gellan in a mono-valent, or mixed, salt form, the gelation and melting processes, andthe properties of the gels, can be complex and difficult to interpret.The purpose of the investigation leading to the results shown in


Fig.18 was to avoid such complexity by using solutions inwhich theonly cation present initially was Me4Nþ at a concentration(27.8 mM) about an order of magnitude lower than the criticalgelling concentration (Fig. 16b) of w264 mM, thus allowing theeffect of subsequent incorporation of divalent cations to be studiedin isolation from binding of monovalent metal ions.

3.7. Effect of excess salt or low pH

It was shown in early studies of gellan (Sanderson & Clark,1984),and in a number of subsequent investigations (e.g. Kasapis et al.,1999; Moritaka, Fukuba, Kumeno, Nakahama, & Nishinari, 1991;Morris & Brownsey, 1995; Sanderson & Clark, 1984), that the initialincrease in gel strength on progressive addition of salt is followedby a decrease at higher salt concentrations. This effect is illustratedin Fig. 19 for mixtures of 0.8 wt % (w12 mM) Kþ gellanwith KCl andof 0.3 wt % (w4.5 mM) Naþ gellan with MgCl2 (Milas & Rinaudo,1996). Both show maxima in Young’s modulus (E) and force atbreak (proportional to breaking stress, sb), but the molar concen-trations at which these occur are w30 times higher for Kþ than forMg2þ (i.e. w15 times higher on the basis of normality). For bothsalts, themaximum in break stress comes at substantially lower saltconcentration than the maximum in modulus. Similar behaviourwas observed (Morris, E.R. et al., 1999) for Naþ gellan with addedNaCl when the salt-dependence of sb was compared with that of G0.

In the investigation by Milas and Rinaudo (1996) three differentpatterns of fracture under compression were observed as theconcentration of added KCl or MgCl2 was increased. These areshown schematically in Fig.19, and the regions of salt concentrationover which they occurred are identified as a, b and c. In region a,which corresponds to salt concentrations below the maximum inbreak stress, the gels were weak and broke sharply along a singlefracture plane. The stiffer gels formed in region b, which corre-sponds roughly to salt concentrations between the maximum inbreak stress and the maximum in modulus, formed multiple smallfractures during break. In region c, at salt concentrations above the

Fig. 19. Changes in Young’s modulus, E (circles) and maximum force (Fm) at break(squares), from compression testing (25 mm/min) of cylindrical samples (17 mmheight; 17 mm diameter) at 25 �C, on addition of MgCl2 to 0.3 wt % Naþ gellan (filledsymbols) or KCl to 0.8 wt % Kþ gellan (open symbols). The different patterns of failureobserved in regions a, b and c for each salt are shown schematically between the tracesfor MgCl2 (from Milas & Rinaudo, 1996).

maximum in modulus, the gels were softer and their disintegrationresembled a coagulation or phase-separation process.

Gelation of gellan can also be induced by reduction in pH, and intheir study of the effect of monovalent and divalent cationsGrasdalen and Smidsrød (1987) described HCl as “the most potentgel-former”. However, the variation in gel strength with increasingconcentration of acid is not monotonic. Initial acidification fromneutral pH to pH 3.5 causes a large increase in break stress (Picone& Cunha, 2011), but on further decrease in pH below the pKa of theglucuronate residues of gellan (Fig. 1), at wpH 3.4 (Haug, 1964),break stress decreases (Norton, Cox, & Spyropoulos, 2011) and bypH 2 the gels are extremely weak and turbid, and show phaseseparation of polymer and solvent (Moritaka, Nishinari, Taki, &Fukuba, 1995). Indeed, precipitation by acid can be used asa method for isolation and purification of gellan (Sanderson, 1990).

Initial increase and subsequent decrease in Young’s modulus,giving curves similar to those shown in Fig. 19, has also beenobserved on progressive addition of alkali metal salts to kappacarrageenan (Watase & Nishinari, 1982), and maxima in gelstrength on varying salt concentration and/or pH are commonlyobserved in thermogelation of globular proteins (Foegeding,Bowland, & Hardin, 1995). Indeed, such maxima seem to beuniversal, and are perhaps to be expected. Biopolymer gels formpart of a continuum from solutions of individual molecules at oneextreme to close-packed solids at the other as the extent of inter-molecular association is increased (by varying conditions such aspH and ionic environment). Optimum crosslinking will occursomewhere along the continuum; less association will givea weaker network; greater association will give larger aggregates,with consequent reduction in the effective number of individualjunctions, until ultimately the network collapses into a solidprecipitate. This interpretation is consistent with the apparent“phase separation” observed for gellan gels at very low pH or witha large excess of salt (region c in Fig. 19), and with the observation(Ohtsuka & Watanabe, 1996) that the gels become turbid at thepoint where their strength begins to decrease.

3.8. Summary and interpretation

The first step in gelation of gellan is conversion of the polymerfrom the disordered coil state to the double-helix form. However,conformational ordering does not, in itself, give a cohesive network.Formation of true gels requires association of double helices intostable aggregates. Aggregation is inhibited by electrostatic repul-sion between the helices. One way in which the repulsion can besuppressed, allowing gels to form, is reduction in pH, whichreduces the charge on the helices by conversion of glucuronatecarboxyl groups (Fig. 1) from the negatively-charged COO� form tothe uncharged COOH form.

Aggregation can also be promoted by salt. The simple anions andcations from dissolved salt screen electrostatic repulsion betweenthe gellan helices. Cations cause further reduction in repulsion byclustering around the helices and thus lowering their effectivenegative charge. Both of thesemechanisms are non-specific: chargescreening depends solely on ionic strength, determined by theconcentration and charge of both anions and cations, and theextent to which the (negatively-charged) helices are surroundedpreferentially by (positively-charged) cations is determined by theconcentration and charge of the cations. Group I (alkali metal)cations, however, can cause further reduction in the effectivenegative charge of the gellan double helices by attaching to them inspecific binding sites. Site binding is triggered initially by electro-static attraction of cations to the carboxylate groups of the polymer,but is augmented and stabilised by formation of a coordinationcomplex with appropriately-spaced oxygen atoms from both

Fig. 20. DSC traces recorded (Miyoshi & Nishinari, 1999a) on cooling (left) and heating(right) at 0.5 �C/min for Naþ gellan (NaGG-3; Table 1) in water at the concentrationsshown to the right of the heating curves (1.0e5.5 wt %).


strands of the double helix. The equilibrium between bound andunbound cations is determined by the efficiency of coordination,which, as discussed in Section 3.4, seems to decrease withdecreasing ionic size (Csþ > Rbþ > Kþ > Naþ > Liþ).

Stabilisation of gellan double helices by association intoaggregates leads to thermal hysteresis between the temperaturesat which helices form on cooling and aggregates melt on heating.On gelation with alkali metal (Group I) cations, the onset ofhysteresis is accompanied by the onset of formation of true gels,and occurs at progressively lower cation concentration as ionicsize increases.

At low concentrations of monovalent cations, solutions ofordered gellan give mechanical spectra similar to those of solutionsof disordered coils (Fig. 10b or c). On further increase in saltconcentration there is a region of “weak gel” response (Section 3.3)before reaching the threshold concentration for formation of truegels. This progression, which can be attributed to progressivesuppression of electrostatic repulsion between the gellan doublehelices, is observed for both alkali metal and M4Nþ cations. The saltconcentrations over which the three types of rheology (solution,“weak gel” and true gel) occur are, however, much higher for M4Nþ

than for Group I cations. This can be readily explained by theinability of organic cations such as M4Nþ to form coordinationcomplexes, which makes them incapable of reducing the effectivenegative charge of the gellan double helices by the site binding thatoccurs with Group I cations.

Gelation with divalent cations (Ca2þ and Mg2þ) is qualitativelydifferent. The thermal hysteresis associatedwith stable aggregationbegins immediately (Fig. 18) on progressive incorporation of CaCl2(or MgCl2) in solutions of M4Nþ gellan, and is accompanied byimmediate massive increase in G0 and G00. Maximum gel strength isattained (Tang et al., 1996) when the concentration of divalentcations reaches w100% of stoichiometric equivalence to thecarboxyl groups of the polymer (for gellan concentrations rangingfrom 0.6 to 2.2 wt %), which argues against “salting out” as thecause of reduction in gel strength after the initial increase (Fig. 19).A proposed interpretation (Morris et al., 1996; Tang et al., 1996) isthat divalent metal ions promote aggregation by site bindingbetween pairs of carboxylate groups on neighbouring helices,rather than by suppressing electrostatic repulsion by binding toindividual helices. Computer modelling (Chandrasekaran &Thailambal, 1990) has shown that direct bridging between gellandouble helices by Ca2þ is sterically feasible.

In Ca2þ-gelation of alginate and pectin, the poly-L-guluronatesequences of alginate (Morris et al., 1978) and poly-D-galacturonatesequences of pectin (Morris et al., 1982) both form highly-stabledimeric junctions in which the participating chains adopta buckled, 2-fold conformation with site-bound Ca2þ cationsbridging between them. Larger assemblies, involving more chainsand more layers of site-bound cations can be built up in the sameway, but the dimer structure is particularly stable. This can beexplained by simple electrostatic considerations: incorporation ofthe first layer of divalent cations causes a gross reduction innegative charge density, so that binding between dimers is farweaker than initial binding within individual dimers.

Since the participating chains have 2-fold symmetry and onlythe inner faces are involved in chelation (coordination) of cations,the Ca2þ content of polyguluronate and polygalacturonate dimers ishalf the full stoichiometric requirement of the polymer carboxylgroups. Formation of analogous Ca2þ-mediated dimeric junctionsbetween gellan double helices, which have 3-fold symmetry(Fig. 2b), would therefore reach completion when the content ofsite-bound cations is 1/3 of stoichiometric equivalence. The sharpminimum in tan d at this point (Fig. 18a) can then be explained bytransition from a primary mechanism of association through

highly-stable helixehelix dimers to a secondary process involvingmuch weaker association between dimers.

Finally, as described in Section 3.6, excess salt or low pH canweaken gellan networks by promoting excessive aggregation,leading to collapse of gel structure and, ultimately, precipitation ofthe polymer.

4. Gelation of gellan in water, with no added salt

Fig. 20 shows DSC traces recorded on cooling and heating at0.5 �C/min for essentially pure Naþ gellan (NaGG-3, Table 1) atconcentrations ranging from 1.0 to 5.5 wt % inwater, with no addedsalt (Miyoshi & Nishinari, 1999a). As would be expected, themagnitude of the thermal transitions increases with increasingpolymer concentration. The cooling traces show a single exothermwhich moves to progressively higher temperature as the concen-tration of gellan is raised. At gellan concentrations up to 4 wt %, theheating traces show a single endotherm which is essentially equaland opposite to the corresponding exotherm on cooling. At 4.5 wt%, however, a small second endotherm can be detected. On furtherincrease in gellan concentration, the second endotherm increasesin magnitude relative to the first, and the separation between themincreases.

The accompanying rheological changes, characterised (Miyoshi& Nishinari, 1999a) by low-amplitude oscillatory measurementsof G0 and G00 at 0.1 rad/s during cooling and heating at 0.5 �C/min areshown in Fig. 21. The initial sharp increase in G00 during coolingcoincides with the exothermic peaks for the same samples in DSC(Fig. 20) and with conformational ordering, as monitored(Matsukawa et al., 1999) by circular dichroism at 204 nm; thetemperature at which it occurs can therefore be regarded (Section3.2) as the coilehelix transition temperature (Tch). G0 also increases

Table 2Variation of coilehelix and solegel transition temperatures, Tch and Tsg (Fig. 21),with concentration (C) of Naþ gellan (NaGG-3, Table 1) in water, with no added salt(Miyoshi & Nishinari, 1999a).

C (wt %) 0.5 1.0 2.0 2.5 3.0 3.5 4.5

Tch (�C) 20 26.5 34 37.7 38.2 42 43Tsg (�C) Nonea Nonea 9.4 26 30.5 42 43

a G0 remained below G00 to the lowest temperature at which measurements weremade (5 �C).


sharply below Tch, but at gellan concentrations less thanw2 wt % itremains lower than G00 (Fig. 21a and b). At higher concentrations,however, G0 rises above G00, and, at the low frequency of oscillationused (0.1 rad/s), the temperature at which the two curves cross oneanother can be taken (Section 3.2) as the solegel transitiontemperature (Tsg). Both Tch and Tsg increased (Table 2) withincreasing concentration of gellan, paralleling the increase oftransition temperatures in DSC (Fig. 20).

Over the concentration-range where the DSC heating curvesshowed a single endotherm at the same temperature as the singleexotherm on cooling (up to w4 wt %) the rheological changes onheating followed the same temperature-course as those observedon cooling (Fig. 21aef). However, at 4.5 wt %, the concentration atwhich a second, higher temperature, endothermwas first observedin DSC, the final steep decrease in G0 and G00 on heating alsooccurred at higher temperature (Fig. 21g) than the initial steepincrease on cooling.

Mechanical spectra recorded (Miyoshi & Nishinari, 1999a) atselected temperatures during cooling are shown in Fig. 22 forNaGG-3 in water at concentrations of 1.0, 2.0, 2.5, 3.0 and 3.5 wt %.As described in the figure legend, the spectra are shifted along thehorizontal axis (frequency) and/or along the vertical axis (moduli)to avoid overlap and allow the relative magnitudes of G0 and G00, andtheir response to frequency (u), to be compared at each concen-tration and temperature.

For the 1% solution (Fig. 22a) in the disordered coil form at 30 �C,G0 was too low to measure, and log G00 increased linearly as log u

was increased, with the slope of þ1 typical (Section 3.1) of a dilutepolymer solution. At 25 �C, which is just below the onset ofconformational ordering at Tch ¼ 26.5 �C (Table 2), the slope of logG00 versus log u remains the same (þ1) and G0 is now detectable,with a slope of þ2 on the double logarithmic plot. As described inSection 3.1, this is again the behaviour typical of a dilute solution ofpolymeric species free to move independently. On further reduc-tion in temperature, to 15, 5 and 0 �C, however, there is a progres-sive decrease in the frequency-dependence of both moduli towardsthe lower slopes typical of semi-dilute solutions (Fig. 10b), whichcan be explained by progressive entanglement between growing

Fig. 21. Temperature-dependence of G0 (circles) and G00 (triangles), measured at 0.1 rad/s du(NaGG-3; Table 1) in water at concentrations (wt %) of (a) 0.5, (b) 1.0, (c) 2.0, (d) 2.5, (e) 3.0,start of the steep increase in G00 on cooling, and the solegel transition temperature (Tsg) as

clusters of chains crosslinked through double helices. Formation ofa continuous network, with gel-like mechanical response (Fig. 10a),was not, however, observed for this concentration of gellan in theabsence of salt, even at 0 �C.

A similar progression towards increasing entanglement astemperature was decreased below Tch (Table 2) is evident at 2 wt %(Fig. 22b), but at this concentration a solegel transition wasobserved (at 9.4 �C), and the mechanical spectrum recorded atlower temperature (5 �C) has some gel-like character (G0 > G00),although still showing pronounced frequency-dependence of bothmoduli.

The solegel transition temperatures (Table 2), by the criterion ofG0 crossing G00, are 26 �C at 2.5 wt % gellan and 30.5 �C at 3.0 wt %.Mechanical spectra recorded at, or very close to, those tempera-tures, at 26 �C for 2.5 wt % (Fig. 22c) and at 30 �C for 3.0 wt %(Fig. 22d), have the form characteristic of a critically-crosslinkednetwork (Section 3.2): log G0 and log G00 both increase linearlywith log u over the entire range of accessible frequencies and havethe same slope of w0.5. On progressive reduction in temperaturebelow the critical gel point, there is a progressive decrease infrequency-dependence, and G0 rises progressively above G00,showing progressive development of elastic (gel-like) character.

At 3.5 wt % gellan, the sharp increase in G00 during cooling(Fig. 21f) began atw42 �C and was accompanied by a simultaneoussharp increase in G0 to above G00, giving Tch z Tsg z 42 �C (Table 2).The mechanical spectra (Fig. 22e) recorded at higher temperatures(43 and 45 �C) both show solution-like response, although the

ring cooling (open symbols) and heating (filled symbols) at 0.5 �C/min, for Naþ gellan(f) 3.5 and (g) 4.5. The coilehelix transition temperature (Tch in frame c) is taken as thethe point where the traces for G0 and G00 cross (Miyoshi & Nishinari, 1999a).

Fig. 22. Frequency-dependence of G0 and G00 for Naþ gellan (NaGG-3; Table 1) in water at concentrations (wt %) of (a) 1.0, (b) 2.0, (c) 2.5, (d) 3.0, and (e) 3.5 at the temperaturesshown within the frames. At each concentration and temperature the symbols used for the two moduli are the same, but are open for G0 and filled for G00 . To avoid overlap,individual pairs of traces (G0 and G00) are shifted along the horizontal axis by an integral number of decades (a) and/or by b decades on the vertical axis. The values of a and b used areshown to the left of each pair of curves (a first and b below it). Temperatures of the coilehelix and solegel transitions (Tch and Tsg) from Fig. 21 are shownwithin the frames (Miyoshi& Nishinari, 1999a).

Fig. 23. Variation of coilehelix (B) and solegel (C) transition temperatures (Tch andTsg) on cooling (see Fig. 21) with concentration (C) of Naþ gellan (NaGG-3; Table 1) inwater; the region labelled Sol-I corresponds to solutions of disordered coils, Sol-II toordered (helical) structures in solution, and Gel to continuous networks (Miyoshi &Nishinari, 1999a).


spectrum at 45 �C resembles that of a dilute solution (Fig. 10c)whereas the spectrum at 43 �C is similar to that of a semi-dilutesolution of entangled coils (Fig. 10b), suggesting that somelimited association of chains into entangled clusters has alreadyoccurred. The spectrum recorded at 42 �C, by contrast, has the formtypical of a polymer gel (Fig. 10a), with G0 about an order ofmagnitude higher than G00 and little frequency-dependence ofeither modulus. It is evident, therefore, that the critical gel point, bythe criterion (Section 3.2) of constant tan d, must lie within thenarrow window of temperature between 43 and 42 �C but, becauseof the very large changes in moduli within this range, determina-tion of the precise temperature at the gel point was not experi-mentally practicable.

Fig. 23 shows the variation of Tch and Tsg with concentration ofNaGG-3 during cooling in water, with no added salt (as inFigs. 20e22). Below w2 wt % gellan, Tch increased steeply withincreasing polymer concentration, and G0 remained lower than G00,so that no values of Tsg could be observed. At 2 wt % and above, G0

crossed G00 during cooling, and the resulting values of Tsg increasedsteeply with increasing concentration of gellan, until becomingapproximately equal to Tch at w3.5 wt %. At higher concentrations,Tch and Tsg remained approximately equal to one another, andshowed only slight further increase with increasing concentrationof gellan (Miyoshi & Nishinari, 1999a).

Three different physical states of gellan can be distinguished inFig. 23. The region above Tch corresponds to solutions of disorderedcoils, the region between Tch and Tsg to solutions of ordered gellan,and the region below Tsg to continuous gel networks. In the


mechanical spectra of the networks formed at concentrationsbelow 3.5 wt % (Fig. 22bed) there is little separation between G0

and G00 and both show large variations with frequency. Asmentioned in Section 3.3, spectra of this type are commonly seenfor “weak gels”. The mechanical spectra observed (Fig. 22e) for thenetworks formed at 3.5 wt % gellan, by contrast, have the formtypical (Fig. 10a) of true gels (G0 >> G00; little variation withfrequency). The “Gel” region in Fig. 23 can perhaps therefore besubdivided, tentatively, into true gels at gellan concentrations of3.5 wt % and above, and “weak gels” at lower concentrations, downto 2 wt %, below which there is no solegel transition.

The concentrations of NaGG-3 used in the rheological studiesdescribed above ranged (Fig. 23) from 0.20 to 4.75 wt %. Since thesolutions were prepared in water with no added salt, and 1 wt %Naþ gellan corresponds (Section 2.3) to 15 mM, the total concen-tration of Naþ cations therefore ranged from 3.0 to 71.25 mM.Fig. 24 shows the variation of coilehelix transition temperature(Fig. 23) with Naþ concentration plotted according to Eq. (3), indirect comparison with the corresponding values for solutions inwhich the concentration of Naþ gellan was held fixed at 0.05 wt %(Milas & Rinaudo, 1996; Fig. 9) or 1.0 wt % (Manning, 1992; Fig. 12)and the total concentration of Naþ was varied by incorporation ofNaCl. Despite the widely different gellan concentrations, the threefamilies of points lie convincingly on the same straight line,demonstrating that, as would be expected from Eqs. (1)e(3), thetemperature at which gellan converts from coils to double heliceson cooling is determined by the total cation concentration, withcounterions to the polymer having the same effect as the samecations introduced by addition of salt, and is unaffected by theconcentration of the polymer chains themselves.

The changes in DSC heating traces (Fig. 20) on varying concen-tration of Naþ gellan with no added salt bear a striking qualitativeresemblance to the changes observed (Fig. 11) on progressiveaddition of NaCl to a fixed concentration of Naþ gellan (1 wt %).Quantitatively, the gellan concentration of 4.5 wt % at whichsplitting of the DSC heating curve becomes detectable (Fig. 20) isequivalent (Section 2.3) to a counterion concentration of

Fig. 24. Dependence of Tch on total concentration of Naþ (counterions to the polymerplus added NaCl, where present) for 0.20e4.75 wt % Naþ gellan with no added salt (C;Fig. 23) and for 0.05 wt % (B; Fig. 9) and 1.0 wt % (6; Fig. 12) Naþ gellan with orwithout NaCl.

15 � 4.5 ¼ 67.5 mM Naþ, which is in close agreement with thecorresponding values of CT ¼ 67 mM or 65 mM observed by,respectively, Robinson et al. (1991) and Mazen et al. (1999) for 1 wt% Naþ gellan with added NaCl. It seems likely, therefore, that theorderedisorder transition temperatures observed for gellan inwater, like the temperature of the disordereorder transition(Fig. 24), are determined by the concentration of counterions to thecarboxylate groups of the polymer, rather than being affecteddirectly by the concentration of polymer itself.

A similar investigation, using the same procedures and experi-mental conditions, was carried out by Miyoshi, Takaya, andNishinari (1996) for aqueous solutions of the less pure Naþ gellansample NaGG-2 (Table 1). At gellan concentrations of 1 and 2 wt %the changes in G0 and G00 on cooling and heating were broadlysimilar to those observed (Fig. 21b and c) for the same concentra-tions of the purer sodium salt form, NaGG-3. At 3wt %, however, theinitial steep increase in G0 on cooling was followed by a second“wave” of increase at lower temperature, which was not seen forNaGG-3 (Fig. 21e). At concentrations up to 3 wt %, the heatingcurves for NaGG-2 superimposed closely on the cooling curves,with no detectable thermal hysteresis, as was also observed forNaGG-3. On slight increase in the concentration of NaGG-2 to3.2 wt %, however, reduction in both G0 and G00 occurred in twosteps, the first coincident with the increases in moduli observed oncooling, and the second at higher temperature. On further slightincrease in gellan concentration to 3.3 wt % the second meltingprocess was displaced to even higher temperature, and at 3.5 wt %loss of gel structure had not gone to completion by the highesttemperature at which measurements were made (60 �C). The onsetof (slight) thermal hysteresis for NaGG-3, by contrast, was notobserved until the polymer concentration had reached 4.5 wt %(Fig. 21g). The obvious interpretation of the greater thermalstability of the gels formed by NaGG-2 is that the polymer hasa significant content of Kþ and, particularly, Ca2þ and Mg2þ cationswhich are present only in much smaller amounts in NaGG-3(Table 1). The ability of these cations to promote formation ofmore stable junctions than those formed with Naþ is discussed inSections 3.4 and 3.6 and shown in Figs. 13 and 18b.

DSC traces (Fig. 25) recorded in the same investigation (Miyoshi,Takaya, & Nishinari, 1996) also show evidence of the effect ofcations other than Naþ on thermal transitions of NaGG-2. The DSCcooling curves (Fig. 25) are broadly similar to those observed(Fig. 20) for NaGG-3, but are displaced systematically to slightlyhigher temperature, which may reflect the ability of divalentcations to induce conformational ordering of gellan at highertemperatures (Fig. 9) than equivalent concentrations of mono-valent cations.

The main difference in DSC between NaGG-2 and NaGG-3,however, is in the heating curves. Detectable splitting of the DSCendotherm for NaGG-3 did not occur until the polymer concen-tration had reached 4.5 wt % (Fig. 20) and only two endothermicprocesses were then observed up to the highest concentrationstudied (5.5 wt %). For NaGG-2, by contrast, there is obvious split-ting at 3.2 wt % (Fig. 25). As the concentration of NaGG-2 isincreased to 3.5 and 3.6 wt % the separation between the twoendothermic processes increases, and on further increase in poly-mer concentration up to the highest studied (4.2 wt %) multipleendothermic processes are observed, strongly indicating that thevarious cations present in NaGG-2 form aggregated junctions ofdifferent thermal stabilities. As might therefore be expected,addition of extraneous salts increases the complexity of the heatingtraces in DSC (Miyoshi, Takaya, & Nishinari, 1994b). The ability ofthe comparatively small amounts of divalent cations in NaGG-2(Table 1) to cause large enhancements in thermal stabilityemphasises the importance of the higher concentrations normally

Fig. 25. DSC traces recorded (Miyoshi, Takaya, & Nishinari, 1996) on cooling (left) andheating (right) at 0.5 �C/min for Naþ gellan (NaGG-2; Table 1) in water at theconcentrations shown to the right of the heating curves (1.0e4.2 wt %).


present in commercial gellan in yielding thermally-stable gels atlow concentrations of polymer.

5. Topology and properties of gellan networks

5.1. Internal structure of gellan gels

Two schematic models proposed for the internal structure ofgellan gels are shown in Fig. 26. The first (Fig. 26a) was based on

Fig. 26. Models for gelation of gellan proposed by (a) Robinson et al. (1991) and (b) Guaggregation of gellan double helices.

experimental evidence from DSC and rheology (Manning, 1992;Morris et al., 1996; Robinson et al., 1988, 1991); the second(Fig. 26b) came from investigation of M4Nþ gellan by light scat-tering (Gunning & Morris, 1990).

Although rather different visually, the two models have manyfeatures in common. Both envisage formation of true gels byassociation of double helices in the presence of “gel promotingcations” (i.e. metal ions). In the paper by Robinson et al. (1991) theassociated helices are termed “cation-mediated aggregates” and inGunning and Morris (1990) they are described as “crystallinejunction zones”, but the underlying concept of ordered assembliesof double helices incorporating metal cations to balance thenegative charge of the polysaccharide appears to be the same inboth.

The schematic network shown on the right-hand side at the topof Fig. 26a includes both “cation-mediated aggregates” and unag-gregated stretches of double helix, with the unaggregated helicesmelting first on heating, leaving a network crosslinked solely byhelixehelix aggregates. This is an essential feature of the model,invoked to rationalise the two thermal transitions observed in DSCheating scans (Fig. 11). The simultaneous presence of aggregatedand unaggregated double helices in gelled films of Kþ gellan(KGG-1, Table 1) has been observed directly (Nakajima et al., 1996)by scanning tunnelling microscopy (STM). Coexisting stretchesof aggregated and unaggregated double helices are also present inthe network shown bottom-left in Fig. 26b, although the doublehelices are represented by solid black bars, whereas in Fig. 26a theyare shown as two strands wound round one another. Two-stagemelting does not form part of the second model, but is notprecluded by it.

A central feature of the second model, shown schematically onthe right-hand side of Fig. 26b, is formation of long “filaments” oncooling under non-gelling conditions (including specifically M4Nþ

gellan with or without low concentrations of added M4Nþ). Theseare described by Morris, V.J. (1995) as “filamentous aggregates”.However, to avoid confusion with use of the term “aggregates” torefer to structures formed by lateral (side-by-side) association of

nning and Morris (1990). In both models, filled circles denote cations that promote


double helices, we will continue to use the term “clusters” todescribe assemblies of chains that are crosslinked solely by indi-vidual double helices and do not form a continuous network (as inthe discussion of critical gel point in Section 3.2). Transient asso-ciation of soluble clusters (foot of Fig. 26a) was incorporated in thefirst model to explain “weak gel” properties. Although linear clus-terswere not envisaged specificallywhen thismodel was proposed,there was certainly no intention (or experimental evidence) topreclude them. Conversely, the second model (Fig. 26b) includessome branching of the linear filaments. Thus, although theemphasis of the two models is different, there is no fundamentalclash between them.

One genuine difference, however, is that the schematicnetworks shown in Fig. 26a include some stretches of disorderedpolymer chains between the gellan double helices (aggregated ornon-aggregated), while there are no such disordered regions in thesecond model (Fig. 26b). As shown in Fig. 8, Milas and Rinaudo(1996) observed complete loss of detectable 1H NMR signal,implying complete conversion from the disordered to the orderedform, when a non-gelling solution of Naþ gellan was cooledthrough the temperature-range of the coilehelix transitionobserved by other techniques. In a more recent investigation,Hossain and Nishinari (2009) recorded high-resolution 1H NMRspectra for 1 wt % Kþ gellan in the solution state at 50 �C and in thegel state at 20 �C. The spectrum at 50 �C showed the comparativelysharp resonances characteristic (Section 2.3) of disordered polymercoils. The same resonances were still detectable at 20 �C, althoughtheir intensity was decreased tow10% of that at 50 �C. DSC coolingtraces recorded (Tanaka & Nishinari, 2007) for the same concen-tration of the same gellan sample showed a well-defined exothermwith a peak-maximum temperature of w30 �C, and no evidence offurther conformational ordering on continued cooling after thetemperature had reached 20 �C. A possible explanation of thevisible 1H NMR resonances detected (Hossain & Nishinari, 2009) atthis temperature is that they arose from disordered sequences“trapped” by topological constraints in formation of the crosslinkednetwork, which would not occur in the non-gelling solutionstudied by Milas and Rinaudo (1996), and that the ratio of order todisorder in the resulting gel was about 9:1. This conclusion isconsistent with the schematic model in Fig. 26a, although thepresence of disordered sequences was not necessary to explain theexperimental evidence on which the model was based, and couldnot be inferred from that evidence.

The presence or absence of disordered sequences is important inunderstanding the mechanical properties of gels (Nishinari, Koide,& Ogino, 1985). For networks with an appreciable content ofdisordered sequences the main response to applied stress isstretching of these flexible regions, and the elastic resistance todeformation comes predominantly from the consequent reductionin conformational entropy. Since change in entropy (DS) becomesincreasingly significant as temperature (T) is raised (DG¼ DH�TDS),the elastic modulus of “entropic” networks increases withincreasing temperature, as explored for gellan gels by Watase andNishinari (1993). The elasticity of networks formed by lateralassociation of fibrillar strands (Fig. 26b), by contrast, would comepredominantly from the increase in enthalpy (DH) on distortion ofthe aggregated filaments, or the entire fibrillar network (Morris, V.J.et al., 1999).

5.2. Dimensions of strands in gellan networks

Branched fibrillar strands have been visualised directly(Gunning et al., 1996; Gunning, Kirby, Ridout, Brownsey, & Morris,1997) by atomic force microscopy (AFM) for networks formed bydrying very dilute solutions (3 or 10 mg/L) of Kþ or M4Nþ gellan on

freshly-cleaved mica. The length of the linear regions betweenbranch points in the micrographs varies considerably, but is typi-cally around 150 nm. As discussed in Section 2.3, the tetra-saccharide repeating unit of (Naþ) gellan has mass 668 D, givingalmost exactly 2 kD (3 � 0.668) for a full turn (pitch ¼ 5.64 nm) ofeach strand in the double helix (Fig. 2). The mass per unit length ofthe double helix is therefore 0.71 kD/nm (2 � 2/5.64). Thus thetypical length of w150 nm for the linear regions visualised by AFMcorresponds to a mass of 106 kD, which is in perfect agreementwith the value of Mw (Section 2.4) obtained from light-scatteringstudies of well-clarified solutions of ordered gellan by the samegroup (Gunning & Morris, 1990). Although this exact agreement is,of course, fortuitous, it does lend support to the concept (Fig. 26b)of each chain forming “end-to-end” associations with two adjacentchains over its entire length.

Although technically challenging, it has also proved possible touse AFM to obtain images of the surface of fully-formed gellan gelsat molecular resolution. This was first achieved (Gunning et al.,1996, 1997) for very strong gels of 1.2 wt % gellan obtained bycontrolled reduction in pH using D-glucono-d-lactone (GDL),yielding images of a network of long fibrillar strands. Surfaceimages of gels formed by 2 wt % gellan in the presence of 100 mMKCl or CsCl were obtained in a subsequent study by Ikeda, Nitta,Temsiripong, Pongsawatmanit, and Nishinari (2004), againshowing networks of fibrillar strands.

In the same investigation, images were recorded for structuresformed by deposition of very dilute solutions onto freshly-cleavedmica, as in the study by Gunning et al. (1996) described above.Solutions of gellan (KGG-1, Table 1) were prepared at a concentra-tion of 0.2 wt % in 10 mM KCl or CsCl, held at 90 �C for 30 min,cooled to room temperature, and diluted 500-fold (to 4 mg/L)before deposition on the mica surface. Comparison was made withthe same concentration of M4Nþ gellan. The images obtained forthe M4Nþ salt form showed branched clusters of strands with fairlyuniform height of around 0.5 nm, consistent with crosslinkingsolely through unaggregated double helices. Isolated clusters werealso observed for the sample incorporating Kþ, but the individualstrands were longer and their heights extended to w1.2 nm, indi-cating side-by-side association of double helices. For samplesincorporating Csþ, which, as discussed in Section 3.4, is moreeffective than Kþ in promoting gelation of gellan, a continuousnetwork structure was observed, with the height of the strandsagain corresponding to aggregates formed by lateral association ofa few double helices.

Strand dimensions in gellan gels have been explored (Yuguchiet al., 1993, 1996, 1999) by small-angle X-ray scattering (SAXS) foreach of the three samples (Table 1) studied in the Japanesecollaborative research initiative (Section 1). In the first of theseinvestigations (Yuguchi et al., 1993) it was concluded that gelsformed by 1.5 wt % KGG-1 (with no added salt) had (at least) threedifferent populations of strands, with cross-sectional radii of 0.54,1.23 and 1.84 nm; these were attributed to sequences comprising,respectively, one, four or six double helices, present in relativemass-fractions of 63, 7 and 30%.

In the second investigation (Yuguchi et al., 1996) NaGG-2, againwith no added salt, was studied at four concentrations: 1.0, 1.5, 2.9and 5.7 wt %. At the two lower concentrations the increase in cross-sectional radius of gyration observed on cooling from the disor-dered state (at 60 �C) to the ordered state (at 10 �C) was consistentwith conversion of single chains to unaggregated double helices.Rheological studies (Miyoshi, Takaya, & Nishinari, 1996) of the samebatch of gellan showed that these concentrations (1.0 and 1.5 wt %)are non-gelling. At the higher concentrations (2.9 and 5.7 wt %),where a gel is formed, analysis of the SAXS data suggested twopopulations of strands; at 2.9 wt % these had cross-sectional radii of


0.52 and 1.05 nm, with relative mass-fractions of 85% and 15%; thecorresponding values at 5.7 wt % were 0.90 nm (92%) and 1.21 nm(8%). The thicker stands observed (Yuguchi et al., 1993) for KGG-1can reasonably be attributed to its high content of Kþ and diva-lent cations.

In the third investigation (Yuguchi et al., 1999) 1.5 wt % solutionsof NaGG-3 were prepared with no added salt and with 50 mM LiCl,NaCl, KCl or CsCl. As mentioned in Section 3.4, the cross-sectionalradii obtained for these samples in the ordered state (at 10 �C)were, respectively, 0.30, 0.44, 0.50, 1.01 and 1.37 nm. The SAXSscattering profiles were fitted by combining different proportions(Table 3) of calculated profiles for individual double helices and foraggregated strands consisting of 2, 4, 8 or 12 double helices. Asemphasised by the authors, these specific models were used forcomputational convenience and do not imply that there are nostructures consisting of intermediate (or larger) numbers of helices,but they give a useful general indication of the size of the fibrillarstrands in gellan gels, and the way in which they vary with ionicenvironment. At the specific concentration of polymer (1.5 wt %)used by Yuguchi et al. (1999) the strands varied (Table 3) fromindividual double helices in the absence of added salt to aggregatesconsisting predominantly of around 12 double helices in thepresence of 50 mM Csþ.

In a more recent investigation by TEM, Kasapis, Abeysekara,Atkin, Deszczynski, and Mitchell (2002) observed strands withdiameters up to w40 nm and lengths of several hundred nano-metres for Naþ gellan (0.7 wt %) in the presence of Ca2þ ata concentration (10 mN) around stoichiometric equivalence to thecarboxyl groups of the polymer, demonstrating that divalentcations give longer, thicker strands than monovalent (Group I)metal ions.

Finally, measurements of the diffusion coefficient of a probemolecule (pullulan) in gellan gels (Shimizu, Brenner, Liao, &Matsukawa, 2012) have demonstrated that, as would be expected,the size of the void spaces between the polymer chains (mesh size)increases on cooling, as individual molecules associate intoa smaller number of double helices and helixehelix aggregates.

5.3. Gelation by cations at ambient temperature

As described in Sections 2e4, gellan gels are normally formed bycooling solutions from the disordered state at high temperature.Gelation can, however, also be induced (Gibson & Sanderson, 1997;Sanderson & Clark, 1984) by diffusion of cations into solutions ofgellan at ambient temperature, or by the “internal set” procedure(Sime,1990) developed for alginate, inwhich Ca2þ ions are releasedinto solution at a controlled rate by dissociation of an insolublecalcium salt on reduction in pH by GDL.

Introduction of cations at ambient temperature can also be usedto reinforce existing gels. This was demonstrated in a study byNitta, Ikeda, and Nishinari (2006) inwhich gels of Kþ gellan (KGG-1,Table 1) were immersed in 1 M solutions of LiCl, NaCl, KCl or CsClat 25 �C. Large, progressive increases in E0 were observed as

Table 3Relative proportions (%) of model structures used (Yuguchi et al., 1999) to fit SAXSscattering profiles for ordered gellan (1.5 wt % NaGG-3) with no added salt, and with50 mM LiCl, NaCl, KCl or CsCl.

Double helices per strand 1 2 4 8 12

No salt 100LiCl 81 19NaCl 68 32KCl 17 17 66CsCl 27 73

immersion time increased, with moduli after w2 h immersionfollowing the same order of cation-dependence as observed(Section 3.4) for gels formed by cooling hot solutions(Liþ<Naþ< Kþ<Csþ). The rheological changes were accompaniedby large changes in circular dichroism, indicating formation ofdouble helices in addition to cation-induced association of existinghelices.

5.4. Conformational freedom and release of polymer chains

The ability of gellan double helices to form within theconstraints of an existing gel network was also demonstrated in anearlier study by Nitta et al. (2001). The gellan used was againKGG-1, at a concentration of 1.6 wt % in water, with no added salt.The gels formed under these conditions remained intact up to thehighest temperature at which rheological measurements could bemade, which is consistent (Fig. 18b) with the high content ofdivalent cations in the gellan sample (Table 1). However, DSCheating scans showed a sharp endotherm at w28 �C, indicatingmelting of unaggregated double helices, to give disordered chainsequences crosslinked solely by cation-mediated aggregates (asillustrated schematically at the top of Fig. 26a).

On subsequent cooling (Fig. 27), there was a corresponding DSCexotherm, essentially equal and opposite to the endothermobserved on heating. This was accompanied by a sigmoidal increasein E0 and E00 (Fig. 27a), showing reinforcement of the networkstructure remaining after initial heating, and by a sigmoidaldecrease in circular dichroism at 202 nm (Fig. 27b), as seen (Fig. 4)on conversion of gellan chains from disordered coils to doublehelices. It is evident, therefore, that disordered chain sequencesformed during heating of the KGG-1 gel have sufficient freedom ofmovement within the surviving crosslinked network to revert tothe double helix state on cooling.

Two recent investigations (Hossain & Nishinari, 2009; Tanaka &Nishinari, 2007) have shown release of polymer chains from gellangels immersed in excess water. Both studies used the same sampleof Kþ gellan. Cylindrical gels of height 1.5 cm and diameter 1.0 cm(volume z 1.18 mL) were prepared by cooling hot solutions ofgellan in distilled water with no added salt, and immersed in 50 mLdistilled water at 10 �C. The concentration of gellan released intothe surrounding liquid was quantified by phenol-sulphuric acidassay.

In one series of experiments (Hossain & Nishinari, 2009) releaseof Kþ ions was also monitored (by ICP). The gellan concentrationused was 2 wt %; the polymer content of the gel (volume 1.18 mL)was therefore 23.6 mg, which for Kþ gellan (formula weight pertetrasaccharide repeat unit ¼ 684 D) equates to 34.4 mmol.Measurements were made after immersion of the gel for 1, 2, 4, 6and 8 h. At the end of this period, the concentration of Kþ cations inthe surrounding solution (volume 50 mL) was 8.19 ppm, whichcorresponds to release of 10.5 mmol Kþ. The accompanyingconcentration of gellan in the solution was 0.0146 wt %, whichcorresponds to release of 10.7 mmol of tetrasaccharide units, inclose agreement with the value of 10.5 mmol for Kþ (as would beexpected from the requirement to preserve electrical neutrality inboth the gel and the surrounding liquid). Studies by size-exclusionchromatography (Hossain & Nishinari, 2009) showed that thedistribution of molecular mass for the released chains was dis-placed to somewhat lower values than in the correspondingchromatograph for gellan retained in the gel network. However,there was little difference in the DSC cooling traces recorded(Tanaka & Nishinari, 2007) for the two populations, demonstratingthat the chains released from the gellan network were capable offorming double helices.

Fig. 27. Rheological, conformational and thermal changes observed (Nitta et al., 2001)for Kþ gellan gels (sample KGG-1, Table 1; 1.6 wt % in water) on cooling, after heatingfrom 5 to 60 �C. (a) Storage and loss moduli E0 (:) and E00 (6) and loss tangent, tan d

(B) from longitudinal oscillatory deformation, measured after equilibration for 15 minat each temperature. (b) Specific ellipticity (202 nm) from circular dichroism (A) andDSC exotherm (solid line with no symbols), both recorded at a cooling rate of0.5 �C/min.


After immersion for 8 h, the volume of the gel had increased byw19% and w31% (100 � 10.7/34.4) of the polymer had beenreleased into the surrounding solution. Movement of water into thegel and of polymer into the solution is analogous to the processesthat occur during dissolution of solid particles, with equilibriumbeing reached only when the polymer is distributed homoge-neously throughout the solvent. Rapid release of gellan (over 10% ofthe total polymer content of the gel) occurred within the first 1 h ofimmersion, with slower, progressive release at longer times.According to classic theory of polymer gelation (Flory,1953) there isa residual “sol fraction” of polymer chains and/or crosslinkedclusters of chains that do not form part of the continuous network,evenwhen the extent of intermolecular association is much greaterthan at the critical gel point (Section 3.2). It seems likely that theinitial rapid increase in the concentration of gellan in the solutionsurrounding the gel comes predominantly from release of this solfraction, and the subsequent slower increase from progressivedissociation (dissolution) of the continuous network.

Comparative studies (Hossain & Nishinari, 2009) using gelsamples with the same height (1.5 cm) but smaller diameters (0.5or 0.3 cm) also showed initial rapid release of gellan and

progressive slower release on longer immersion, although, sincethe volumes and surface areas of the gels were different, theabsolute concentrations of gellan in the surrounding solutionwere,of course, also different.

Measurements of E0 throughout the 8 h immersion periodshowed an initial increase in modulus over about the first 3 h ofimmersion, and a steep decrease afterw5 h. These were attributed(Hossain & Nishinari, 2009) to, respectively, swelling and erosion ofthe gel. Swelling of chemically-crosslinked (“entropic”) networksleads to a decrease in moduli (Flory, 1953) in response to thedecrease in polymer concentration. For gellan gels, which arecrosslinked (Section 5.1) by extended regions of aggregated doublehelices, the observed increase in modulus may arise from enthalpicresistance to distortion of the polymer network during swelling.

Similar initial increase in modulus was observed (Hossain &Nishinari, 2009) when the gellan concentration was raised from2.0 wt % to 2.5 and 3.0 wt %, but the subsequent decrease at longertimes was much smaller, indicating that the gel networks weremore stable and resistant to dissociation. As found (Section 5.3) byNitta et al. (2006) and confirmed by Hossain and Nishinari (2009),immersion of gellan gels in salt solutions gives a progressiveincrease in modulus towards constant values, abolishing thedownturn observed on immersion in water. It seems likely, there-fore, that increase in stability of gellan networks with increasingconcentration comes, at least in part, from the accompanyingincrease (Section 4) in the concentration of counterions to thepolymer chains.

5.5. Texture of gellan gels

The technique of texture profile analysis (TPA), developedinitially by General Foods in the 1960s, has been used extensivelyby Kelco to characterise gellan gels and to compare their texturalproperties with those obtained using other gelling agents (Gibson &Sanderson, 1997; Sanderson, 1990; Sanderson, Bell, Clark et al.,1988; Sanderson & Clark, 1984; Sanderson & Ortega, 1994; Sworn,2009). It involves subjecting a free-standing gel to two consecu-tive cycles of compression. In the specific procedure used by Kelco(Sanderson, 1990) the sample is compressed to 30% of its initialheight at 2 inches per minute (w0.85 mm/s), the crosshead of theinstrument is then raised, and a second cycle of compression ismade under the same conditions as the first.

Three parameters are obtained from the first compression:modulus, hardness and brittleness. The modulus is identical toYoung’s modulus (E), although usually expressed in different units(pounds force per square inch). Hardness is defined as themaximum force generated in resistance to the first compression,and is usually equivalent to break stress (sb), although againexpressed in different units. For some samples, however, maximumresistance may occur during further compression after initialfailure. The brittleness value is identical to strain at break ( 3b),whichmeans, confusingly, that very brittle materials (i.e. those thatbreak at low strain) have very low “brittleness” values, and veryelastic (non-brittle) materials have high “brittleness”.

Two further parameters can be obtained from the second cycleof compression: elasticity and cohesiveness. Elasticity, whichcharacterises the ability of the sample to recover from compression,is derived from the point at which the descending crossheadtouches the top of the sample and is defined as the height of thesample at this point expressed as a percentage of the initial heightprior to compression. Cohesiveness, which is intended to indicatetoughness during eating, is measured as the area under theforceedistance curve obtained in the second cycle of compressionexpressed as a percentage of the corresponding area from the firstcompression.


Early studies by this technique (Sanderson & Clark, 1984)showed that commercial gellan gum gave gels strong enough to becharacterised by compression testing at polymer concentrations aslow as 0.2 wt %, and that hardness values increased initially andthen decreased on progressive reduction in pH or progressiveaddition of Naþ, Kþ, Mg2þ or Ca2þ, as confirmed (Section 3.7) inlater studies under better-defined ionic conditions. It was alsofound that brittleness values decreased systematically with cationconcentration (i.e. that the gels became more brittle as more saltwas added).

Subsequently it was demonstrated (Sanderson, Bell, Clark et al.,1988) that the modulus, hardness, brittleness, elasticity and cohe-siveness of agar in traditional Japanese food products could bematched well using gellan at much (2e3 times) lower concentra-tions, although agar itself is a very efficient gelling agent. In thesame study it was found that on progressive increase in concen-tration of gellan at fixed concentration of added Ca2þ there wasa progressive reduction in brittleness values (i.e. with the gelsbecomingmore brittle as the polymer concentrationwas increased)and a progressive, large, increase in elasticity, as well as the ex-pected increase in modulus and hardness.

TPA has been particularly useful in comparing gellan with othergelling agents. These can give a wide spectrum of different textures(Sworn, 2009) ranging from firm and brittle for agar and kappacarrageenan to soft and flexible for gelatin and the “synergistic”gels (Morris, E.R., 1995b) formed by mixtures of xanthanwith plantpolysaccharides such as locust bean gum (LBG) or konjac gluco-mannan, with alginate and pectin giving gels of intermediatetexture. The gels formed by commercial deacylated gellan (gellangum) typically have brittleness values of around 25e30%, placingthem at the “firm and brittle” extreme of this continuum.However, as discussed further in Section 7.2, high acyl gellangives gels that lie at the opposite extreme (“soft and flexible”).

5.6. Mobility of water in gellan networks

In an investigation of the dielectric properties of solutions andgels of Naþ gellan (NaGG-2, Table 1) by the time domain reflec-tometry method, Mashimo, Shinyashiki, and Matsumura (1996)observed two relaxation processes, one at high frequency(w10 GHz) and the other at much lower frequency (w3 MHz). Therelaxation time for the high-frequency process agreed well withthat of pure water, and the magnitude of this process demonstratedthat, even in the gel state at low temperature, most of thewater hadthe same mobility as bulk water. The conclusion that most of thewater in hydrated biopolymer networks is present as freewater hasalso been reached for other gelling biopolymers and, indeed, forhigh-moisture food products (Ablett & Lillford, 1991; Ablett,Lillford, Baghdadi, & Derbyshire, 1978; Davies et al., 2010). Theprocess at lower frequency was attributed to bound water. Oncooling through the temperature-range of the disordereordertransition, there was a large (w8-fold) increase in the relaxationstrength of this process, indicating that water molecules bind moretightly to the ordered structure of gellan in the gel state than to thedisordered coils in solution. The same conclusion has been reportedfor agarose (Watase, Nishinari, & Hatakeyama, 1988) and also forKGG-1 (Hatakeyama, Quinn, & Hatakeyama, 1996).

The mobility of water in gellan networks was also investigatedby Ohtsuka and Watanabe (1996) using the pulsed field gradientstimulated echo NMR method. Samples were prepared by coolinghot solutions of 2 wt % Naþ gellan (NaGG-2, Table 1) incorporatinga wide range of concentrations of either KCl or CaCl2. Experimentalvalues of the diffusion coefficient of water were analysed bya simple model in which the polymer is regarded as a series ofpermeable barriers running parallel to one another and separated

by a fixed distance, a. One immediate conclusion was that thediffusional mobility of water in the channels between the barrierswas essentially the same as that of bulk water, consistent with thedielectric studies (Mashimo et al., 1996) summarised above.

On progressive increase in Kþ concentration, the separation (a)of the hypothetical barriers increased sharply, passed througha shallow maximum, decreased sharply, and then levelled outtowards a constant value; permeability of the barriers (p) showedconverse changes over the same range of Kþ concentration (i.e.decreasing initially, passing through a shallow minimum,increasing again, then becoming essentially constant). The initialincrease in a, with accompanying decrease in p, was attributed toconversion from a homogeneous solution to a network structurewith larger void spaces and decreased permeability, and began at[Kþ]z 20 mM. As shown in Fig. 13, thermal hysteresis of Kþ gellan,attributed to aggregation of double helices, also became detectable(Milas & Rinaudo, 1996) at 20 mM Kþ.

The steep decrease in a, with accompanying increase in p,occurred at [Kþ]z 90mM, which coincides with the point at whichthe force required to fracture Kþ gellan gels (Fig. 19) was found todecrease (Milas & Rinaudo, 1996). The corresponding changes ina and p for the gels formedwith CaCl2 occurred at [Ca2þ]z 3.3mM,which again agrees well with the salt concentration (Fig. 19) atwhichMilas and Rinaudo (1996) observed reduction in gel strength(force at break) on addition of MgCl2 to Naþ gellan (bearing in mindthat, as discussed in Section 3.6, Ca2þ and Mg2þ are essentiallyindistinguishable in their interaction with gellan). Thus, althoughthe physical reality of the simple model used by Ohtsuka andWatanabe (1996) may be debatable, it seems clear that thechanges in restricted diffusion of water derived by NMR correlateconvincingly with changes in the macromolecular organisation ofgellan observed by other techniques.

Kþ concentrations around the shallow maximum in a andshallow minimum in p (from w50 to w80 mM) gave transparent,elastic gels. Only this region of high a and low p, with littlechange in either parameter, and the subsequent steep decrease ina and increase in p, were observed for gels formed with CaCl2,which is consistent with the observation (Fig. 18) of immediategelation at very low concentrations of Ca2þ, without the precedingregions of solution and “weak gel” response (Fig. 12) that occur onprogressive addition of monovalent cations.

Finally, Ohtsuka and Watanabe (1996) found that the plots ofa and p versus concentration of Ca2þ superimposed closely on thecorresponding plots for Kþ when moved horizontally along theconcentration axis, indicating that the networks formed by gellanin the presence of monovalent or divalent cations have the sameoverall structure, and the same effect on mobility of water, andchange in the same way when cation concentration is varied,although the concentration-range over which the changes occur ismuch lower for divalent cations than for monovalent (as can also beseen for the changes in rheology shown in Fig. 19).

5.7. Syneresis

Gellan gels are comparatively stable when stored quiescently atambient temperature or under refrigeration, but there may besome release of fluid (syneresis), particularly at polymer concen-trations below w0.2 wt % (Gibson & Sanderson, 1997). Syneresis isundesirable in most food products, and it may be necessary to addthickeners to prevent it.

Syneresis can also cause problems of slippage when gellannetworks are investigated by rheological measurements undershear. This was demonstrated (Morris, E.R. et al., 1999) in aninvestigation of the effect of NaCl on gelation of Naþ gellan.Measurements of G0 and G00 were made using either smooth (cone-


and-plate) geometry, or a novel perforated-cylinder arrangementin which the sample surrounds and penetrates both the movingand stationary elements, making slippage impossible (Richardson &Goycoolea, 1994). At low concentrations of added salt, the cone-and-plate geometry gave plots of G0 and G00 versus temperaturesimilar to those shown in Fig. 21. At higher salt concentrations,where stiffer gels are formed, there was again an initial sharpincrease in G0 and G00 on cooling, but G0 then decreased slightly, andboth moduli fluctuated erratically. On switching to the perforated-cylinder geometry, however, these unusual features were no longerobserved, demonstrating that they were artefacts caused by slip-page of the sample on the smooth surfaces of the cone and plate.

Release of fluid when gellan gels are squeezed can be observedvisually (Gibson & Sanderson, 1997), and was demonstrateddramatically in an investigation by Nakamura, Shinoda, and Tokita(2001). The gellan used was KGG-1 (Table 1); cylindrical gels(11.5 mm diameter; 10 mm height) were prepared at a polymerconcentration of 1.33 wt % in water, with no added salt, and werecompressed at rates ranging from 1000 to 0.005 mm/min. Threedifferent patterns of response were observed over different rangesof compression rate:w1 mm/min and higher; w0.02 mm/min andlower; and intermediate rates. Fig. 28 shows photographs of thegels taken before, during and immediately after compression atrepresentative rates within the three ranges (100, 0.1 and0.005 mm/min), and after the compressed samples had beensoaked in water for two days.

On rapid compression (100 mm/min) the gels fractured cleanly,as represented schematically in region a of Fig. 19, but returnedalmost fully to their original height when applied stress wasremoved. On reduction of compression rate to 0.1 mm/min, thepattern became similar to those shown in regions b and c in Fig. 19:vertical cracks 3e5 mm in height formed at the surface of thecylindrical sample and water flowed from them. There was stillsubstantial recovery towards the initial height of the sample whenthe crosshead of the instrument was raised, but less than occurredafter more rapid compression (as would be expected from loss offluid at the slower compression rate). At the slowest rate ofcompression studied (0.005 mm/min) no fracture was observed.

Fig. 28. Compression of Kþ gellan gels (sample KGG-1, Table 1; 1.33 wt % in water at 22.5 � 1the gels (1) before compression (height 10 mm; diameter 11.5 mm), (2) during compressiooriginal position, and (4) after soaking in water for 2 days. Fluid released from the gels ducompressed gel in 3(c) can be seen as a thin sheet at the bottom of the photograph.

Instead, there was continuous, progressive expulsion of water fromthe gel until it had been compressed to a flat disc 1 mm thick (i.e.10% of the original height), which showed little recovery whenstress was removed. There was no change in diameter duringcompression, so the apparent value of Poisson’s ratio is zero!

These observations indicate that gellan gels respond tocompression by two different mechanisms: (i) elastic deformationup to the point of fracture, and (ii) rearrangement of the polymernetwork to accommodate the imposed strain, with consequentexpulsion of fluid. As the rate of compression is decreased, allowingmore time for rearrangement to occur, the second of these becomesdominant. Time-dependent reduction in the resistance of gellangels to imposed deformation has been observed directly bymeasurements of stress relaxation (Morris, V.J. & Brownsey, 1995).

When the compressed gels (Fig. 28) were soaked in water fortwo days they returned almost completely to their original size.Gellan networks also show substantial recovery (Sanderson, Bell,Clark et al., 1988) after disruption by shear, with both phenomenademonstrating their dynamic nature.

5.8. Flavour release

It is well known in the food industry that perceived intensities offlavour and taste are lower for gelled products than for fluid prod-ucts (such as sauces and drinks) incorporating the same objectiveconcentrations of flavour compounds or “tastants” such as sugar orsalt. Gellan causes less suppression than most other gelling agents,or, as more commonly expressed, it has outstanding “flavourrelease” properties. One possible explanation (Gibson & Sanderson,1997), suggestedby release offluidwhengellan gels are compressed(Section 5.7), is that water is released from the gels during masti-cation, carryingwith it flavour and taste compounds. An alternative,though possibly related, interpretation was suggested (Gothard,1994; Morris, E.R., 1994, 1995a) from sensory and rheologicalcomparisons of gellan with other gelling polysaccharides spanninga wide range of different textures (Section 5.5).

The materials studied were xanthan in combination with LBG(giving flexible, elastic gels), commercial gellan gum (giving stiff,

�C) at (a) 100, (b) 0.1 or (c) 0.005 mm/min (Nakamura et al., 2001). Photographs shown, (3) after release of applied stress by raising the crosshead of the instrument to itsring compression was removed before the photographs in column 3 were taken; the


brittle gels) and two others (calcium alginate and the potassiumsalt form of kappa carrageenan) that gave gels of intermediatetexture. Polysaccharide concentrations were varied across the fullrange at which measurable gels could be obtained and fixedconcentrations of sucrose and flavouring were included in allsamples. The gels were rated for perceived firmness, sweetness andintensity of flavour by a sensory panel, and characterised bycompression testing to give Young’s modulus (E), breaking stress(sb) and breaking strain ( 3b). As found in earlier studies of thickenedsystems (Baines & Morris, 1989), sweetness and flavour were sup-pressed to the same extent. The flavour/taste intensities forsamples of equivalent perceived firmness differed widely betweenthe different gel systems, with highest intensities for gellan andlowest for xanthaneLBG.

No correlation was found between panel scores for sweetnessand flavour and instrumental measurements of modulus and breakstress. However, when flavour/taste intensity was plotted double-logarithmically against breaking strain a good linear relationshipwas observed, with progressive reduction in perceived intensity asthe deformation required to break the network increased, and nosystematic discrepancies between the results obtained for thedifferent gelling agents. It was therefore concluded that flavourrelease is determined by the extent of deformation required tofracture the gel and create new surfaces from which release canoccur. The same conclusion was reached in a later investigation(Bayarri, Rivas, Izquierdo, & Costell, 2007) in which gellan wascompared with only one other gelling agent (kappa carrageenan)but two different sweeteners were studied and panellists assessedchanges in perceived sweetness over time. There is no necessaryconflict between this interpretation and the proposal (Gibson &Sanderson, 1997) that flavour release comes from expulsion offluid under compression, since release of water could providea mechanism for transport of flavour/taste compounds across thefresh surfaces created by fragmentation of the gel network.

5.9. Gellan liquid crystals

In a recent investigation by Nitta, Takahashi, and Nishinari(2010), Naþ gellan was partially depolymerised by alkaline hydro-lysis (10e45 mM NaOH; 12 h at 50 �C). Three samples, denoted asG-10, G-15 and G-30 in order of increasing molecular weight (i.e.decreasing concentration of NaOH used in their preparation), wereselected for investigation. When a concentrated (8 wt %) solution ofthe sample of lowest molecular weight (G-10) was held quiescentlyin a vial at low temperature, it gradually resolved into two layers.Examination by polarised light microscopy showed that the upperlayer was an anisotropic liquid crystalline phase. The concentrationat which birefringence, diagnostic of anisotropy, became detectabledecreased with increasing molecular weight, as has also beenobserved for xanthan (Inatomi, Jinbo, Sato, & Teramoto, 1992; Lee &Brant, 2002) and for kappa carrageenan in solutions with sodiumiodide (Borgström, Egermayer, Sparrman, Quist, & Piculell, 1998).

When solutions of G-10 were cooled through the temperature-range of the disordereorder transition, G00 increased steeply, asexpected, with an accompanying increase in G0. At concentrationsabove w2.5 wt %, however, the initial increase was followed bya large decrease on further cooling. Similar behaviour was observedfor G-15 and G-30, but reduction in moduli occurred at progres-sively lower concentration as molecular weight increased, beingclearly evident at 2 wt % G-30. The highest moduli recordedwere atleast an order of magnitude lower than those required to trigger theslippage artefacts described in Section 5.7, strongly indicating thatthe observed reductions were caused by formation of an aniso-tropic phase, rather than by slippage. Similar changes in modulihave been reported for other polysaccharides known to form

anisotropic liquid crystals, including xanthan (Lee & Brant, 2002;Maret, Milas, & Rinaudo, 1981; Milas & Rinaudo, 1983), schizo-phyllan (Fang, Takemasa, Katsuta, & Nishinari, 2004) and methyl-cellulose (Yin, Nishinari, Zhang, & Funami, 2006). Like thesesystems, the solutions studied by Nitta et al. (2010) were all non-gelling, which is an essential requirement for formation of liquidcrystals.

6. Effect of sugars

The ability of high concentrations of sucrose, or other sugars, toinduce gelation of high-methoxy pectin under acidic conditions inproduction of jams and jellies is well documented (e.g. Rolin, 1993).Incorporation of sugars has also been shown to enhance thestrength and thermal stability of networks formed by gellingbiopolymers such as kappa carrageenan (Nishinari, Watase,Williams, & Phillips, 1990), starch (Katsuta, Nishimura, & Miura,1992), oxidised starch (Evageliou, Richardson, & Morris, 2000),agarose and gelatin (Nishinari et al., 1992).

The effect of sugars on solutions of Naþ gellan was studied ininvestigations by Miyoshi, Takaya, and Nishinari (1998) andMiyoshi and Nishinari (1999b). In both of these the concentration ofgellan was held fixed at 1 wt %, and sugar concentrations werevaried within the range 0e72 wt %. The experimental proceduresused were the same as those described in Section 4, where gellanconcentrationwas varied in the absence of sugar. Concentrations ofsugar were expressed as molarities. Since the molecular weight ofmonosaccharides such as glucose and fructose is 180, a 1M solutioncorresponds to 18.0 wt %; for disaccharides such as sucrose(molecular weight ¼ 342), 1 M ¼ 34.2 wt %.

In the first of these investigations (Miyoshi et al., 1998) thegellan used was NaGG-2 (Table 1) and the sugars studied wereglucose and mannose. When 1 wt % NaGG-2 was cooled in theabsence of sugar, there was a sharp increase in G00 at Tch z 30 �C,with an accompanying exotherm in DSC (Fig. 25); G0 was too low tobe measured, but became detectable on incorporation of a lowconcentration of glucose (0.01 M ¼ 0.18 wt %). Addition of 1.6 M(28.8 wt %) glucose to 1 wt % NaGG-2 gave cooling and heatingcurves of G0 and G00 virtually identical to those observed for 2 wt %NaGG-2 in water, with increase in Tch tow37 �C and G0 rising aboveG00 at Tsg z 7 �C. On further increase in glucose concentration, Tchcontinued to increase, with an accompanying steeper increase inTsg, as seen (Fig. 23) when gellan concentration was varied in theabsence of sugar, until conformational ordering and gelationoccurred simultaneously at Tch ¼ Tsg ¼ 41 �C when the concentra-tion of glucose had reached 3.5 M (63 wt %). Thermal hysteresisbetween cooling and heating scans became apparent at a glucoseconcentration of 1.8 M (32.4 wt %) and increased in magnitude asthe concentration of glucose was raised further.

Qualitatively similar changes in Tch and Tsg were observed withmannose as cosolute, but the sugar concentrations required weresystematically higher than for glucose, and no thermal hysteresiswas observed even at the highest concentration of mannosestudied (63 wt %). Two central conclusions from this investigationare therefore (i) incorporation of sugars promotes conformationalordering and gelation of gellan, and (ii) glucose is more effectivethan mannose.

In the second investigation (Miyoshi & Nishinari, 1999b) thegellan used was NaGG-3 (Table 1) and the sugars studied wereglucose, fructose, sucrose and trehalose. The changes in Tch withincreasing concentration of glucosewere virtually identical to thoseseen (Miyoshi et al., 1998) for NaGG-2, but the accompanyingincrease in Tsg was less steep: at 3.3 M (63 wt %) glucose, whereconformational ordering and gelation of NaGG-2 occurred simul-taneously, gelation of NaGG-3 (crossover of G0 and G00) was not

Fig. 29. Stressestrain curves from compression testing of gellan gels: (a) 1.2 wt %gellan with no added sucrose; (b) 0.8 wt % gellan with 60% sucrose. Symbols showexperimental data; the solid lines are fits calculated using a modified reel-chain model.Stretch ratio, l ¼ d/do, where d is the diameter of the sample during compression anddo is the initial diameter prior to compression (Kawai et al., 2008).


observed until w9 �C below Tch, and even at the highest concen-tration of glucose studied (4M¼ 72 wt %) Tsg was still slightly lowerthan Tch. The most likely explanation for the earlier gelation ofNaGG-2 is that it arises from the higher content of divalent cationsin NaGG-2 than in NaGG-3 (Table 1).

DSC scans showed a systematic increase in peak-maximumtemperatures as concentration of sugars was increased, with nothermal hysteresis between cooling and heating. The disaccharidesstudied gave greater increases than the monosaccharides. Theincreases were slightly greater with sucrose thanwith trehalose, andmuch greater with glucose than with fructose. Taken together withthe comparison between glucose and mannose by Miyoshi et al.(1998), the order of effectiveness in promoting ordered associationof gellan is: sucrose > trehalose > glucose > mannose >> fructose.

One obvious way in which high concentrations of sugars, orother cosolutes, can promote association of polymer chains is byreplacing most of the solvent. Miyoshi and Nishinari (1999b)explored this effect by expressing concentrations of gellan rela-tive to the mass of residual water, rather than total mass, andmaking comparisons of Tch and Tsg with those observed for thesame concentrations of gellan in water (with no sugars present).Much of the increase in transition temperatures with increasingconcentration of sugar could be explained in this way, but thevalues of Tch and Tsg for mixtures of gellan with the disaccharides(sucrose and trehalose) were still substantially higher than forequivalent concentrations of gellan alone, those for mixtures withglucose were slightly higher, and at high concentrations of fructosethey were lower.

The enhancements observed with sucrose, trehalose andglucose were ascribed to sugarewater associations in competitionwith interactions between water and gellan, and the order ofeffectiveness was correlated with “dynamic hydration number”,which is determined by the number of equatorial hydroxyl groupsin the sugar (Katsuta et al., 1992; Nishinari & Watase, 1992;Nishinari et al., 1990). It was suggested that the inhibitory effect offructose might be due to attachment of fructose molecules to gellanby hydrogen bonding, with consequent inhibition of self-association of the polymer. Direct attachment of fructose to poly-mer chains has also been proposed for mixtures with high-methoxy pectin (Tsoga, Richardson, & Morris, 2004b), on thebasis of an intense exothermic process on heating that was notobserved for mixtures with other sugars or polyols (Tsoga,Richardson, & Morris, 2004a).

Other investigations have focussed on the effect of sugars oncommercial gellan (Kelcogel) at concentrations where gels areformed in the absence of sugar. Bayarri, Costell, and Durán (2002)used compression testing to study the effect of sucrose at concen-trations in the range 0e25 wt % on three gelling concentrations ofKelcogel: 0.30, 0.75 and 1.2 wt %. In all cases there was a systematicincrease in Young’s modulus (E), break stress (sb), and strain atbreak ( 3b) with increasing concentration of sucrose (i.e. with thegels becoming stronger and less brittle). At the lowest concentra-tion of gellan studied (0.3 wt %) the changes were very small, butthey became clearly evident at the higher concentrations. For themaximum concentrations of gellan and sucrose studied (1.2 and25 wt %, respectively), E was w38% higher than in the absence ofsugar, sb wasw72% higher and 3b increased from the very low valueof w17% strain at break to w23%.

Small increases in gel strength with accompanying smallincreases in breaking strain continue (Sworn & Kasapis,1998) as theconcentration of sucrose is raised to 40 wt %, where the strain atbreak is w32%. On further increase in sucrose concentration to60 wt %, however, there is a massive increase in 3b, with the gelsremaining intact up to w65% strain. Fig. 29 shows illustrativecompression curves for gellanwith no added sugar (Fig. 29a) and in

the presence of 60 wt % sucrose (Fig. 29b). The extent ofcompression is characterised by the accompanying increase in thewidth of the sample (Section 3.1). The curves were fitted (Kawai,Nitta, & Nishinari, 2008) by a “reelechain” model proposed previ-ously to explain the temperature-dependence of the elasticmodulus of thermoreversible gels (Nishinari et al., 1985). The fitsare so precise that the calculated curves, shown as solid lines inFig. 29, are almost entirely obscured by the experimental points(shown as symbols). The reduction in brittleness on incorporationof 60 wt % sucrose (Fig. 29b) is evident from the much greaterdeformation at break (stretch ratio z 1.7) in comparison with thesample (Fig. 29a) with no added sugar (stretch ratio z 1.16). Theconcentration of gellan (0.8 wt %) in the mixture with sucrose issomewhat lower than in the sample without sucrose (1.2 wt %) butthe break stress is substantially higher (w32 kPa in comparisonwith w7 kPa), demonstrating the effectiveness of sucrose in rein-forcing gellan networks.

Partial replacement of sucrose by either glucose or fructose ata total sugar concentration of 60 wt % gives stronger gels than at60 wt % sucrose alone (Gibson & Sanderson, 1997). This wasdetected in an experiment intended to test for possible degradationof gellan (0.5 wt %) when held (2 h) at 85 �C under acidic conditions(pH 3.4) in the presence of 60 wt % sucrose. The gel formed oncooling was found to be stronger than before exposure to hightemperature and low pH. This unexpected behaviour was traced topartial hydrolysis (inversion) of sucrose to glucose and fructose. Itwas also found (Gibson & Sanderson, 1997) that values of modulusat break (i.e. sb/ 3b) were higher for gellan gels incorporating fruc-tose than for those incorporating equivalent concentrations ofglucose. The apparent order of effectiveness of sucrose and itshydrolysis products in strengthening gellan gels is therefore


fructose > glucose > sucrose, which is the reverse of the orderfound by Miyoshi and Nishinari (1999b).

It seems likely that the difference arises from the divalentcations that are present in much larger amounts in normalcommercial gellan gum than in the NaGG-2 and NaGG-3 samplesstudied by, respectively, Miyoshi et al. (1998) and Miyoshi andNishinari (1999b). Sworn and Kasapis (1998) explored the effectof small additions of CaCl2 on the modulus (E) of gels formed by0.5 wt % Kelcogel in the absence of sugar and in the presence ofsucrose, glucose or fructose at concentrations of 20, 40 or 60 wt %.As discussed in Section 3.7 and illustrated in Fig. 19, the modulishowed an initial increase and subsequent decreasewith increasingconcentration of salt. The maximum modulus was displaced toprogressively lower concentration of Ca2þ as the concentration ofsugar was increased. The Ca2þ concentration at which the modulibegan to fall were lowest for the gels incorporating sucrose andhighest for those incorporating fructose.

The apparent clash of experimental evidence mentioned abovecould perhaps, therefore, be explained as follows. Sugars promoteassociation of gellan in the order sucrose> glucose> fructose.Whenthe extent of association in the absence of sugar is well below thatrequired for optimum gelation, as in the Naþ gellan systems studiedby Miyoshi and coworkers, gel strength increases in the same order.However, commercial gellan contains sufficient divalent cations togive gels that are already at, or close to, the optimum degree ofcrosslinking, so that further association in response to the presenceof sugar can cause excessive aggregation (Section 3.7) and thusweaken the network rather than strengthening it. Gel strength in thepresence of different sugars would then decrease with increasingextent of further association, giving the reverse order of effective-ness: fructose > glucose > sucrose, as observed.

Mixtures of commercial gellan with low or moderate concen-trations (up tow40 wt %) of sucrose or other cosolutes give coolingand heating curves similar in form to those observed for NaGG-2 atconcentrations above w3.2 wt % in the absence of cosolute: G0 andG00 are extremely low in the solutions state at high temperature butincrease sharply at the coilehelix transition temperature (Tch) oncooling, and show substantial thermal hysteresis on heating.Entirely different behaviour, however, has been reported (Kasapiset al., 2002; Papageorgiou, Kasapis, & Richardson, 1994) formixtures of Kelcogel (0.5 wt %) with very high concentrations(80e85 wt %) of cosolute. Experimentally, these concentrationswere attained by using 50 wt % sucrose and adding the requiredfurther amount of cosolute as corn syrup of dextrose equivalent(DE) 42, which corresponds to a number-average degree of poly-merisation of w2.4.

At high temperature (90 �C) these mixtures gave mechanicalspectra similar to those of polysaccharide gels (Fig. 10a). On coolingto 5 �C there was no indication of the sharp increase in moduliobserved in the absence of cosolute, and no detectable thermalhysteresis on heating. Instead, there was a smooth, progressiveincrease in moduli during cooling, with G00 rising above G0. Suchbehaviour is typical (Haward & Young, 1997; Sworn & Kasapis,1999) of a material that undergoes vitrification, transformingfrom a rubbery solid to a high-viscosity liquid (glass). In mechanicalspectra recorded on completion of cooling to 5 �C (Papageorgiou,Kasapis et al., 1994), G00 remained higher than G0 at all values offrequency (u) studied (0.01e10 Hz). At the upper end of thisfrequency range, log G00 versus log u had the slope of þ1 charac-teristic (Section 3.1) of a liquid, with log G0 versus log u runningroughly parallel. At lower frequencies, however, the slope of bothplots decreased and G0 rose towards G00, indicating survival ofa crosslinked network within the vitrified sample.

Crystalline materials do not undergo vitrification. For vitrifica-tion to occur, the sample must be largely amorphous. This led to the

proposal (Sworn & Kasapis, 1999) that the networks formed bygellan in the presence of high concentrations of cosolute consistpredominantly of flexible, disordered chains, with only occasionaljunctions between them. As discussed in Section 5.1, the resistanceto deformation of such networks would be predominantly entropicin origin, contrasting with the networks of aggregated strands(Fig. 26b) formed in the absence of cosolute, where resistancewould be dominated by enthalpy.

Changes in G0 and G00 during cooling suggest that both types ofnetwork are formed at cosolute concentrations between w40 andw80 wt % (Whittaker, Al-Ruqaie, Kasapis, & Richardson, 1997).Throughout this range, G0 is already higher than G00 at 95 �C,demonstrating the presence of the entropic network. On initialcooling, both moduli show the smooth, progressive increaseobserved at higher concentrations of cosolute and attributed tovitrification. On reaching the coilehelix transition temperature,however, the moduli show the steep, sigmoidal increase observedin the absence of cosolute, demonstrating formation of an enthalpicnetwork. As the concentration of cosolute is increased, themagnitude of the sigmoidal transition decreases (Sworn & Kasapis,1999), until eventually, at w80 wt % cosolute, it becomes unde-tectable, leaving only the smooth increases from vitrification of theentropic network.

In anattempt to rationalise the effect of awide range of sugars andpolyols on gelation of high-methoxy pectin, Tsoga et al. (2004a,b)proposed that the effect of replacement of water by cosolute mightbe partially offset by cosolute molecules clustering around the poly-mer chains and thus hampering association of chains into a gelnetwork (i.e. by enthalpically-favourable polymerecosolute interac-tions competing with polymerepolymer interactions). Althoughspeculative, this proposal is consistent with the observed behaviourof gellan in the presence of sugars and other cosolutes such as cornsyrup, as summarised below.

(i) Inhibition of formation and aggregation of gellan double helicesby “condensation” of cosolute molecules around the polymerchains would explain the progressive loss of the enthalpicnetwork as concentration of cosolute is increased, and theaccompanying large decrease in transition enthalpy (DH)observed by DSC (Al-Marhoobi & Kasapis, 2005; Kasapis, 2006).

(ii) Similarly, it is consistent with only very limited residualassociation of gellan chains in the surviving entropic networksat very high concentrations of cosolute.

(iii) In the presence of soluble solids at concentrations abovew15 wt % commercial gellan gives “crystal clear gels”(Sanderson, 1993), without the slight haze that can sometimesbe observed in the absence of cosolute. Increased clarity withincreasing concentration of cosolute (0e35 wt % sucrose orfructose) has been demonstrated objectively (Tang, Mao, Tung,& Swanson, 2001) by instrumental measurements of turbidity,and can again be explained by decreased aggregation frompolymerecosolute interactions competing with interactionsbetween the gellan double helices.

Finally, Nickerson, Paulson, and Speers (2004) carried out aninvestigation under experimental conditions essentially identical tothose used by Papageorgiou, Kasapis et al. (1994) and obtainedbroadly similar results. They proposed, however, that gellan in thepresence of high concentrations of cosolute forms “gel particles” or“gel islands” embedded within the cosolute matrix, rather thana continuous network. This interpretation was strongly challengedin a spirited response by Kasapis (2006). One of the most directlines of rebuttal was that TEMmicrographs (Kasapis et al., 2002) forgellan (0.7 wt %) in the presence of 80 wt % cosolute showed nofeatures that could be attributed to gel particles, although, as


mentioned in Section 5.2, extended fibrillar strands were success-fully visualised in the same investigation for 0.7 wt % gellan in thepresence of 5 mM CaCl2 with no cosolute.

7. Effect of acyl substituents

7.1. Acyl groups in native gellan

The helical structure of gellan with attached acyl groups (Fig. 1)was first explored by using computer modelling to extrapolate fromthe known solid-state geometry (Fig. 2) of the deacylated polymer(Chandrasekaran & Thailambal, 1990). The main conclusion wasthat acetyl substituents would lie on the periphery of the duplex(Fig. 2b), with no modification of the underlying helix geometry,but that the glyceryl group would lie in the interior of the helix andforce the carboxylate group of the adjacent glucuronate residue toswing round through an angle of w30� about the C(5)eC(6) bond,with consequent major changes in the pattern of hydrogen bondingwithin and between the participating strands. When diffractionpatterns of the quality needed for detailed analysis were subse-quently obtained (Chandrasekaran, Radha, & Thailambal, 1992) itwas found that the actual rotation of the carboxylate group is lessthan predicted (w14�), but that the required separation from theglyceryl substituent is achieved by the glucuronate residue itselfalso undergoing a significant rotation (17� 3�) about the glycosidicbonds linking it to the two adjacent glucose residues (Fig. 1).

In normal commercial production of gellan gum, acyl groups areremoved by brief exposure to alkali (KOH) at high temperature(w95 �C). The reaction can be expressed as:

ReCOOeR0 þ KOH/ReCOOKþHOR0

where ReCOO and R0 denote, respectively, the substituent and thepolymer chain. Partial removal can be achieved by limiting theamount of alkali used. When hydrolysis is carried out in this way, atelevated temperature with the polymer in the disordered form,glyceryl substituents are liberated somewhat more rapidly thanacetyl groups. An alternative procedure is to hydrolyse for muchlonger times at lower temperature, where the polymer is con-formationally ordered. Under these conditions, release of acetylgroups from the periphery of the double helix occurs far morerapidly than removal of glyceryl substituents embedded within thehelix. Thus by appropriate manipulation of temperature, alkaliconcentration, and hydrolysis time (Baird, Talashek, & Chang,1992),it is possible to obtain samples differing widely in the proportion ofrepeat units carrying residual acetyl or glyceryl substituents(denoted here as, respectively, fa and fg). To take account of possibleunintended loss of acyl groups, it is more correct to refer to gellanprepared without deliberate deacylation as “high acyl” rather than“native”.

In the investigation where the presence of L-glyceryl substitu-ents in high acyl gellan was first detected (Kuo et al., 1986) it wasreported that every repeat unit carried a glyceryl group, but thatonly half the repeating units were acetylated (i.e. fg ¼ 1.0; fa ¼ 0.5).Other investigators have found different values. Baird et al. (1992)reported mass-fractions of 10e12% glycerate and 4% acetate,which correspond approximately to fg ¼ 0.72e0.87 and fa z 0.52.In an investigation of mutant strains cultured in their laboratory,Jay et al. (1998) reported fg ¼ 0.49 and fa ¼ 0.60 for gellan fromnormal (“wild type”) bacteria, and Mazen et al. (1999) reportedfg ¼ 0.80 and fa ¼ 0.80 for high acyl gellan that was also producedand purified in their own laboratory.

A possible explanation for the variability is that the polymer isexpressed initially with stoichiometric content of both substitu-ents, but that some loss of acyl groups occurs during extraction and

post-fermentation processing (and, perhaps, in the culture brothduring continued fermentation), which seems more likely thaninitial biosynthesis with varying contents of acyl substituents.Consistent with this interpretation, Jay et al. (1998) detectedsubstantial amounts of free glycerate in preparations with unusu-ally low values of fg.

7.2. High acyl gellan

Comparison (Sanderson, Bell, Clark et al., 1988) by TPA (Section5.5) has shown that high acyl gellan gives gels with lower modulusand lower hardness than those formed by commercial gellan gum,but the elasticity is much greater, and the “brittleness” values(strain at break) are also much greater (typically around 65%, incomparison with w30%). In contrast to the ease with which gellangum gels release fluid (Section 5.7), the gels formed by high acylgellan show no syneresis (Sworn, 2009). As described below, thehigh acyl and deacylated forms also differ substantially in theirresponse to changes in temperature and ionic environment.

As shown in Fig. 30a, the conformational transitions (monitoredby optical rotation) that accompany formation and melting of highacyl gellan gels (1.0 wt %) show no detectable thermal hysteresis,either in water or in 100 mM NaCl (Morris et al., 1996), and thereare only small variations (Fig. 30b) in melting temperature of thegels and in transition midpoint temperature from DSC heatingscans (Mazen et al., 1999) on varying concentration of NaClbetween 10 and 100 mM, in contrast to the massive changes inmelting temperature observed (Fig. 12) for deacylated gellan overthe same range of NaCl concentrations.

The comparative insensitivity of high acyl gellan to changes inionic environment was also observed by Huang, Singh, Tang, andSwanson (2004) who studied the effect of monovalent cations(10e200 mM Naþ or Kþ) and divalent cations (2e80 mM Mg2þ orCa2þ) on the temperature of the solegel transition (Tsg). There wasno significant difference between Naþ and Kþ, or between Mg2þ

and Ca2þ, but the divalent cations gave somewhat higher transitiontemperatures than the same normalities of the monovalent ions.Both showed good linearity (Fig. 9) when plotted according to Eq.(3), as observed (Milas & Rinaudo, 1996) for deacylated gellan, butthe plots for the high acyl form were less steep and the transitionsoccurred at much higher temperature than for the deacylatedpolymer.

Higher transition temperatures are also evident in the DSCcooling scans (Morris et al., 1996) shown in Fig. 31a for high acylgellan (Naþ salt form; 1.0 wt % in water) in comparison withdeacylated gellan under the same conditions. The other noticeabledifference is that the DSC peak is substantially wider for the highacyl form, indicating that the transition is less co-operative. Heatingcurves for the same samples, recorded in the same investigation butreported separately (Baird et al., 1992), were essentially equal andopposite to the cooling curves in Fig. 31a (i.e. with again no indi-cation of thermal hysteresis).

7.3. Blends of high acyl and deacylated gellan

As discussed further in Section 8, mixtures of high acyl anddeacylated gellan give gels with textures that lie between theextreme brittleness of the deacylated form and the extremeextensibility of the high acyl form (Morrison et al., 1999). Oncooling from high temperature the mixtures show two regions ofsteep increase in G0 (Kasapis et al., 1999), the first coincident withthe solegel transition of high acyl gellan at high temperature andthe second with the corresponding transition of the deacylatedpolymer at much lower temperature.

-680

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Fig. 30. (a) Variation of optical rotation (436 nm) with temperature (Morris et al.,1996) for high acyl gellan (1.0 wt %) on heating (filled symbols) and cooling (opensymbols) in deionised water (circles) and in 100 mM NaCl (triangles); (b) effect of salt(NaCl) on the midpoint temperature of the orderedisorder transition (B), as charac-terised by DSC heating scans at 0.3 �C/min, and on the gelesol transition temperature(C), taken as the crossover of G0 and G00 (1 Hz; 10% strain) on heating, for 1.0 wt % highacyl gellan in the Naþ salt form (Mazen et al., 1999).

Fig. 31. (a) DSC scans for high acyl and deacylated gellan (1.0 wt% in deionised water)on cooling at 0.1 �C/min. Both samples were in the Naþ salt form. (b) DSC cooling scans(0.7 �C/min) for gellan samples devoid of acetyl substituents, but with L-glycerylgroups present in the following percentages of stoichiometric substitution: A: 61.5; B:43.4; C: 19.4; D: 8.3; E: 3.8. The minor exotherms observed at low temperature forsamples A and B are identified by the corresponding lower-case letters (Morris et al.,1996).


Two separate transitions are also observed by DSC (Kasapiset al., 1999; Morris et al., 1996), with peaks in the same positionsas those shown in Fig. 31a for the individual constituents, and bythe temperature-course of reduction in intensity of detectable 1HNMR resonances on cooling and accompanying changes in circulardichroism spectra (Matsukawa & Watanabe, 2007). It seems clear,therefore, that high acyl and deacylated gellan do not form doublehelices incorporating strands of both types.

7.4. Partially deacylated gellan

In an investigation by Noda et al. (2008) four samples of gellanwith different degrees of deacylation were studied by oscillatoryrheological measurements and AFM. The content of glyceryl andacetyl groups in these samples is shown in Table 4, expressed bothon a weight basis and as the fraction of repeat units carryingsubstituents (i.e. as fg and fa). Solutions (1.0 wt %) of these materialsin water, with no added salt, showed a sharp, sigmoidal increase inG0 on cooling, with no thermal hysteresis on heating. The temper-ature at the onset of the rise in G0 decreased progressively withdecreasing content of acyl substituents, towards the value ofTch z 26.5 �C observed (Table 2) for the same concentration (1.0 wt

%) of fully deacylated gellan. AFM micrographs for dilute aqueoussolutions (1 g/L) dried on mica showed fibrillar strands, but nocontinuous network was observed for any of the four samples.

On incorporation of 100 mM KCl, there was again a sharp,sigmoidal increase in G0 during cooling, which moved to progres-sively lower temperatures as acyl content decreased (Fig. 32), butreduction in modulus on heating moved in the opposite direction,giving progressively greater thermal hysteresis, which wasaccompanied by progressive development of continuous networkstructure in the micrographs from AFM. Thus the ability of fullydeacylated gellan to form fibrillar networks by cation-mediatedassociation of double helices is progressively restored as acylgroups are removed from the native polymer.

7.5. Individual roles of glyceryl and acetyl groups

Fig. 33 shows changes in G0 observed (Morris et al., 1996) oncooling and heating for two gellan samples with, respectively,fg ¼ 0.43, fa ¼ 0.03, and fg ¼ 0.52, fa ¼ 0.50. Although both havea similar, substantial, content of residual glycerate, the first sample,which is virtually devoid of acetate, shows pronounced thermalhysteresis (Fig. 33a), but no hysteresis is detectable (Fig. 33b) for thesecond sample, which has a high content of acetate. It wouldtherefore appear that the presence of acetyl groups on theperiphery of the double helix is the dominant factor in blockinghelixehelix aggregation. However, the temperature-course ofgelation is closely similar for both samples, indicating that acetategroups have little influence on the formation of individual double

Table 4Content of acyl groups in gellan samples studied (Fig. 32) by Noda et al. (2008).

GG1 GG2 GG4 GG6

Glyceryl (wt %) 10.0 9.1 7.2 5.6Acetyl (wt %) 3.0 2.8 2.7 2.5fg 0.72 0.65 0.50 0.38fa 0.39 0.36 0.34 0.31

Data are presented as means of duplicate determinations. All samples were in theNaþ salt form, with negligible content of other cations.


helices, and that the enhanced stability of the helix structure inhigh acyl gellan is due predominantly, or solely, to glycerate.

The effect of glyceryl substituents on the thermal stability ofthe gellan double helix was explored in greater detail (Morriset al., 1996) by using DSC to monitor conformational orderingof five gellan samples (identified as A, B, C, D and E) which wereprepared by Kelco from the same fermentation broth byprogressively longer exposure to alkali (25 mM KOH) at 40 �C,where the polymer is in the ordered form. The shortest hydro-lysis time used (4 h) was sufficient to give almost completeremoval of acetyl groups from the periphery of the helix. Theglycerate level, however, decreased systematically over time,giving fg values of 0.615, 0.434, 0.194, 0.083 and 0.038 forsamples A, B, C, D and E, respectively.

Fig. 32. Temperature-dependence of G0 (bold lines; left-hand axis) and tan d (faint lines; rig1.0 wt % in 0.1 M KCl on cooling from 90 �C to 20 �C at 0.2 �C/min, and re-heating to 90 �C aframes (Noda et al., 2008).

Fig. 31b shows the DSC cooling scans recorded for these mate-rials. At glycerate levels abovew40% stoichiometric (samples A andB) the traces show a large exotherm at high temperature, widelyseparated from a much smaller peak centred close to 25 �C. Onfurther reduction of glycerate content to below w20% stoichio-metric (samples C and D) the two peaks overlap, but with obviousincrease in the relative contribution of the second transition tooverall thermal change. Finally, at low glycerate content (<4%stoichiometric; sample E) the peaks merge, but the position andwidth of the exotherm suggest that it is still a composite of two(heavily overlapping) transitions.

As shown in Fig. 31b, the first, major, transition moves toprogressively lower temperature with decreasing content ofglycerate, whereas the temperature of the second, smaller, tran-sition remains essentially constant. The interpretation proposedby Morris et al. (1996) was that the first process corresponds toformation of the ‘high acyl’ structure, with progressive reductionin stability as the proportion of missing glyceryl groups increases,and that the second comes from ordering of chain sequencestotally devoid of glycerate. If the fraction of tetrasaccharide unitslacking glyceryl substituents (1�fg) is denoted as f, then theprobability of finding n such units adjacent to one another in thepolymer chain is f n. This was compared (Morris et al., 1996) withthe relative contribution of the second transition to overallthermal change to give an approximate value of the minimum

ht-hand axis) for Naþ gellan samples (a) GG1, (b) GG2, (c) GG4 and (d) GG6 (Table 4) att the same rate; the direction of temperature change is indicated by arrows within the

0

1

10

100

0 20 40 60 80 100

Temperature (°C)

G' (

Pa)

a

0

1

10

100

0 20 40 60 80 100

Temperature (°C)

G' (

Pa)

b

Fig. 33. Variation of G0 (10 rad/s; 2% strain) on cooling (B) and heating (:) at1 �C/min for partially deacylated gellan preparations (0.5 wt % in deionised water) withacyl contents (% stoichiometric) of (a) 40% glyceryl, 3% acetyl and (b) 52% glyceryl, 50%acetyl (Morris et al., 1996).


sequence length required for adoption of the fully-deacylatedhelix structure.

The two DSC peaks (Fig. 31b) for sample C (fg ¼ 0.194; f ¼ 0.806)are particularly well resolved, and could be quantified with goodprecision. The lower temperature transition contributes w26% ofthe total heat change (i.e. f n z 0.26), which is in close agreementwith the calculated value for n ¼ 6 (0.8066 ¼ 0.27). A consecutiverun of six glycerate-free repeating units on each of the participatingstrands would correspond to two full turns of the deacylateddouble-helix structure (Fig. 2), which seems a physically realisticlength for stable association.

In summary, L-glyceryl groups in high acyl gellan increase thestability of the double helix by forming additional hydrogenbonds within and between the participating strands, but destroythe binding site for metal cations by changing the orientation ofthe adjacent carboxyl group (Fig. 2b). The consequent loss ofcation-mediated aggregation (Sections 3.4 and 3.6) reduces gelstrength and eliminates thermal hysteresis. In mixtures of highacyl and deacylated gellan, each forms its own type of doublehelix, with no intermediate structures involving both acylatedand non-acylated chains. Sequences of six or more repeat unitsdevoid of glycerate terminate the ’high acyl’ structure, but aresufficient for stable association in the deacylated arrangement.Shorter runs of glycerate-free units can be accommodated in the‘high acyl’ structure, but reduce its stability. Acetyl substituents

remain conformationally mobile on the periphery of the doublehelix (Fig. 2b), introducing a steric and entropic barrier toaggregation.

8. Mixtures and applications

In many practical applications, gelling agents are present inmixtures with other biopolymers, present as natural constituents offood materials such as fruit, vegetables, meat, fish and eggs, oradded as industrial hydrocolloids to modify texture, createcomposite structures, or control syneresis. Applications of gellanand its behaviour in combination with other biopolymers aretherefore discussed together in this section.

Interactions between two different polymers can be classified as“associative” if they are thermodynamically more favourable thaninteractions between the individual polymers of each type and“segregative” if they are less favourable. The most commonmechanism of association is electrostatic attraction between poly-anions, such as negatively-charged polysaccharides, and poly-cations, such as proteins below their isoelectric point. Lesscommonly, associative junctions can be formed between polymerswith the same chain geometry, as is believed to occur in mixtures ofalginate and high-methoxy pectin at low pH, and in “synergistic”gelation of xanthan with konjac glucomannan (KGM) or galacto-mannans such as LBG which, like xanthan, have backbones con-sisting of (1 / 4)-diequatorially-linked residues. Segregativeinteractions can cause separation into a dispersed phase containingmost of one polymer surrounded by a continuous phase containingmost of the second polymer. The two phases may, however, runthrough one another, giving a bicontinuous structure like a jelly setin the pores of a sponge. Alternatively, the two polymers mayremain intimately mixed (without being confined to only part ofthe total volumes as in phase-separated bicontinuous networks)and form two separate gels permeating through one another, togive an “interpenetrating network” (IPN) structure (Morris, E.R.,1990; Morris, V.J., 1986).

Mixtures of Naþ gellan (NaGG-2, Table 1) with KGM (MW950 kD)at a total polymer concentration of 0.8 wt % in water were observed(Miyoshi, Takaya, Williams, & Nishinari, 1996, 1997; Nishinari,Miyoshi, Takaya, & Williams, 1996) to give a sharp maximum in G0

at a mixing ratio of 3:5 (i.e. 0.3 wt % gellan plus 0.5 wt % KGM) aftercooling to low temperature (0 �C). Mechanical spectra recordedunder these conditions had the form typical (Fig. 10a) of a gelnetwork, although the individual constituents of themixture showedonly solution-like response. On addition of monovalent cations (Naþ

or Kþ) G0 and G00 of themixtures increased and the solegel transitionmoved to higher temperatures. Low concentrations of divalentcations (Mg2þ or Ca2þ) also promoted gelation of the gellaneKGMmixtures, but higher concentrations decreased the moduli, as found(Fig. 19) for gellan alone. The proposed interpretation was that KGMenhances the strength and thermal stability of gellan networks bybinding to the surface of aggregated assemblies of double helices. Asimilar interpretation has been proposed for mixtures of KGM (orgalactomannans) with agarose, furcellaran or kappa carrageenan (asreviewed by, for example, Morris, E.R., 1990, 1995b).

Dried films cast from mixtures of commercial gellan (Kelcogel)with KGM (MW 1320 kD) were studied by Xu, Li, Kennedy, Xie, andHuang (2007). Maximum tensile strength was observed when theKGM content of the films was around 70%, which is in reasonableagreement with themixing ratio of 3:5 gellan:KGM found (Miyoshi,Takaya, Williams et al., 1996; Miyoshi et al., 1997; Nishinari,Miyoshi et al., 1996) to give maximum gel strength. Wide-angleX-ray diffraction analysis of the dried films (Xu et al., 2007) indi-cated that the crystalline packing found for gellan or KGM alonewas inhibited by hydrogen bonding between the two polymers, and


Fourier-transform infrared (FT-IR) spectra suggested that thebonding was predominantly between the carboxyl groups of gellanand the acetyl and hydroxyl groups of KGM. It was suggested (Xuet al., 2007) that gellaneKGM films incorporating an anti-microbial agent (nisin) could be used in “active packaging” offood products to extend their shelf-life.

Tamarind xyloglucan has the same (1 / 4)-diequatorial back-bone geometry as KGM, but is solubilised by monosaccharide anddisaccharide sidechains rather than by acetyl substituents.Mixtures of this material with Naþ gellan were examined (Nittaet al., 2003; Nitta & Nishinari, 2005) at a total polymer concentra-tion of 1.0 wt %, where the individual polysaccharides are non-gelling. The mixtures converted from solutions to gels on cooling.The temperature at the critical gel point (Section 3.2), where tan d isindependent of frequency (u), increased with increasing content ofxyloglucan, from 21 �C at 0.6 wt % gellan plus 0.4 wt % xyloglucan to25 �C at 0.3 wt % gellan plus 0.7 wt % xyloglucan. Values of thecommon slope of log G0 and log G00 versus log uwithin this range ofcompositions varied between 0.31 and 0.42, closely similar to thevalues of 0.32e0.43 observed (Hossain et al., 1997) for iotacarrageenan.

The solegel transition of the gellanexyloglucan mixtures wasaccompanied by an exothermic transition in DSC, at highertemperature than the exotherm from the coilehelix transition ofgellan alone. As the concentration of xyloglucan in the mixtureswas increased, the magnitude of the higher-temperature transitionalso increased, until ultimately it became the only thermal processdetectable by DSC. The same progressive replacement of theconformational transition of gellan alone by a new transition athigher temperature was also seen in DSC heating scans. Circulardichroism spectra showed an increase in ellipticity at 202 nm onheating through the temperature-range of the first transition inDSC, as observed (Fig. 4) for gellan alone, but on further heatingthrough the range of the second transition the ellipticity decreased,indicating dissociation of a different type of intermolecularstructure.

It would appear, therefore, that both KGM and tamarind xylo-glucan associate with gellan to give “coupled networks” (Morris,V.J., 1986) in which the participating polysaccharides are directlyattached to one another. In the mixtures with KGM, gellan retainsits double-helix structure, whereas in the mixtures with tamarindxyloglucan self-association of gellan through double helicesappears to be replaced by a new type of association between gellanand xyloglucan chains.

Direct association between gellan and gelatin by electrostaticattraction (“complex coacervation”) was explored by Chilvers andMorris (1987), using commercial gellan (Gelrite). The gelatin wasobtained from pig skin by extraction with acid, giving an isoionicpoint of pH 8. Mixtures were prepared at a temperature (60 �C) wellabove the solegel transition temperatures of the individual poly-mers. Coacervate droplets (1e100 mmdiameter) were formed at pHvalues in the range 3.5e5.0, where the gelatin had a net positivecharge, and the gellan a negative charge. The droplets gelled oncooling.

The potential use of gellanegelatin coacervates to encapsulateoils or solid particles was demonstrated by addingmodel substrates(paraffin oil, sunflower oil, aluminium powder or beads of ionexchange resin) to the biopolymer mixtures in the solution state,reducing pH into the range required for coacervation, and cooling togel the coacervate layer that had formed around the core material.The resulting beads were then fixed with glutaraldehyde, washed,and dehydrated with isopropanol to yield concentrated slurries orfree-flowing powders.

Mixtures of Kþ gellanwith kappa carrageenan, also in the Kþ saltform, were found (Nishinari, Watase, Rinaudo, & Milas, 1996) to

give two separate transitions in DSC, one at about the sametemperature as for carrageenan alone and the other at highertemperature, about the same as for gellan alone, which arguesagainst any association between the two polymers. However,values of jh�j obtained for solutions prepared at a total polymerconcentration of 0.25 wt % with varying proportions of gellan andkappa carrageenan were substantially higher than those of theindividual polymers, reaching a maximum at w2:8 gellan:carra-geenan. This was attributed (Nishinari, Watase et al., 1996) to phaseseparation, with consequent increase in the effective concentrationof each polymer when confined to only part of the total volume. Athigher total concentrations of polymer (2e5 wt %), where gels wereformed, E0 dropped below the values observed for the individualpolysaccharides, reaching minimum values at w1:1 gellan:carra-geenan. This can again be explained by phase separation, with theweaker network forming the continuous phase and the stronger gelconfined to dispersed particles.

Phase separation has also been reported (Papageorgiou, Gothardet al., 1994) for mixed gels of commercial gellan (Kelcogel) withcalcium alginate. Incorporation of moderate concentrations ofgellan (0.1e0.3 wt %) increased the strength (break stress) of thegels formed by 2.0 wt % alginate, but the breaking strain wasunaffected, indicating that the brittle gellan network was distrib-uted as dispersed particles embedded in a more elastic calciumalginate matrix. At higher concentrations of gellan (above w0.7 wt%), however, the rheological response to addition of Mg2þ cationssuggested a bicontinuous (phase-separated) network structure,although the possibility of phase inversion to a continuous gellannetwork surrounding dispersed particles of calcium alginate gelcould not be eliminated conclusively.

Mixtures of Kþ gellan with agarose (Nishinari, Takaya, & Watase,1994) showed separate DSC transitions for the two polysaccharides,as was also seen (Nishinari, Watase et al., 1996) for kappacarrageenegellan mixtures, and a phase-separated network wasagain proposed. A subsequent study by Amici, Clark, Normand, andJohnson (2000), however, found no evidence of the large increasein turbidity expected to occur on phase separation, or of biphasicstructures in images obtained by confocal microscopy or TEM.Instead, the TEM micrographs showed a homogeneous distributionof both polymers, and rheological analysis was entirely consistentwith interpenetrating networks of gellan and agarose.

Initial formation of an interpenetrating network structure wasalso proposed (Clark, Eyre, & Ferdinando, & Lagarrique, 1999) formixtures of commercial gellan (Kelcogel) with gelling potato mal-todextrin (Paselli SA2), although interpretationwas complicated bysubsequent self-association of the maltodextrin component intolarge aggregates.

Mixtures of high acyl and deacylated gellan show a smoothprogression of modulus and brittleness values from one extreme tothe other as the relative proportions of the two components isvaried from high acyl alone to deacylated alone (Morrison et al.,1999). There is no indication of the discontinuity in propertiesthat would be expected from phase separation, which againstrongly indicates an interpenetrating network structure.

Mixtures of Naþ gellan (NaGG-3, Table 1) with sodium andcalcium salts of hyaluronic acid were studied by Mo, Kubota, &Nishinari (2000). Incorporation of Naþ hyaluronate raised thevalues of both G0 andG00 observed (Fig. 21a) for 0.5 wt % NaGG-3, buthad little effect on the changes that occurred on cooling throughthe temperature-range of the disordereorder transition, and themixtures did not gel. Gelation was, however, observed for mixturesof NaGG-3 (0.1e0.3 wt %) with Ca2þ hyaluronate. At pH 7, there wasa slow, progressive increase in G0 and G00 when the mixtures wereheld for 2 h at 25 �C, and mechanical spectra recorded at the end ofthis holding period had the form typical (Fig. 10a) of true gels. On

Table 5Typical food applications of gellan.

Major food area Typical products

Confectionery Starch jellies, pectin jellies, fillings, marshmallowJams and jellies Reduced calorie jams, imitation jams, bakery

fillings, jelliesFabricated foods Fabricated fruits, vegetables, meatsWater-based gels Dessert gels, aspicsPie fillings and puddings Instant desserts, canned puddings, pre-cooked

puddings, pie fillingsIcings and frostings Bakery icings, canned frostingsDairy products Ice cream, gelled milk, yogurt, milkshakes,

low-fat spreads, dipsBeverages Fruit, milk-based, soy and carbonated drinksFilms/coatings Batters, breadings, coatings, adhesion systems


reduction in pH to 2.5, the moduli increased more rapidly, reachingconstant values withinw30 min, and the resulting gels were much(w20 times) stronger. They were also more transparent than thoseformed at pH 7, although gellan alone gives turbid gels at low pH(Section 3.7). Gelation of mixtures of Naþ gellan with Ca2þ hya-luronate can, of course, be explained, at least in part, by interactionbetween the gellan component and Ca2þ cations from the hyalur-onate, but substantial further research would be required tounravel the complexity of other possible macromolecular and ionicinteractions in this system.

Mixtures of commercial gellan (Gelrite) with a wide range ofstarches (native and chemically-modified) were studied bySanderson, Bell, Burgum, Clark, and Ortega (1988), using a standardBrabender Amylograph procedure in which samples are heatedfrom 50 to 95 �C over 30min, held at 95 �C for 30min, and cooled to50 �C over a further 30 min period, with measurement of viscositythroughout. Gellan (0.5 wt %) had little effect on the maximumviscosity generated by 4.5 wt % starch during heating, in contrast toother hydrocolloids such as xanthan and carboxymethylcellulosewhich can give undesirably high values of peak viscosity. The starchpastes formed in the Amylograph were cooled overnight, and thensheared. The viscosities of the pastes containing gellan werereduced significantly by shearing, but remained higher than thoseof the corresponding pastes of starch alone, suggesting that gellancould be useful in preparation of a variety of starch-based fillings,which are generally prepared by cooking, stored in bulk, shearedduring filling into their final containers, and allowed to recoverviscosity on standing.

Most gelling agents had well-established areas of applicationlong before there was any proper molecular understanding of theirmechanisms of gelation. Fundamental research on gellan, bycontrast, has proceeded alongside development of practical appli-cations (Sanderson, 1990; Sanderson & Clark, 1984).

The first applications of commercial gellan (Gelrite) were ingrowth media for microbial cultures, as a replacement for agar(Harris, 1985; Lin & Cassida, 1984; Morris, V.J., 1995). Gellansubstrates have the advantages of clarity, purity, and the ability towithstand prolonged incubation at high temperature. Gellan wasalso identified (Shimomura & Kamada, 1986) as a promisingsubstitute for agar in plant tissue culture, because of the absence ofimpurities found in agar, the lower polysaccharide concentrationsneeded, and the greater clarity of the gels, allowing clearer obser-vation of the development of roots and tissue.

Typical food applications of gellan are summarised in Table 5,which is taken directly from the CPKelco booklet available on-lineat the web address shown at the end of Section 1. Examples givenbeloware from the same source, unless other references are cited. Alist of potential food applications broadly similar to that in Table 5was published by Sanderson and Clark (1984) before gellan wascleared for use in food (Section 1).

Preparation of gellan solutions at factory scale is more difficultthan in the laboratory. The following concise description of theproblem and the way in which it can be addressed is quotedverbatim from Sanderson (1993). “In low solid systems, calciumions need to be removed to allow gellan gum to hydrate. This iseasily done using a sequestrant such as sodium citrate. Unlike lowlevels of calcium, low levels of sodium ions from the sequestrantdo not inhibit hydration. When gelation subsequently takes place,usually brought about by cooling, it is sometimes but not alwaysnecessary to add back ions to obtain optimal gelation. For acidicproducts such as jams, jellies and confectionary, acidification ofthe hot solution liberates calcium that is bound to the citrate toboost gel strength. Simply stated, the essence of Kelcogel gellangum technology is calcium ion control through the use ofsequestrants”.

The same difficulty of hydration/dissolution does not, however,occur in production of confectionary, where high concentrationsof sugar and/or other soluble solids are present and gellan formsonly a sparingly-crosslinked network (Section 6). Gellan can beused as a replacement for gelatin in marshmallows and sweets(candies) such as “Gummi bears”, giving products acceptable tovegetarians and followers of religions that forbid materials frompigs or cattle. Another advantage is heat-stability. Marshmallowsprepared from gellan and starch do not melt when added to hotcocoa or when baked in cake mixes (Sanderson, 1993). Sweetsstructured by gellan, alone or in combination with other gellingagents such as xanthan/LBG, do not have the undesirable tendencyto stick to one another when exposed to high ambient tempera-ture, as often happens when gelatin is used. Sworn (2009) hasreported detailed formulations for jelly sweets incorporatinggellan alone or with partially-hydrolysed (“thin boiling”) starch,and for gellan-based low-calorie jam, dessert jelly, fruit juice jelly,and fruit preparations suitable for use in yogurt or in bakedproducts.

Gellan can also be used, in complete or partial replacement ofgelatin, to improve the characteristics of savoury gels or aspics inmeat, fish or vegetable products, and to raise the setting-temperature of gelatin desserts, removing the need for refrigera-tion. Incorporation of gellan in starch-based puddings gives addi-tional structure and stability, while still retaining the characteristic“thick and pasty” consistency. Gellan can also be used in productionof low-fat spreads, to replace oil or fat with structured (gelled)water.

As mentioned in Section 5.5, Sanderson, Bell, Clark et al. (1988)found that gellan could match the texture of much (2e3 times)higher concentrations of agar in some traditional Japanese foods.The products evaluated weremitsumame jelly cubes, hard red beanjelly, soft red bean jelly, and tokoroten noodles, and the concen-trations of gellan requiredwere, respectively, 0.4, 0.5, 0.1 and 0.4 wt%. Gellan can also be used to replace agar in decorative icings,frostings, and glazes for baked goods.

Procedures for preparing fluid “weak gel” dispersions of orderedgellan (Section 3.3) have been described by Oomoto, Uno, & Asai(1999) and by Sworn (2009), who also reported fluid-gel formula-tions for beverages prepared with and without incorporation offruit juice. Low concentrations (w0.12 wt %) of ordered gellan arealso effective in stabilising soy milk and soy beverages.

Novel beverages can be obtained by incorporating beads ofgellan gel. Beads can be prepared easily (Section 5.3) by drippinga solution of gellan, with appropriate flavouring and colour, intoa solution containing Ca2þ. A similar procedure can be used to formcarbonated beads, by dispersing calcium carbonate in a gellansolution and dripping the dispersion into a solution of citric acid, orinto fruit juice. The consequent reduction in pH triggers gelation of


the gellan, and converts calcium carbonate into carbon dioxide,which is trappedwithin the gelled beads, and released on storage ina stoppered bottle or other sealed container to give a carbonateddrink. Gellan beads can also be used as carriers of savoury ingre-dients such as soy sauce, garlic juice or onion juice in, for example,salad dressing, instant gravy or soup mixes.

Gellan dissolves in hot milk (above w80 �C), without seques-trant, and the network that forms on cooling can suspend cocoaparticles in chocolate milk drinks, and stabilise products such asmilkshakes, ice cream, sour cream and yogurt. Low concentrationsof gellan (w0.05 wt %) can extend the shelf-life of acid milk drinks,probably by electrostatic association of acid-casein fragments to thegellan “weak gel” network, restricting sedimentation of the casein(Kiani, Mousavi, Razavi, & Morris, 2010).

Gellan can also be used to form a protective film around mate-rials such as chicken, fish, cheese, potatoes and other vegetables,and dough-coated products such as egg rolls, to reduce absorptionof oil during frying. Breaded products can be prepared in a similarway, by dipping into, or sprayingwith, a solution of gellan, followedby breading, then freezing or partial baking. Subsequent heating ina conventional or microwave oven gives a product similar to thoseobtained by frying, but with lower fat content. Surface coating withgellan has also been suggested (Nussinovitch & Hershko, 1996) asaway of extending the shelf-life of vegetables, including specificallygarlic.

A related application of gellan is as an “adhesion system” forsurface coating of products such as crackers (biscuits), cookies(buns), potato chips (crisps), corn chips, pretzels and rice cakes. Adilute solution of gellan is sprayed, as a finemist, onto the surface ofthe product. Spices, flavouring or sweeteners can be incorporatedin the solution, or applied to the surface of the product immediatelyafter spraying. The adhesion system can also be used to attach othermaterials, such as fruit pieces, herbs, or dairy-based powders.

Gellan has recently (January 2011) been listed as an allowedingredient for use in organic foods and beverages by the USDANational Organic Program (http://www.foodingredientsfirst.com/news/KELCOGEL-Gellan-Gum-Approved-for-Use-in-Organic-Foods-and-Beverages.html), which should add to its existing use by thefood industry.

Gellan also has a wide range of established or potential non-food uses, in addition to the applications in microbiology andtissue culture mentioned above. The “weak gel” properties of gel-lan, coupled with clarity and preservation of network structure athigh temperature, can be exploited in creams and lotions, includingsuntan lotions and sunscreens, hair-care products such as sham-poos and conditioners, toothpaste, and gelled air-fresheners. Gellancan also be used in paper, to increase its strength and improve itsperformance in ink-jet printing, and in pharmaceutical formula-tions for sustained release of drugs (Alhaique et al., 1996; Kubo,Miyazaki, & Attwood, 2003).

It has also been suggested (Norton et al., 2011) that acid-inducedgelation of gellan at gastric pH could induce satiety by structuringthe contents of the stomach, and thus be useful in combatingobesity.

In view of its short history relative to that of other gelling agents,it seems likely that many further advances will be made beforegellan comes close to realising its full potential. These willundoubtedly include identification of new areas of application, andformulation of novel or improved products. An additionalapproach, however, might be informed manipulation of acylcontent, by variant cultures or controlled deacylation (Section 7.1),to tailor the properties of gellan to the requirements of specificapplications. Although the feasibility of this approach has alreadybeen demonstrated (Sections 7.4 and 7.5), it has not yet beenexploited on a commercial scale.

References

Ablett, S., & Lillford, P. J. (1991). Water in foods. Chemistry in Britain, 27, 1024e1026.Ablett, S., Lillford, P. J., Baghdadi, S. M., & Derbyshire, W. (1978). Nuclear magnetic

resonance investigations of polysaccharide films, sols, and gels: I. Agarose.Journal of Colloid and Interface Science, 67, 355e377.

Al-Marhoobi, I. M., & Kasapis, S. (2005). Further evidence of the changing nature ofbiopolymer networks in the presence of sugar. Carbohydrate Research, 340,771e774.

Alhaique, F., Santucci, E., Carafa, M., Coviello, T., Murtas, E., & Riccieri, F. M. (1996).Gellan in sustained release formulations: preparation of gel capsules andrelease studies. Biomaterials, 17, 1981e1986.

Amici, E., Clark, A. H., Normand, V., & Johnson, N. B. (2000). Interpenetratingnetwork formation in gellaneagarose gel composites. Biomacromolecules, 1,721e729.

Annaka, M., Honda, J.-I., Nakahira, T., Seki, H., & Tokita, M. (1999). Multinuclear NMRstudy on the solegel transition of gellan gum. Progress in Colloid and PolymerScience, 114, 25e30.

Arnott, S., Scott, W. E., Rees, D. A., & McNab, C. G. A. (1974). i-Carrageenan:molecular structure and packing of polysaccharide double helices in orientedfibres of divalent cation salts. Journal of Molecular Biology, 90, 253e267.

Baines, Z. V., & Morris, E. R. (1989). Suppression of perceived flavour and taste byfood hydrocolloids. In R. D. Bee, P. Richmond, & J. Mingins (Eds.), Food colloids(pp. 184e192). Cambridge, U.K.: Royal Society of Chemistry, RSC Special Publi-cation No. 75.

Baird, J. K., Talashek, T. A., & Chang, H. (1992). Gellan gum: effect of composition on gelproperties. In G. O. Phillips, P. A. Williams, & D. J. Wedlock (Eds.), Gums andstabilisers for the food industry 6, (pp. 479e487). Oxford, UK: IRL Press.

Bayarri, S., Costell, E., & Durán, L. (2002). Influence of low sucrose concentrations onthe compression resistance of gellan gum gels. Food Hydrocolloids, 16, 593e597.

Bayarri, S., Rivas, I., Izquierdo, L., & Costell, E. (2007). Influence of texture on thetemporal perception of sweetness of gelled systems. Food Research Interna-tional, 40, 900e908.

Bohdanecký, M., & Kovár, J. (1982). Viscosity of polymer solutions. Amsterdam:Elsevier.

Borgström, J., Egermayer, M., Sparrman, T., Quist, P. O., & Piculell, L. (1998). Liquidcrystallinity versus gelation of k-carrageenan in mixed salts: effects of molec-ular weight, salt composition, and ionic strength. Langmuir, 14, 4935e4944.

Brownsey, G. J., Chilvers, G. R., I’Anson, K., & Morris, V. J. (1984). Some observations(or problems) on the characterisation of gellan gum solutions. InternationalJournal of Biological Macromolecules, 6, 211e214.

Caggioni, M., Spicer, P. T., Blair, D. L., Lindberg, S. E., & Weitz, D. A. (2007). Rheologyand microrheology of a microstructured fluid: the gellan gum case. Journal ofRheology, 51, 851e865.

Chambon, F., & Winter, H. H. (1985). Stopping of crosslinking reaction in a PDMSpolymer at the gel point. Polymer Bulletin, 13, 499e503.

Chandrasekaran, R., Millane, R. P., Arnott, S., & Atkins, E. D. T. (1988). The crystalstructure of gellan. Carbohydrate Research, 175, 1e15.

Chandrasekaran, R., Puigjaner, L. C., Joyce, K. L., & Arnott, S. (1988). Cation inter-actions in gellan: an X-ray study of the potassium salt. Carbohydrate Research,181, 23e40.

Chandrasekaran, R., Radha, A., & Thailambal, V. G. (1992). Roles of potassium ions,acetyl and L-glyceryl groups in native gellan double helix: an X-ray study.Carbohydrate Research, 224, 1e17.

Chandrasekaran, R., & Thailambal, V. G. (1990). The influence of calcium ions,acetate and L-glycerate groups on the gellan double-helix. Carbohydrate Poly-mers, 12, 431e442.

Chilvers, G. R., & Morris, V. J. (1987). Coacervation of gelatinegellan gum mixturesand their use in microencapsulation. Carbohydrate Polymers, 7, 111e120.

Clark, A. H., Eyre, S. C. E., Ferdinando, D. P., & Lagarrique, S. (1999). Interpenetratingnetwork formation in gellanemaltodextrin gel composites. Macromolecules, 32,7897e7906.

Clark, A. H., & Ross-Murphy, S. B. (1985). The concentration dependence ofbiopolymer gel modulus. British Polymer Journal, 17, 164e168.

Cox, W. P., & Merz, E. H. (1958). Correlation of dynamic and steady-flow viscosities.Journal of Polymer Science, 28, 619e622.

Crescenzi, V., Dentini, M., & Dea, I. C. M. (1987). The influence of side-chains on thedilute-solution properties of three structurally related bacterial anionic poly-saccharides. Carbohydrate Research, 160, 283e302.

Dai, L., Liu, X., Liu, Y., & Tong, Z. (2008). Concentration dependence of criticalexponents for gelation in gellan gum aqueous solutions upon cooling. EuropeanPolymer Journal, 44, 4012e4019.

Davies, E., Huang, Y., Harper, J. B., Hook, J. M., Thomas, D. S., Burger, I. K., et al. (2010).Dynamics of water in agar gels. International Journal of Food Science and Tech-nology, 42, 2502e2507.

Dentini, M., Coviello, T., Burchard, W., & Crescenzi, V. (1988). Solution properties ofexocellular microbial polysaccharides. 3. Light scattering from gellan and fromthe exocellular polysaccharide of Rhizobium trifolii (strain TA-1) in the orderedstate. Macromolecules, 21, 3312e3320.

Durand, D., Delsanti, M., Adam, M., & Luck, J. M. (1987). Frequency dependence ofviscoelastic properties of branched polymers near gelation threshold. Euro-physics Letters, 3, 297e301.

Evageliou, V., Richardson, R. K., & Morris, E. R. (2000). Effect of sucrose, glucose andfructose on gelation of oxidised starch. Carbohydrate Polymers, 42, 261e272.


Fang, Y., Takemasa, M., Katsuta, K., & Nishinari, K. (2004). Rheology of schizophyllansolutions in isotropic and anisotropic phase regions. Journal of Rheology, 48,1147e1166.

Flory, P. J. (1953). Principles of polymer chemistry. Ithaca, New York: CornellUniversity Press.

Foegeding, E. A., Bowland, E. L., & Hardin, C. C. (1995). Factors that determine thefracture properties and microstructure of globular protein gels. Food Hydro-colloids, 9, 237e249.

Garcia, M. C., Alfaro, M. C., Calero, N., & Muñoz, J. (2011). Influence of gellan gumconcentration on the dynamic viscoelasticity and transient flow of fluid gels.Biochemical Engineering Journal, 55, 73e81.

Gibson, W., & Sanderson, G. R. (1997). Gellan gum. In A. Imeson (Ed.), Thickening andgelling agents for food (2nd ed.). (pp. 119e143) London: Blackie.

Gothard, M. G. E. (1994). Functional properties of gellan gum. PhD thesis, CranfieldUniversity, Silsoe College, Silsoe, Bedford, UK.

Graessley, W. W. (1974). The entanglement concept in polymer rheology. Advancesin Polymer Science, 16, 1e179.

Grant, G. T., Morris, E. R., Rees, D. A., Smith, P. J. C., & Thom, D. (1973). Biologicalinteractions between polysaccharides and divalent cations: the egg-box model.FEBS Letters, 32, 195e198.

Grasdalen, H., & Smidsrød, O. (1987). Gelation of gellan gum. Carbohydrate Polymers,7, 371e393.

Gunning, A. P., Kirby, A. R., Ridout, M. J., Brownsey, G. J., & Morris, V. J. (1996).Investigation of gellan networks and gels by atomic force microscopy. Macro-molecules, 29, 6791e6796.

Gunning, A. P., Kirby, A. R., Ridout, M. J., Brownsey, G. J., & Morris, V. J. (1997).Correction to paper in preceding reference. Macromolecules, 30, 163e164.

Gunning, A. P., & Morris, V. J. (1990). Light scattering studies of tetramethylammonium gellan. International Journal of Biological Macromolecules, 12,338e341.

Haque, A., Richardson, R. K., Morris, E. R., & Dea, I. C. M. (1993). Xanthan-like “weakgel” rheology from dispersions of ispaghula seed husk. Carbohydrate Polymers,22, 223e232.

Harris, J. E. (1985). Gelrite as an agar substitute for cultivation of mesophilicMethanobacterium and Methanobrevibacter species. Applied EnvironmentalMicrobiology, 50, 1107e1109.

Harris, P., & Pointer, S. J. (1986). Edible gums. UK Patent GB 2,128,871B.Hatakeyama, T., Quinn, F. X., & Hatakeyama, H. (1996). Changes in freezing bound

water in wateregellan systems with structure formation. Carbohydrate Poly-mers, 30, 155e160.

Haug, A. (1964). Composition and properties of alginate. Report No. 30. Trondheim,Norway: Norwegian Institute of Seaweed Research.

Haward, R. N., & Young, R. J. (1997). The physics of glassy polymers (2nd ed.). London:Chapman & Hall.

Hossain, K. S., Nemoto, N., & Nishinari, K. (1997). Dynamic viscoelasticity of iota-carrageenan gelling system near solegel transition. Nihon Reoroji Gakkaishi,25, 135e142.

Hossain, K. S., & Nishinari, K. (2009). Chain release behaviour of gellan gels. Progressin Colloid and Polymer Science, 136, 177e186.

Huang, Y., Singh, P. P., Tang, J., & Swanson, B. G. (2004). Gelling temperatures of highacyl gellan as affected by monovalent and divalent cations with dynamicrheological analysis. Carbohydrate Polymers, 56, 27e33.

Ikeda, S., Nitta, Y., Temsiripong, T., Pongsawatmanit, R., & Nishinari, K. (2004).Atomic force microscopy studies on cation-induced network formation ofgellan. Food Hydrocolloids, 18, 727e735.

Inatomi, S., Jinbo, Y., Sato, T., & Teramoto, A. (1992). Isotropic-liquid crystal phaseequilibrium in semiflexible polymer solutions. Effects of molecular weight andionic strength in polyelectrolyte solutions. Macromolecules, 25, 5013e5019.

Jansson, P., Lindberg, B., & Sandford, P. A. (1983). Structural studies of gellan gum, anextracellular polysaccharide elaborated by Pseudomonas elodea. CarbohydrateResearch, 124, 135e139.

Jay, A. J., Colquhoun, I. J., Ridout, M. J., Brownsey, G. J., Morris, V. J., Fialho, A. M., et al.(1998). Analysis of structure and function of gellans with different substitutionpatterns. Carbohydrate Polymers, 35, 179e188.

Kang, K. S., Veeder, G. T., Mirrasoul, P. J., Kaneko, T., & Cottrell, I. W. (1981). Agar-likepolysaccharide produced by a Pseudomonas species: production and basicproperties. Applied Environmental Microbiology, 4, 1086e1091.

Kasapis, S. (2006). The morphology of the gellan network in a high-sugar envi-ronment. Food Hydrocolloids, 20, 132e136.

Kasapis, S., Abeysekara, R., Atkin, N., Deszczynski, M., & Mitchell, J. R. (2002).Tangible evidence of the transformation from enthalpic to entropic gellannetworks at high levels of co-solute. Carbohydrate Polymers, 50, 259e262.

Kasapis, S., Giannouli, P., Hember, M. W. N., Evageliou, V., Poulard, C., Tort-Bourgeois, B., et al. (1999). Structural aspect and phase behaviour in deacylatedand high acyl gellan systems. Carbohydrate Polymers, 38, 145e154.

Katchalsky, A. (1971). Polyelectrolytes. Pure and Applied Chemistry, 26, 327e373.Katsuta, K., Nishimura, A., & Miura, M. (1992). Effects of saccharides on stabilities of

rice starch gels. Food Hydrocolloids, 6, 387e398.Kavanagh, G. M., & Ross-Murphy, S. B. (1998). Rheological characterisation of

polymer gels. Progress in Polymer Science, 23, 533e562.Kawai, S., Nitta, Y., & Nishinari, K. (2008). Model study for large deformation of

physical polymeric gels. Journal of Chemical Physics, 128, 134903.Kiani, H., Mousavi, M. E., Razavi, H., & Morris, E. R. (2010). Effect of gellan, alone and

in combination with high-methoxy pectin, on the structure and stability ofdoogh, a yogurt-based Iranian drink. Food Hydrocolloids, 24, 744e754.

Kubo, W., Miyazaki, S., & Attwood, D. (2003). Oral sustained delivery of paracetamolfrom in situ-gelling gellan and sodium alginate formulations. InternationalJournal of Pharmaceutics, 258, 55e64.

Kuo, M. S., Mort, A. J., & Dell, A. (1986). Identification and location of L-glycerate, anunusual acyl substituent in gellan gum. Carbohydrate Research, 156, 173e187.

Lee, H.-C., & Brant, D. A. (2002). Rheology of concentrated isotropic and anisotropicxanthan solutions. 1. A rodlike low molecular weight sample. Macromolecules,35, 2212e2222.

Lin, C. C., & Cassida, L. E., Jr. (1984). Gelrite as a gelling agent for the growth ofthermophilic microorganisms. Applied Environmental Microbiology, 47, 427e429.

Lopes da Silva, J. A., Gonçalves, M. P., Doublier, J. L., & Axelos, M. A. V. (1996). Effectof galactomannans on the viscoelastic behaviour of pectin/calcium networks.Polymer Gels and Networks, 4, 65e83.

Manning, C.E. (1992). Formation and melting of gellan polysaccharide gels. PhD thesis,Cranfield Institute of Technology, Silsoe College, Silsoe, Bedford, UK.

Manning, G. S. (1969a). Limiting laws and counterion condensation in poly-electrolyte solutions I. Colligative properties. Journal of Chemical Physics, 51,924e933.

Manning, G. S. (1969b). Limiting laws and counterion condensation in poly-electrolyte solutions II. Self-diffusion of the small ions. Journal of ChemicalPhysics, 51, 934e938.

Maret, G., Milas, M., & Rinaudo, M. (1981). Cholesteric order in aqueous solutions ofthe polysaccharide xanthan. Polymer Bulletin, 4, 291e297.

Martin, J. E., Adolf, D., & Wilcoxon, J. P. (1989). Viscoelasticity near the solegeltransition. Physical Review A, 39, 1325e1332.

Mashimo, S., Shinyashiki, N., & Matsumura, Y. (1996). Water structure in gellangumewater system. Carbohydrate Polymers, 30, 141e144.

Matsukawa, S., Tang, Z., & Watanabe, T. (1999). Hydrogen-bonding behaviour ofgellan in solution during structural change observed by 1H NMR and circulardichroism methods. Progress in Colloid and Polymer Science, 114, 15e24.

Matsukawa, S., & Watanabe, T. (2007). Gelation mechanism and network structureof mixed solution of low- and high-acyl gellan studied by dynamic viscoelas-ticity, CD and NMR measurements. Food Hydrocolloids, 21, 1355e1361.

Mazen, F., Milas, M., & Rinaudo, M. (1999). Conformational transition of native andmodified gellan. International Journal of Biological Macromolecules, 26, 109e118.

Milas, M., & Rinaudo, M. (1983). Properties of the concentrated xanthan gumsolutions. Polymer Bulletin, 4, 271e273.

Milas, M., & Rinaudo, M. (1996). The gellan solegel transition. Carbohydrate Poly-mers, 30, 177e184.

Milas, M., Shi, X., & Rinaudo, M. (1990). On the physicochemical properties of gellangum. Biopolymers, 30, 451e464.

Miyoshi, E., & Nishinari, K. (1999a). Rheological and thermal properties near thesolegel transition of gellan gum aqueous solutions. Progress in Colloid andPolymer Science, 114, 68e82.

Miyoshi, E., & Nishinari, K. (1999b). Effect of sugar on the solegel transition in gellangum aqueous solutions. Progress in Colloid and Polymer Science, 114, 83e91.

Miyoshi, E., Takaya, T., & Nishinari, K. (1994a). Gelesol transition in gellan gumsolutions. 1. Rheological studies on the effects of salts. Food Hydrocolloids, 8,505e527.

Miyoshi, E., Takaya, T., & Nishinari, K. (1994b). Gelesol transition in gellan gumsolutions. 2. DSC studies on the effects of salts. Food Hydrocolloids, 8, 529e542.

Miyoshi, E., Takaya, T., & Nishinari, K. (1995). Effects of salts on the gelesol tran-sition of gellan gum by differential scanning calorimetry and thermal scanningrheology. Thermochimica Acta, 267, 269e287.

Miyoshi, E., Takaya, T., & Nishinari, K. (1996). Rheological and thermal studies ofgelesol transition in gellan gum aqueous solutions. Carbohydrate Polymers, 30,109e119.

Miyoshi, E., Takaya, T., & Nishinari, K. (1998). Effects of glucose, mannose and konjacglucomannan on the gelesol transition in gellan gum aqueous solutions byrheology and DSC. Polymer Gels and Networks, 6, 273e290.

Miyoshi, E., Takaya, T., Williams, P. A., & Nishinari, K. (1996). Effects of sodiumchloride and calcium chloride on the interaction between gellan gum andkonjac glucomannan. Journal of Agricultural and Food Chemistry, 44, 2486e2495.

Miyoshi, E., Takaya, T., Williams, P. A., & Nishinari, K. (1997). Rheological and DSCstudies of mixtures of gellan gum and konjac glucomannan. MacromolecularSymposia, 120, 271e280.

Mo, Y., Kubota, K., & Nishinari, K. (2000). Rheological evidence of the gelationbehaviour of hyaluronanegellan mixtures. Biorheology, 37, 401e408.

Moritaka, H., Fukuba, H., Kumeno, K., Nakahama, N., & Nishinari, K. (1991). Effect ofmonovalent and divalent cations on the rheological properties of gellan gels.Food Hydrocolloids, 4, 495e507.

Moritaka, H., Nishinari, K., Taki, M., & Fukuba, H. (1995). Effects of pH, potassiumchloride, and sodium chloride on the thermal and rheological properties ofgellan gum gels. Journal of Agricultural and Food Chemistry, 43, 1685e1689.

Morris, E. R. (1985). Rheology of hydrocolloids. In G. O. Phillips, D. J. Wedlock, &P. A. Williams (Eds.), Gums and stabilisers for the food industry 2, (pp. 57e78).Oxford, UK: Pergamon Press.

Morris, E. R. (1990). Mixed polymer gels. In P. Harris (Ed.), Food gels (pp. 291e359).London: Elsevier.

Morris, E. R. (1991). Pourable gels: polysaccharides that stabilise emulsions anddispersions by physical trapping. International Food Ingredients, No. 1, Jan/Feb,32e37.

Morris, E. R. (1994). Rheological and organoleptic properties of food hydrocolloids.In K. Nishinari, & E. Doi (Eds.), Food hydrocolloids: Structures, properties, andfunctions (pp. 201e210). New York: Plenum Press.


Morris, E. R. (1995a). Polysaccharide rheology and in-mouth perception. InA. M. Stephen (Ed.), Food polysaccharides and their applications (pp. 517e546).New York: Marcel Dekker.

Morris, E. R. (1995b). Polysaccharide synergism e more questions than answers? InS. E. Harding, S. E. Hill, & J. R. Mitchell (Eds.), Biopolymer mixtures (pp. 247e288)Nottingham, UK: Nottingham University Press.

Morris, E. R., Gothard, M. G. E., Hember, M. W. N., Manning, C. E., & Robinson, G.(1996). Conformational and rheological transitions of welan, rhamsan andacylated gellan. Carbohydrate Polymers, 30, 165e175.

Morris, E. R., Powell, D. A., Gidley, M. J., & Rees, D. A. (1982). Conformations andinteractions of pectins I. Polymorphism between gel and solid states of calciumpolygalacturonate. Journal of Molecular Biology, 155, 507e516.

Morris, E. R., Rees, D. A., Thom, D., & Boyd, J. (1978). Chiroptical and stoichiometricevidence of a specific primary dimerisation process in alginate gelation.Carbohydrate Research, 66, 145e154.

Morris, E. R., Richardson, R. K., & Whittaker, L. E. (1999). Rheology and gelation ofdeacylated gellan polysaccharide with Naþ as the sole counterion. Progress inColloid and Polymer Science, 114, 109e115.

Morris, V. J. (1986). Multicomponent gels. In G. O. Phillips, D. J. Wedlock, &P. A. Williams (Eds.), Gums and stabilisers for the food industry 3, (pp. 87e99).London: Elsevier Applied Science Publishers.

Morris, V. J. (1995). Bacterial polysaccharides. In A. M. Stephen (Ed.), Food poly-saccharides and their applications (pp. 341e375). New York: Marcel Dekker.

Morris, V. J., & Brownsey, G. J. (1995). Physical chemistry of heterogeneous andmixed gels. In E. Dickinson, & D. Lorient (Eds.), Food macromolecules and colloids(pp. 376e389). Cambridge, U.K.: Royal Society of Chemistry, RSC SpecialPublication No. 156.

Morris, V. J., Kirby, A. R., & Gunning, A. P. (1999). A fibrous model for gellan gels fromatomic force microscopy studies. Progress in Colloid and Polymer Science, 114,102e108.

Morrison, N. A., Sworn, G., Clark, R. C., Chen, Y. L., & Talashek, T. (1999). Gelatin alter-natives for the food industry. Progress in Colloid and Polymer Science, 114, 127e131.

Nakajima, K., Ikehara, T., & Nishi, T. (1996). Observation of gellan gum by scanningtunneling microscopy. Carbohydrate Polymers, 30, 77e81.

Nakamura, K., Harada, K., & Tanaka, Y. (1993). Viscoelastic properties of aqueousgellan solutions: the effects of concentration on gelation. Food Hydrocolloids, 7,435e447.

Nakamura, K., Shinoda, E., & Tokita, M. (2001). The influence of compression velocityon strength and structure for gellan gels. Food Hydrocolloids, 15, 247e252.

Nickerson, M. T., Paulson, A. T., & Speers, R. A. (2004). Timeetemperature studies ofgellan polysaccharide gelation in the presence of low, intermediate and highlevels of co-solutes. Food Hydrocolloids, 18, 783e794.

Nishinari, K. (1996). Properties of gellan gum. In G. O. Phillips, D. J. Wedlock, &P. A. Williams (Eds.), Gums and stabilisers for the food industry 8, (pp. 371e383).Oxford, UK: IRL Press.

Nishinari, K., Koide, S., & Ogino, K. (1985). On the temperature dependence ofelasticity of thermoreversible gels. Journal de Physique (France), 46, 793e797.

Nishinari, K., Miyoshi, E., & Takaya, T. (1998). Gelesol transition of gellan aqueoussolutions by rheology and DSC: effects of salts. In K. te Nijenhuis, & W. J. Mijs(Eds.), The Wiley polymer networks group review I: Chemical and physicalnetworks, formation and control of properties (pp. 79e90). Chichester, UK: JohnWiley & Sons.

Nishinari, K., Miyoshi, E., Takaya, T., & Williams, P. A. (1996). Rheological and DSCstudies on the interaction between gellan gum and konjac glucomannan.Carbohydrate Polymers, 30, 193e207.

Nishinari, K., Takaya, T., & Watase, M. (1994). Rheology and DSC of gellaneagarosemixed gels. In K. Nishinari, & E. Doi (Eds.), Food hydrocolloids: Structures,properties, and functions (pp. 473e476). New York: Plenum Press.

Nishinari, K., & Watase, M. (1992). Effect of sugars and polyols on the gelesoltransition of k-carrageenan gels. Thermochimica Acta, 206, 149e162.

Nishinari, K., Watase, M., Kohyama, K., Nishinari, N., Koide, S., Ogino, K., et al. (1992).The effect of sucrose on the thermoreversible gelesol transition in agarose andgelatin. Polymer Journal, 24, 871e877.

Nishinari, K., Watase, M., Rinaudo, M., & Milas, M. (1996). Characterization andproperties of gellanek-carrageenan mixed gels. Food Hydrocolloids, 10,277e283.

Nishinari, K., Watase, M., Williams, P. A., & Phillips, G. O. (1990). k-Carrageenan gels:effects of sucrose, glucose, urea and guanidine hydrochloride on the rheologicaland thermal properties. Journal of Agricultural and Food Chemistry, 38,1188e1193.

Nitta, Y., Ikeda, S., & Nishinari, K. (2006). The reinforcement of gellan gel network bythe immersion into salt solution. International Journal of Biological Macromole-cules, 38, 145e147.

Nitta, Y., Ikeda, S., Takaya, T., & Nishinari, K. (2001). Helixecoil transition in gellangum gels. Transactions of MRS-J, 26, 621e624.

Nitta, Y., Kim, B. S., Nishinari, K., Shirakawa, M., Yamatoya, K., Oomoto, T., et al.(2003). Synergistic gel formation of xyloglucan/gellan mixture as studied byrheology, DSC and circular dichroism. Biomacromolecules, 4, 1654e1660.

Nitta, Y., & Nishinari, K. (2005). Gelation and gel properties of polysaccharidesgellan gum and tamarind xyloglucan. Journal of Biological Macromoleules, 5,47e52.

Nitta, Y., Takahashi, R., & Nishinari, K. (2010). Viscoelasticity and phase separation ofaqueous Na-type gellan solution. Biomacromolecules, 11, 187e191.

Noda, S., Funami, T., Nakauma, M., Asai, I., Takahashi, R., Al-Assaf, S., et al. (2008).Molecular structures of gellan gum imaged with atomic force microscopy in

relation to the rheological behaviour in aqueous systems. 1. Gellan gum withvarious acyl contents in the presence and absence of potassium. Food Hydro-colloids, 22, 1148e1159.

Norton, A. B., Cox, P. W., & Spyropoulos, F. (2011). Acid gelation of low acyl gellangum relevant to self-structuring in the human stomach. Food Hydrocolloids, 25,1105e1111.

Norton, I. T., Goodall, D. M., Frangou, S. A., Morris, E. R., & Rees, D. A. (1984).Mechanism and dynamics of conformational ordering in xanthan poly-saccharide. Journal of Molecular Biology, 175, 371e394.

Norton, I. T., Jarvis, D. A., & Foster, T. J. (1999). A molecular model for the formationand properties of fluid gels. International Journal of Biological Macromolecules,26, 255e261.

Nussinovitch, A., & Hershko, V. (1996). Gellan and alginate vegetable coatings.Carbohydrate Polymers, 30, 185e192.

Ogawa, E. (1993). Osmotic pressure measurements for gellan gum aqueous solu-tions. Food Hydrocolloids, 7, 397e405.

Ogawa, E., Matsuzawa, H., & Iwahashi, M. (2002). Conformational transition ofgellan gum of sodium, lithium, and potassium types in aqueous solutions. FoodHydrocolloids, 16, 1e9.

Ogawa, E., Takahashi, R., Yajima, H., & Nishinari, K. (2006). Effects of molar mass onthe coil to helix transition of sodium-type gellan gums in aqueous solutions.Food Hydrocolloids, 20, 378e385.

Ohtsuka, A., & Watanabe, T. (1996). The network structure of gellan gum hydrogelsbased on the structural parameters by the analysis of the restricted diffusion ofwater. Carbohydrate Polymers, 30, 135e140.

Okamoto, T., Kubota, K., & Kuwahara, N. (1993). Light scattering study of gellan gum.Food Hydrocolloids, 7, 363e371.

O’Neill, M. A., Selvendran, R. R., & Morris, V. J. (1983). Structure of the acidicextracellular gelling polysaccharide produced by Pseudomonas elodea. Carbo-hydrate Research, 124, 123e133.

Oomoto, T., Uno, Y., & Asai, I. (1999). The latest technologies for the application ofgellan gum. Progress in Colloid and Polymer Science, 114, 123e126.

Papageorgiou, M., Gothard, M. G., Willoughby, L. E., Kasapis, S., Richardson, R. K., &Morris, E. R. (1994). Rheology and structure of gellanealginate co-gels. InG. O. Phillips, P. A. Williams, & D. J. Wedlock (Eds.), Gums and stabilisers for thefood industry 6, (pp. 345e365). Oxford, UK: IRL Press.

Papageorgiou, M., Kasapis, S., & Richardson, R. K. (1994). Glassy-state phenomena ingellanesucroseecorn syrup mixtures. Carbohydrate Polymers, 25, 101e109.

Picone, C. S. F., & Cunha, R. L. (2011). Influence of pH on formation and properties ofgellan gels. Carbohydrate Polymers, 84, 662e668.

Picout, D. R., & Ross-Murphy, S. B. (2003). Rheology of biopolymer solutions andgels. The Scientific World, 3, 105e121.

Pollock, T. J. (1993). Gellan-related polysaccharides and the genus Sphingomonas.Journal of General Microbiology, 139, 1939e1945.

Rees, D. A., Morris, E. R., Thom, D., & Madden, J. K. (1982). Shapes and interactions ofcarbohydrate chains. In G. O. Aspinall (Ed.), The polysaccharides, Vol. 1 (pp.195e290). New York: Academic Press.

Richardson, R. K., & Goycoolea, F. M. (1994). Rheological measurement of k-carra-geenan during gelation. Carbohydrate Polymers, 24, 223e225.

Richardson, R. K., & Ross-Murphy, S. B. (1987). Non-linear viscoelasticity of poly-saccharide solutions. 2: xanthan polysaccharide solutions. International Journalof Biological Macromolecules, 9, 257e264.

Rinaudo, M. (2009). Polyelectrolyte properties of a plant and animal polysaccharide.Structural Chemistry, 20, 277e298.

Rinaudo, M., & Milas, M. (2000). Gellan gum, a bacterial gelling polymer. InG. Doxastaxis, & V. Kiosseoglou (Eds.), Novel macromolecules in food systems (pp.239e263). Amsterdam: Elsevier.

Robinson, G., Manning, C. E., & Morris, E. R. (1991). Conformation and physicalproperties of the bacterial polysaccharides gellan, welan and rhamsan. InE. Dickinson (Ed.), Food polymers, gels and colloids (pp. 22e33). Cambridge, UK:Royal Society of Chemistry.

Robinson, G., Manning, C. E., Morris, E. R., & Dea, I. C. M. (1988). Side-chainemainchain interactions in bacterial polysaccharides. In G. O. Phillips,P. A. Williams, & D. J. Wedlock (Eds.), Gums and stabilisers for the food industry 4(pp. 173e181). Oxford, UK: IRL Press.

Rochas, C., & Rinaudo, M. (1980). Activity coefficients of counterions and confor-mation in kappa-carrageenan gels. Biopolymers, 19, 1675e1687.

Rochas, C., Rinaudo, M., & Vincedon, M. (1983). Spectroscopic characterisation andconformation of oligo kappa carrageenans. International Journal of BiologicalMacromolecules, 5, 111e115.

Rodriguez-Hernández, A. I., Durand, S., Garnier, C., Tecante, A., & Doublier, J. L.(2003). Rheologyestructure properties of gellan systems: evidence of networkformation at low gellan concentrations. Food Hydrocolloids, 17, 621e628.

Rolin, C. (1993). Pectin. In R. L. Whistler, & J. N. BeMiller (Eds.), Industrial gums:Polysaccharides and their derivatives (3rd ed.). (pp. 257e293) San Diego, USA:Academic Press.

Ross-Murphy, S. B. (1984). Rheological methods. In H. W.-S. Chan (Ed.), Biophysicalmethods in food research. Critical reports on applied chemistry, (pp. 195e290).London, UK: SCI.

Ross-Murphy, S. B. (2008). Lecture course at Department of Polymer Chemistry,Kyoto University, Japan, March 2008.

Sanderson, G. R. (1990). Gellan gum. In P. Harris (Ed.), Food gels (pp. 201e232).London: Elsevier.

Sanderson, G. R. (1993). Gellan gum yields new vigor for sweets; US approval allowsuse of gellan gum for variety of uses. Candy Industry, Thursday, July 1, 1993.


Sanderson, G. R., Bell, V. L., Burgum, D. R., Clark, R. C., & Ortega, D. (1988). Gellangum in combination with other hydrocolloids. In G. O. Phillips, P. A. Williams, &D. J. Wedlock (Eds.), Gums and stabilisers for the food industry 4, (pp. 301e308).Oxford, UK: IRL Press.

Sanderson, G. R., Bell, V. L., Clark, R. C., & Ortega, D. (1988). The texture of gellangum gels. In G. O. Phillips, P. A. Williams, & D. J. Wedlock (Eds.), Gums andstabilisers for the food industry 4, (pp. 219e229). Oxford, UK: IRL Press.

Sanderson, G. R., & Clark, R. C. (1984). Gellan gum, a new gelling polysaccharide. InG. O. Phillips, P. A. Williams, & D. J. Wedlock (Eds.), Gums and stabilisers for thefood industry 2, (pp. 201e210). Oxford, UK: Pergamon Press.

Sanderson, G., & Ortega, D. (1994). Alginates and gellan gum: complementarygelling agents. In K. Nishinari, & E. Doi (Eds.), Food hydrocolloids: Structures,properties, and functions (pp. 83e89). New York: Plenum Press.

Shimazaki, T., & Ogino, K. (1993). Viscoelastic properties of gellan gum aqueoussolutions. Food Hydrocolloids, 7, 417e426.

Shimizu, M., Brenner, T., Liao, R., & Matsukawa, S. (2012). Diffusion of probe polymerin gellan gum solutions during gelation process studied by gradient NMR. FoodHydrocolloids, 26, 28e32.

Shimomura, K., & Kamada, H. (1986). Roles of gelling agents in plant tissue culture.Plant Tissue Culture, 3, 38e41.

Sime, W. J. (1990). Alginates. In P. Harris (Ed.), Food gels (pp. 53e78). London:Elsevier.

Smidsrød, O., & Haug, A. (1971). Estimation of the relative stiffness of the molecularchain in polyelectrolytes from measurements of viscosity at different ionicstrengths. Biopolymers, 10, 1213e1227.

Sworn, G. (2009). Gellan gum. In G. O. Phillips, & P. A. Williams (Eds.), Handbook ofhydrocolloids (2nd ed.). (pp. 204e227) Cambridge, UK: Woodhead Publishing.

Sworn, G., & Kasapis, S. (1998). Effect of conformation and molecular weight ofcosolutes on the mechanical properties of gellan gum gels. Food Hydrocolloids,12, 283e290.

Sworn, G., & Kasapis, S. (1999). Molecular origin of the rheology of high-sugar gellansystems. Progress in Colloid and Polymer Science, 114, 116e122.

Sworn, G., Sanderson, G. R., & Gibson, W. (1995). Gellan gum fluid gels. FoodHydrocolloids, 9, 265e271.

Takahashi, R., Tokunou, H., Kubota, K., Ogawa, E., Oida, T., Kawase, T., et al. (2004).Solution properties of gellan gum: change in chain stiffness between single-and double-stranded chains. Biomacromolecules, 5, 516e523.

Tanaka, S., & Nishinari, K. (2007). Unassociated molecular chains in physicallycrosslinked gellan gels. Polymer Journal, 39, 397e403.

Tanaka, Y., Sakurai, M., & Nakamura, K. (1993). Ultrasonic velocities in aqueousgellan solutions. Food Hydrocolloids, 7, 407e415.

Tanaka, Y., Sakurai, M., & Nakamura, K. (1996). Ultrasonic velocities and circulardichroism in aqueous gellan solutions. Food Hydrocolloids, 10, 133e136.

Tang, J., Mao, R., Tung, M. A., & Swanson, B. G. (2001). Gelling temperature, gelclarity and texture of gellan gels containing fructose or sucrose. CarbohydratePolymers, 44, 197e209.

Tang, J., Tung,M. A., & Zeng, Y. (1996). Compression strength and deformation of gellangels formed with mono- and divalent cations. Carbohydrate Polymers, 29, 11e16.

Te Nijenhuis, K. (1997). Thermoreversible networks: viscoelastic properties andstructure of gels. Advances in Polymer Science, 130, 1e252.

Te Nijenhuis, K., & Winter, H. H. (1989). Mechanical properties at the gel pointof a crystallizing poly (vinyl chloride) solution. Macromolecules, 22,411e414.

Tsoga, A., Richardson, R. K., & Morris, E. R. (2004a). Role of cosolutes in gelation ofhigh-methoxy pectin. Part 1 e comparison of sugars and polyols. Food Hydro-colloids, 18, 907e919.

Tsoga, A., Richardson, R. K., & Morris, E. R. (2004b). Role of cosolutes in gelation ofhigh-methoxy pectin. Part 2 e anomalous behaviour of fructose: calorimetricevidence of site-binding. Food Hydrocolloids, 18, 921e932.

Tsutsumi, A., Ya, D., Hiraoki, T., Mochiku, H., Yamaguchi, R., & Takahashi, N. (1993).ESR studies of Mn(II) binding to gellan and carrageenan gels. Food Hydrocolloids,7, 427e434.

Upstill, C., Atkins, E. D. T., & Atwool, P. T. (1986). Helical conformations of gellangum. International Journal of Biological Macromolecules, 8, 275e288.

Valli, R., & Clark, R. (2010). Gellan gum. In A. Imeson (Ed.), Food stabilizers, thickenersand gelling agents (pp. 145e166). Chichester, UK: WileyeBlackwell.

Watase, M., & Nishinari, K. (1982). Effect of alkali metal ions on the viscoelasticity ofconcentrated kappa-carrageenan and agarose gels. Rheologica Acta, 21,318e324.

Watase, M., & Nishinari, K. (1993). Effect of potassium ions on the rheological andthermal properties of gellan gum gels. Food Hydrocolloids, 7, 449e456.

Watase, M., Nishinari, K., & Hatakeyama, T. (1988). DSC study on properties of waterin concentrated agarose gels. Food Hydrocolloids, 2, 427e438.

Whittaker, L. E., Al-Ruqaie, I. M., Kasapis, S., & Richardson, R. K. (1997). Developmentof composite structures in the gellan polysaccharide/sugar system. Carbohy-drate Polymers, 33, 39e46.

Winter, H. H., & Chambon, F. (1986). Analysis of linear viscoelasticity of a cross-linking polymer at the gel point. Journal of Rheology, 30, 367e382.

Winter, H. H., & Mours, M. (1997). Rheology of polymers near liquidesolid transi-tions. Advances in Polymer Science, 134, 165e234.

Xu, X., Li, B., Kennedy, J. F., Xie, B. J., & Huang, M. (2007). Characterisation of konjacglucomannanegellan gum blend films and their suitability for release of nisinincorporated therein. Carbohydrate Polymers, 70, 192e197.

Yin, Y., Nishinari, K., Zhang, H., & Funami, T. (2006). A novel liquid crystalline phasein dilute aqueous solutions of methyl cellulose. Macromolecular RapidCommunications, 27, 971e975.

Yoshida, H., & Takahashi, M. (1993). Structural change of gellan hydrogel induced byannealing. Food Hydrocolloids, 7, 387e395.

Yuguchi, Y., Mimura, M., Kitamura, S., Urakawa, H., & Kajiwara, K. (1993).Structural characteristics of gellan in aqueous solution. Food Hydrocolloids, 7,373e385.

Yuguchi, Y., Mimura, M., Urakawa, H., Kitamura, S., Ohno, S., & Kajiwara, K. (1996).Small angle X-ray characterization of gellan gum containing a high content ofsodium in aqueous solution. Carbohydrate Polymers, 30, 83e93.

Yuguchi, Y., Urakawa, H., Kitamura, S., Wataoka, I., & Kajiwara, K. (1999). Progress inColloid and Polymer Science, 114, 41e47.

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