Review of Gelation of Gellan Polysaccharide

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
a
Department of Food and Nutritional Sciences, University College Cork, Cork, Ireland
Department of Food and Nutrition, Graduate School of Human Life Science, Osaka City University, Sumiyoshi, Osaka 558-8585, Japan
c
Centre de Recherches sur les Macromolecules Vgtales (afliated 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
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 August 2011
Accepted 3 January 2012
Gellan is an anionic extracellular bacterial polysaccharide discovered in 1978. Acyl groups present in the
native polymer are removed by alkaline hydrolysis in normal commercial production, giving the
charged 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 double
helices. The coilehelix transition temperature (Tm) is raised by salt in the way expected from polyelectrolyte theory: equivalent molar concentrations of different monovalent cations (Group I and
Me4N) 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 counterions to the charged groups of the polymer have the same effect as those introduced by addition of
salt. Increasing polymer concentration raises Tm because of the consequent increase in concentration of
the counterions, but the concentration of polymer chains themselves does not affect Tm. Gelation occurs
by aggregation of double helices. Aggregation stabilises the helices to temperatures higher than those at
which they form on cooling, giving thermal hysteresis between gelation and melting. Melting of
aggregated 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 polymer
and thus decreasing electrostatic repulsion between the helices. Group I cations decrease repulsion by
binding to the helices in specic coordination sites around the carboxylate groups of the polymer.
Strength of binding increases with increasing ionic size (Li < Na < K < Rb < Cs); the extent of
aggregation and effectiveness in promoting gel formation increase in the same order. Me4N cations,
which cannot form coordination complexes, act solely by non-specic screening of electrostatic
repulsion, and give gels only at very high concentration (above w0.6 M). At low concentrations of
monovalent cations, ordered gellan behaves like a normal polymer solution; as salt concentration is
increased there is then a region where uid weak gels are formed, before the cation concentration
becomes sufcient to give true, self-supporting gels. Aggregation and consequent gelation with Group II
cations occurs by direct site-binding of the divalent ions between gellan double helices. High
concentrations 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 gellan
carboxylate groups. Much higher concentrations of monovalent cations are required to attain maximum
gel strength. The content of divalent cations in commercial gellan is normally sufcient to give cohesive
gels at polymer concentrations down to w0.15 wt %. Gellan gels are very brittle, and have excellent
avour release. The networks are dynamic: gellan gels release polymer chains when immersed in water
and 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-crosslinked
networks which vitrify on cooling. Gellan forms coupled networks with konjac glucomannan and
tamarind xyloglucan, phase-separated networks with kappa carrageenan and calcium alginate, interpenetrating networks with agarose and gelling maltodextrin, and complex coacervates with gelatin
under 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 substituents
does not change the overall double helix structure, but has profound effects on gelation. L-Glyceryl
Keywords:
Gellan
Gelation
High acyl
Aggregation
Double helix
Polyelectrolyte
* Corresponding author. Tel.: 81 6 6605 2818; fax: 81 6 6605 3086.

E-mail address: katsuyoshi.nishinari@gmail.com (K. Nishinari).
0268-005X/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2012.01.004
374
E.R. Morris et al. / Food Hydrocolloids 28 (2012) 373e411
groups stabilise the double helix by forming additional hydrogen bonds within and between the two
strands, giving higher gelation temperatures, but abolish the binding site for metal ions by changing the
orientation of the adjacent glucuronate residue and its carboxyl group. The consequent loss of cationmediated aggregation reduces gel strength and brittleness, and eliminates thermal hysteresis. Aggregation is further inhibited by acetyl groups located on the periphery of the double helix. Gellan with
a high content of residual acyl groups is available commercially as high acyl gellan. Mixtures of high
acyl and deacylated gellan form interpenetrating networks, with no double helices incorporating
strands of both types. Gellan has numerous existing and potential practical applications in food,
cosmetics, toiletries, pharmaceuticals and microbiology.
2012 Elsevier Ltd. All rights reserved.
Contents
1.
2.
3.
4.
5.
6.
7.
8.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .374
Conformation in the solid state and in solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .376
2.1.
Structure of gellan in the solid state . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376
2.2.
Interactions of cations with anionic polyelectrolytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376
2.3.
Conformational transitions of gellan in solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377
2.4.
Light scattering, osmometry and small-angle X-ray scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
Cation-induced gelation of gellan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
3.1.
Rheology of solutions and gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
3.2.
Critical gel point . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
3.3.
Weak gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
3.4.
Gelation of gellan with Group I (alkali metal) cations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
3.5.
Gelation of gellan with Me4N cations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
3.6.
Gelation of gellan with Group II (alkaline earth) cations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
3.7.
Effect of excess salt or low pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
3.8.
Summary and interpretation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
Gelation of gellan in water, with no added salt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
Topology and properties of gellan networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
5.1.
Internal structure of gellan gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
5.2.
Dimensions of strands in gellan networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394
5.3.
Gelation by cations at ambient temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
5.4.
Conformational freedom and release of polymer chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
5.5.
Texture of gellan gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396
5.6.
Mobility of water in gellan networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
5.7.
Syneresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
5.8.
Flavour release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398
5.9.
Gellan liquid crystals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
Effect of sugars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
Effect of acyl substituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402
7.1.
Acyl groups in native gellan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402
7.2.
High acyl gellan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402
7.3.
Blends of high acyl and deacylated gellan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402
7.4.
Partially deacylated gellan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
7.5.
Individual roles of glyceryl and acetyl groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
Mixtures and applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 408
1. Introduction
Gellan is the most recent addition to the range of gelling
agents available commercially for use in food (Gibson &
Sanderson, 1997; Sanderson, 1990). It is an extracellular bacterial polysaccharide synthesised (Pollock, 1993) by Sphingomonas
elodea (ATCC31461), formerly known as Auromonas elodea or
Pseudomonas elodea, and was identied as having commercial
potential (Sanderson, 1990) in 1978, during an extensive
screening programme of soil and water bacteria by Kelco (San
Diego, USA), the company that was also the rst to produce
xanthan as an industrial polysaccharide.
Gellan, which was known initially as polysaccharide S-60, is
a linear anionic polymer with a tetrasaccharide repeating sequence
(Jansson, Lindberg, & Sandford, 1983; ONeill, Selvendran, & Morris,

1983) which consists of two residues of b-D-glucose, one of b-Dglucuronate and one of a-L-rhamnose (Fig. 1). The native polysaccharide, 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 at
O(6) of the same residue (Kuo, Mort, & Dell, 1986). In normal
commercial production, however, both types of substituent are
removed by treatment of the fermentation broth with hot alkali.
The resulting deacylated polymer is known generically as gellan
gum or by the proprietary names of Kelcogel (food-grade) or
Gelrite (for non-food applications). Most (or perhaps all) of the acyl
groups can be preserved by use of milder extraction procedures,
giving high acyl gellan, which is now also available commercially.
375
Fig. 1. Tetrasaccharide repeating unit of deacylated gellan. The sites of attachment of glyceryl and acetyl substituents in high acyl (native) gellan are indicated.
Throughout this review, however, we will use the term gellan to

refer to the deacylated polysaccharide, unless otherwise specied.
Gellan was granted approval for food use in the USA in
November 1992, followed by EU approval as E418, and is now
allowed as a food additive in many other countries worldwide
(including Canada, Australia, South Africa, and most countries of
South America and Southeast Asia). Approval for food use in Japan
came 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 Japanese companies and researchers, and formation of a group focussing specically on the conformation, gelation and industrial
applications of gellan within the Research Group on Polymer Gels
afliated to the Society of Polymer Science, Japan.
It is well known in the history of the development of rheology
that collaborative research on NBS (National Bureau of Standards,
USA) polyisobutylene played an important role. Using the same
sample, various groups participating in the collaboration compared
their results, and thus made a great contribution to the establishment of a timeetemperature superposition principle and a reduced
variable method. Following this approach, the research initiative in
Japan used different, complementary, experimental techniques to
study a single batch of gellan, thus avoiding complications from
variations between different samples.
One possible source of variation is molecular weight. Plant and
algal polysaccharides show very large differences in molecular
weight from sample to sample, reecting differences in, for
example, botanic source, growth conditions, maturity at harvest,
and methods of extraction of the polymer from the tissue matrix. As
a product from fermentation of a well-dened medium by a pure
bacterial culture, with strict control of process parameters (Kang,
Veeder, Mirrasoul, Kaneko, & Cottrell, 1981) such as pH, temperature, aeration and agitation, and release from a uid broth rather
than from cohesive tissue, gellan would be expected to show far
less variability. Nonetheless, various groups have reported widely
different molecular weights, although, as discussed in Section 2.4,
this may reect differences in experimental procedure, rather than
genuine differences in molecular weight between gellan samples.
A more signicant factor is the nature of the counterions that
balance the charge from the carboxylate groups of the polymer chains
(Fig.1). In food-grade gellan, these are conned to sodium, potassium,
magnesium and calcium cations, present in the nutrient salts
required for growth of the synthesising bacteria and/or introduced
during post-fermentation processing. It was established by early
studies within Kelco (Sanderson, Bell, Clark, & Ortega, 1988) and
externally (Grasdalen & Smidsrd, 1987) that divalent cations (Ca2
and Mg2) are much more effective in promoting gelation of gellan
than monovalent cations (Na and K) and that K is more effective
than Na. The cation content of gellan samples is therefore crucial.
In the rst phase of the Japanese collaborative research initiative,

the principal cation in the common sample used (KGG-1) was K
(Table 1), but with an appreciable content of other cations, particularly Ca2. This material was distributed to the collaborating laboratories, and the results obtained were published in a special issue of
this Journal (Food Hydrocolloids, Volume 7, number 5, December
1993). 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 in
the Na salt form, although the content of other cations, particularly K and Ca2, could still not be regarded as negligible (Table 1).
This sample was studied by 17 collaborating laboratories and the
results were published in a special issue of the international journal
Carbohydrate Polymers (Volume 30, No. 2/3, June/July 1996), which
also 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, was
specially prepared by San-Ei-Gen FFI (based on advice from Dr. G.R.
Sanderson of Kelco) for use in the Japanese collaborative research
programme, can be regarded as essentially pure Na gellan, having
only a negligible content of other cations (Table 1). The results from
work on this sample were again presented in a special issue of an
international 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, 1999
and Sworn & Kasapis, 1999).
During the course of the collaborative research initiative, two
international meetings were held in Osaka, Japan, to promote an
active exchange of information and ideas between scientists,
industrialists and food technologists. The rst was the International
Workshop on Gellan and Related Polysaccharides (14e15 November,
1994), preceding publication of the special issue of Carbohydrate
Polymers in 1996. The second, four years later, was the 4th
Table 1
Cation content (wt %) of the common samples used in Japanese collaborative
research.
Sample
Na
Ca2
Mg2
KGG-1 (1993)
NaGG-2 (1996)
NaGG-3 (1999)
0.19
3.03
2.59
2.08
0.19
0.009
0.512
0.11
0.02
0.146
0.02
0.001
376
International Hydrocolloids Conference. This included an informal

workshop attended by many of the participants in the Japanese
collaborative programme and by scientist working on gellan in other
countries (M. Dentini, E.R. Morris, V.J. Morris and M. Rinaudo),
where there was a frank (and sometimes heated) exchange of views
e which 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 several
changes of ownership, and eventually the part of the company
dealing with biogums such as xanthan and gellan was split from
the original alginate business and merged with Copenhagen Pectin
as CPKelco. An overview of the properties and applications of
gellan 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 bres obtained by slow stretching of gels to promote
alignment and lateral packing before drying (Upstill, Atkins, &
Atwool, 1986) gave X-ray bre diffraction patterns of very high
quality, the pattern for the Li salt form being arguably the best that
had ever been obtained for any polysaccharide. Models proposed
from initial analysis of the diffraction data (Upstill et al., 1986) were
unconvincing, but subsequent re-examination (Chandrasekaran,
Millane, Arnott, & Atkins, 1988) showed conclusively that the
ordered structure of gellan in the solid state is a coaxial double
helix (Fig. 2). In this structure, each strand is a 3-fold, left-handed
helix with a pitch of 5.64 nm. The two chains run parallel to one
another and are exactly half-staggered (i.e. with each chain rotated
by 180 and translated by half a pitch relative to the other), so that
the repeat distance (pitch) of the double helix is half that of the
individual strands (2.82 nm), an arrangement similar to that
observed (Arnott, Scott, Rees, & McNab, 1974) for iota carrageenan.
Three of the four glycosidic linkages in the tetrasaccharide
repeating sequence of gellan (Fig. 1) involve equatorial bonds at C(1)
and C(4) of the participating residues. Polysaccharide chains in
which all the linkages are (1 / 4)-diequatorial (such as cellulose, or
the mannan backbone of galactomannans) adopt at, ribbon-like
structures 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 and
promotes helical geometry, in the same way as the (1 / 3) linkages
in the alternating (1 / 3), (1 / 4)-linked disaccharide repeating
sequences of polysaccharides in the agar/carrageenan series.
2.2. Interactions of cations with anionic polyelectrolytes
When anionic polysaccharides such as gellan are dissolved in
water, the only cations present in the resulting solution are, of
course, those present as counterions to the charged groups of the
polymer chains. The effective concentration (activity) of these
positively-charged ions in the bulk of the solution is reduced
(Katchalsky, 1971; Manning, 1969a,b) by electrostatic attraction to
the negatively-charged polymer chains. The strength of attraction
is determined by the linear charge density (charge per unit length)
of the polyanion and the charge of the individual cations, with
divalent cations therefore being attracted twice as strongly as
monovalent. Cation activity can be quantied experimentally by
measurement of the activity coefcient (g) by potentiometry, the
osmotic coefcient (f) from measurements of osmotic pressure, or
the transport coefcient (f) from conductivity or free diffusion of
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. The
sites of attachment of glyceryl and acetyl substituents in high acyl gellan, relative to
the position of the carboxyl group on the neighbouring glucuronate residue, are
indicated in (b).
377
counterions. These three parameters are not normally identical to

one another, but there are known quantitative relationships
between them (Rinaudo, 2009) which allow the activity of the
cations to be derived by any of the investigative techniques
mentioned.
When extraneous salt is added, the total concentration of
cations (CT) becomes:
CT Cp CS
(1)
and the total cation activity (aT) in dilute solution (assuming the
activity coefcient of small ions is equal to 1) is given by:
aT gCp 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 of
thermodynamically-free counterions.
For anionic polysaccharides such as carrageenans that convert
from a disordered coil to an ordered (double helix) conformation on
cooling (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:
d1=Tm =dlog aT Rfc fh =DH
(3)
The slope of 1/Tm versus log aT is determined by two factors: the

transition enthalpy (DH) and the decrease in osmotic coefcient
from fc when the polymer is in the coil form to fh for the ordered
form (Milas, Shi, & Rinaudo, 1990). This decrease arises from the
higher linear charge density of the double-stranded helix,
promoting greater reduction in cation activity than the single
chains in the coil form.
In solutions where the concentration of added salt (CS) is at least
comparable to the polymer concentration (CP) and the activity
coefcient is high or moderate (g > w0.5), aT in Eq. (3) can be
replaced by the total cation concentration (CT) without any serious
loss of linearity, although the resulting plots have somewhat
steeper slope than those obtained using aT.
2.3. Conformational transitions of gellan in solution

The procedure adopted in many investigations of gellan (and
other anionic polysaccharides) to avoid complications from the
presence of different cations is to convert the polymer to a single
salt form and to use a salt of the same cation to vary the total cation
concentration. For studies of gellan in solution, use of the tetramethylammonium (M4N) salt form avoids further complications
from gel formation, since, as reported by Grasdalen and Smidsrd
(1987) and described further in Section 3.5, gelation of gellan
with M4N 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. Thus
a 1 M (1000 mM) solution of Na gellan has a concentration of
668 g/L 66.8 wt %; a 1 wt % solution therefore corresponds to
1000/66.8 z 15 mM. The higher mass of the M4N cation raises the
formula weight per repeat unit to 719 (645 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 monitor
conformational transitions of polysaccharides is optical rotation
(OR). Fig. 3 shows plots of OR (at 302 nm) versus temperature
obtained by Crescenzi, Dentini, and Dea (1987) for dilute solutions
of M4N gellan (1.2 mM z 0.086 wt %) in water and in the presence
of M4NCl at concentrations ranging from 30 to 250 mM. The plot for
the solution in water is featureless, showing only a slight,
Fig. 3. Temperature-dependence of optical rotation (302 nm) for 1.2 mM (0.086 wt %)

Me4N gellan on heating (B) and cooling (C) in the presence of Me4NCl at concentrations (mM) of (a) 0; (b) 30; (c) 75; (d) 92; (e) 120; (f) 150 and (g) 250 (Crescenzi
et al., 1987).
approximately linear, increase in (negative) optical rotation with

decreasing temperature. For the solutions with added M4NCl, by
contrast, there is a well-dened sigmoidal change, which moves to
progressively higher temperature with increasing concentration of
salt, and which shows no thermal hysteresis between values
recorded on cooling and on heating. Similar sigmoidal changes in
OR have been observed for other polysaccharides and shown to
arise from a conformational transition between an ordered structure at low temperature and a disordered coil state at high
temperature (Rees et al., 1982).
The thermally-reversible changes in optical rotation of M4N
gellan are accompanied by large changes in spectra (Fig. 4) obtained
by the related chiroptical technique of circular dichroism (CD).
Maximum change in CD between the disordered state at high
temperature and the ordered state at low temperature comes at
w202 nm, and measurements of CD ellipticity at or near this
wavelength can also be used to follow the temperature-course of
conformational 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 monitoring
thermally-induced conformational transitions is differential scanning calorimetry (DSC). Unlike OR and CD, DSC does not require
optically-clear samples, and can therefore be used at much higher
concentrations of polymer. Manning (1992) observed well-dened
378
Fig. 4. Circular dichroism spectra recorded (Manning, 1992) for 1.0 wt % (13.9 mM)
Me4N 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 heating

for solutions of M4N gellan (1.0e2.0 wt %) in water and in the
presence of various concentrations of M4NCl. For each solution,
the transition midpoint temperatures from cooling and heating
scans extrapolated to the same value at zero scan rate, consistent
with the absence of detectable thermal hysteresis in OR (Fig. 3).
In the same investigation (Manning, 1992) the conformational
transition of M4N gellan in water and with added M4NCl was
monitored by measurements of optical rotation at higher wavelength (436 nm) and much higher concentration of polymer (1.0 wt
%) than in the study by Crescenzi et al. (1987), and by circular
dichroism (Fig. 4). Despite the differences in experimental
approaches and polymer concentration, the values of Tm from the
two investigations agree well (Fig. 5) and their variation with total
concentration of M4N (counterions to the polymer plus added
M4NCl) gives good linearity when plotted according to Eq. (3).
As shown in Fig. 6, Crescenzi et al. (1987) found that the
conformational changes described above were accompanied by
a massive increase in reduced specic viscosity (hsp/C, where C is
polymer concentration) on cooling and a corresponding reduction
on heating, with no thermal hysteresis. Since intrinsic viscosity, [h],
which is directly related to hydrodynamic volume in solution
(Bohdaneck & Kovr, 1982), is dened as reduced specic viscosity
extrapolated to C 0, the values of hsp/C in Fig. 6, which were
obtained at very low polymer concentration (0.05 wt % in 30 mM
M4NCl), will approximate closely to [h], and the much higher values
at low temperature therefore demonstrate that the ordered structure of M4N gellan is stiffer, and has a much greater hydrodynamic
volume, than the disordered state.
In a more recent study, Ogawa et al. (2006) measured the
temperature-dependence of intrinsic viscosity (in 25 mM NaCl) for
six samples of Na gellan ranging in molecular weight from 120 to
17 kD. For the sample of highest molecular weight there was
a sharp, sigmoidal increase in [h] between the disordered state at
high temperature and the ordered state at low temperature (Fig. 7),
closely similar to the increase shown in Fig. 6. For the samples of
progressively lower molecular weight the increase in [h] on cooling
became progressively smaller (Fig. 7), and the sample of lowest
molecular weight (17 kD) showed no sigmoidal change, suggesting
that the chains were too short to adopt the ordered conformation,
Fig. 5. Salt (Me4NCl) dependence of transition midpoint temperature (Tm) for Me4N
gellan. Results obtained by Crescenzi et al. (1987) using optical rotation (Fig. 3) at
302 nm with a polymer concentration of Cp 0.087 wt % (B) are compared with
values obtained by Manning (1992) using optical rotation at 436 nm (C), circular
dichroism (6) and DSC (-) with values of Cp in the range 1e2 wt %.
even at the lowest temperature studied (10 C). DSC exotherms

observed (Ogawa et al., 2006) for the same gellan samples on
cooling (at 1 wt % in 25 mM NaCl) also showed progressive
reduction in intensity with decreasing molecular weight. Decrease
in the extent of conformational ordering with decreasing chainlength has also been observed (Rochas, Rinaudo, & Vincedon, 1983)
for kappa carrageenan.
Solutions of small molecules, such as sugars, give sharp lines in
high-resolution nuclear magnetic resonance (NMR) spectra. For
solutions of disordered polysaccharide coils the lines are broader, but
still discernable. On conversion of the polymer to a rigid, ordered
structure, however, the lines become so broad that they are effectively attened into the baseline, and can no longer be detected. Loss
Fig. 6. Temperature-dependence of reduced specic viscosity on heating (:) and

cooling (B) for 0.05 wt % Me4N gellan in 30 mM Me4NCl (Crescenzi et al., 1987).
Fig. 7. Temperature-dependence of intrinsic viscosity, [h], for Na gellan in 25 mM

NaCl (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.
379
salts of the same cations, and measuring midpoint temperature of

the disordereorder transition by optical rotation. As shown in
Fig. 9, the variation of Tm with total cation activity (aT) gave good
linearity when plotted according to Eq. (3). Divalent cations
induced conformational ordering at higher temperature than
monovalent, but with no evident difference in effectiveness
between Ca2 and Mg2. There was also no evidence of selectivity
between the Group I metal ions studied (K, Na and Li) or
between them and M4N, in contrast to the pronounced selectivity
observed (Grasdalen & Smidsrd, 1987) in the ability of different
monovalent cations to induce formation of gellan gels. Conductivity
measurements of the decrease in concentration of free Na or K
cations on conversion of the polymer from the disordered to the
ordered form gave values of Tm in close agreement with those
obtained by OR (Milas et al., 1990).
These observations are fully consistent with polyelectrolyte
theory (Katchalsky, 1971; Manning, 1969a,b) in which attraction of
cations to anionic polyelectrolytes depends solely on the charge per
unit length of the polyanion and the concentration and charge of
the cations, and not on the chemical nature of either.
2.4. Light scattering, osmometry and small-angle X-ray scattering

of detectable high-resolution NMR signal can therefore be used to
monitor conformational ordering (Rees et al., 1982).
As shown in Fig. 8, Milas and Rinaudo (1996) observed
a sigmoidal reduction in intensity of 1H high-resolution NMR
spectra with decreasing temperature for solutions of Na gellan
(0.4 wt % in D2O). The reduction was quantied by measurement of
the intensity of the resonance from the methyl group of rhamnose
(Fig. 1), which is well resolved from resonances of other hydrogen
atoms in gellan, relative to the NMR intensity of an external standard (sodium succinate) of known concentration. Loss of detectable
signal (Fig. 8) conrms that the conformational transition observed
by other techniques does indeed correspond to conversion from
a disordered state at high temperature to a rigid, ordered structure
at low temperature. It was also found (Milas & Rinaudo, 1996) that
the transition midpoint temperature for solutions of gellan in D2O
was w6 C higher than in water, indicating stabilisation of double
helices by hydrogen bonding within/between the constituent
strands and to surrounding water molecules.
The effectiveness of different cations in promoting conformational ordering of gellan in solution was explored (Milas & Rinaudo,
1996) by converting the polymer to specic salt forms by cation
exchange, adjusting total cation content by addition of chloride
In an early investigation, Brownsey, Chilvers, IAnson, and

Morris (1984) used static light scattering to determine the
weight-average molecular weight (Mw) of gellan. The sample
studied was a mixed salt form, containing w2% K, w0.5% Mg2,
and smaller amounts (w0.1%) of Na and Ca2. Solutions were
prepared in a mixed solvent of 90% DMSO and 10% water, and
passed through a 0.5 mm lter. Light scattering measurements were
made at 25 C, yielding values of Mw 880 and 960 kD from
replicate determinations.
In subsequent studies by light scattering, Dentini, Coviello,
Burchard, and Crescenzi (1988) obtained Mw 434 kD for M4N
gellan in the ordered state (in 75 mM M4NCl at 25 C) for solutions
Visible signal ( %)
100
80
60
40
20
0
20
40
60
Temperature (C)
80
Fig. 8. Loss of detectable high-resolution 1H signal on conformational ordering of Na

gellan (0.4 wt % in D2O); changes were quantied by the intensity of the well-resolved
resonance of the rhamnosyl methyl group (Fig. 1) relative to an external standard
(sodium succinate) of known concentration (Milas & Rinaudo, 1996).
Fig. 9. Inverse of transition midpoint temperature (Tm) on cooling as a function of the

activity of K (B), Na (6), Li (C), Me4N (:), Ca2 (,) and Mg2 (-) counterions in
solutions of deacylated gellan. Gellan samples were converted to these specic salt forms
by ion exchange, and the total activity (aT) of each cation was adjusted by addition of the
corresponding chloride salt (Milas & Rinaudo, 1996). Corresponding values for the
solegel transition temperature (Tsg) of high acyl gellan with monovalent (Na, K) and
divalent (Ca2, Mg2) cations (Huang et al., 2004) are shown by dashed lines.
380
claried using a 0.45 mm lter, but Okamoto et al. (1993), using

a different sample of M4N gellan but essentially identical experimental conditions, reported a substantially lower value of Mw
(238 kD). The number-average molecular weight (Mn) of the M4N
gellan used by Okamoto et al. (1993) was also characterised
by osmotic pressure measurements (Ogawa, 1993), yielding
Mn 55 kD. This value cannot, however, be compared directly with
the value of Mw 238 kD from light scattering, since the
measurements of osmotic pressure were for the disordered form at
40 C, rather than the ordered form characterised in the light
scattering experiments (Okamoto et al., 1993) at 25 C. Somewhat
lower values of Mn from osmometry were reported by Ogawa,
Matsuzawa, and Iwahashi (2002) for a single batch of commercial
gellan converted by cation exchange to the Li, Na and K salt
forms (48, 43 and 49 kD, respectively, in the disordered state at
45 C).
In a further light scattering study of (ordered) M4N gellan in
75 mM M4NCl at 25 C, Gunning and Morris (1990) compared values
of Mw and Rg (radius of gyration) for solutions that had been passed
through lters with a pore size of either 0.45 mm (as used by Dentini
et al., 1988) or 3 mm. Filtration at 3 mm yielded Mw 4500 100 kD
and Rg 159 10 nm; on reduction in pore size to 0.45 mm, both
values decreased dramatically, to Mw 106 6 kD and
Rg 72 4 nm. It seems likely, therefore, that much of the variation
between reported values of the molecular weight of gellan may have
arisen from differences in the experimental procedure used in
preparation of solutions, with the presence of residual intermolecular aggregates giving spuriously high values.
Differences in sample preparation do not, of course, arise when
the same solutions are measured at different temperatures. In
a study of M4N gellan (in 25 mM M4NCl) by light scattering, Milas
et al. (1990) observed an approximate doubling in molecular
weight (from 250 to 490 kD) on going from the disordered form (at
36 C) to the ordered form (at 24 C), with an accompanying
increase in Rg from 69.5 to 127 nm and in persistence length from
5.9 to 71.2 nm. In a more recent investigation, Takahashi et al.
(2004) studied nine samples of Na gellan (in 25 mM NaCl) by
static and dynamic light scattering. In the disordered form (at
40 C) the molecular weights (Mw) of these samples ranged from
34.7 to 115 kD; there was again an approximate doubling in Mw on
going to the ordered state (at 25 C), accompanied by an increase in
persistence length (determined by application of unperturbed
wormlike chain models to data from hydrodynamic measurements) from 9.4 nm at 40 C to 98 nm at 25 C. For all nine samples,
the ratio of Mw for the ordered state to that for the disordered state
was within the range 1.99e2.07. Furthermore, doubling of mass per
unit length and cross-sectional radius of gyration on going from the
disordered form to the ordered form was observed by small-angle
X-ray scattering in a study of Na gellan (NaGG-2, Table 1) by
Yuguchi et al. (1996).
Taken together, these investigations give compelling evidence
that the ordered form adopted by gellan on cooling in the solution
state is the double-stranded helical structure (Fig. 2) characterised
in the solid state by X-ray bre diffraction, which then reverts to the
single-stranded disordered coil state on heating, with no thermal
hysteresis 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, usually
carried out on cylindrical samples. The three main parameters that
can be derived from the variation of resistance to deformation
(stress, s) as the extent of compression (strain, 3 ) is increased are

the initial slope of the compression curve, which gives Youngs
modulus (E s/3 ), and the values of stress and strain at the point
where the gel breaks (sb and 3 b). For materials whose volume
remains constant during compression, reduction in height is
accompanied by lateral expansion. The change in diameter divided
by the change in height is known as Poissons ratio. Lateral
expansion can also be characterised by the stretch ratio, dened
as the diameter at any particular degree of compression divided by
the original diameter of the sample.
A more versatile and informative procedure, which can be
applied to both solutions and gels and can be used to follow the
formation and melting of gel networks, is measurement of resistance to low-amplitude oscillatory deformation (Morris, 1985;
Ross-Murphy, 1984; Te Nijenhuis, 1997). Elastic (solid-like) resistance is greatest at the extremes of the oscillatory cycle, where the
displacement (strain) is greatest, and drops to zero in the middle of
the cycle. The rate of deformation, which determines the resistance
of ideal liquids, is greatest in the middle of the oscillatory cycle and
drops to zero at the extremes (where the direction of movement is
reversed). Thus the stress generated by perfect solids is exactly in
phase with the oscillatory strain, whereas for perfect liquids it is
exactly (90 ) out of phase.
For viscoelastic materials, such as polysaccharide solutions
and gels, the total stress can be resolved into an in-phase component and an out-of-phase component; dividing these by the applied
strain gives, respectively, the storage modulus, which characterises the solid-like (elastic) response of the sample, and the loss
modulus which characterises the liquid-like response. For longitudinal oscillation (i.e. alternate compression and extension of the
sample), the storage and loss moduli are termed E0 and E00
(following Youngs modulus, E, for unidirectional extension or
compression). It is more common, however, to use shear strain, and
the moduli are then denoted by G0 (storage modulus) and G00 (loss
modulus). The ratio of the total, unresolved stress to the applied
strain is known as the complex modulus, jG j, which is related to G0
and G00 by:
jG j
G02 G002
1=2
(4)
Dividing jG j by the frequency of oscillation (u) gives the

complex dynamic viscosity, jh j, which can be regarded as the
oscillatory analogue of steady-shear viscosity (h) from rotational
measurements or capillary viscometry.
jh j jG j=u
G02 G002
1=2
(5)
jG j and jh j are often written simply as G* and h*, with no
change in meaning. Another informative parameter is the loss
tangent, tan d, which is given by:
tan d G00 =G0
(6)
G0
G00
The variation of
and
with frequency (normally plotted on
logarithmic axes) is known as the mechanical spectrum of the
material; jh j is often also included in the spectrum. Typical
mechanical spectra for polysaccharide solutions and gels are shown
in Fig. 10.
For gels (Fig. 10a), solid-like character (G0 ) predominates over
liquid-like, viscous response (G00 ), usually by at least an order of
magnitude. There is little change in either modulus on varying
frequency (u), from which it follows (Eq. (5)) that log jh j decreases
linearly as log u is increased, with a slope close to 1. Formation of
a continuous gel network occurs only when the polymer concentration (C) reaches a minimum critical value, Co. At concentrations
381
oscillation is increased. As polymer concentration is raised, a point

is reached at which the individual coils are forced to interpenetrate
one another and form an entangled network (Graessley, 1974). The
concentration at which this occurs is known as C*, and solutions of
higher concentration are termed semi-dilute.
At low frequencies, where there is sufcient time for entanglements to come apart within the period of oscillation, semi-dilute
solutions respond predominantly by ow, and their mechanical
spectra (Fig. 10b) are similar to those of dilute solutions (Fig. 10c).
However, at higher frequencies, where there is less time for
disentanglement, the predominant response to oscillatory strain
becomes elastic distortion of the entangled network, and the
mechanical spectra (Fig. 10b) become similar to those observed for
gels (Fig. 10a). The frequency-dependence of jh j superimposes
closely on the shear-rate dependence of steady-shear viscosity
(h s=g_ , where s shear stress and g_ shear rate) at equivalent
values of g_ =s1 and u/rad s1, reecting the same dependence of
rheology on the timescale of molecular rearrangement (entanglement/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 obey
the CoxeMerz rule (Cox & Merz, 1958) and include simple liquids
and dilute polymer solutions, as well as entangled networks.
3.2. Critical gel point
Fig. 10. Typical mechanical spectra of (a) a true gel, (b) a semi-dilute solution of
entangled polymer coils, and (c) a dilute polymer solution.
well above Co, plots of log G0 versus log C for typical gelling
biopolymers have a constant slope of w2 (i.e. G0 w C2); C2dependence is also commonly observed for E and E0 . There is then,
however, a progressive increase in slope (i.e. progressively steeper
concentration-dependence of modulus) as C is decreased towards
Co (Clark & Ross-Murphy, 1985).
For dilute solutions of disordered coils free to move independently (Fig. 10c) G00 predominates over G0 , since resistance to
deformation arises mainly from movement (ow) of polymer
molecules through the solvent; log G00 increases linearly on
increasing log u, with a slope of 1 (i.e. G00 w u). The variation of log
G0 with log u is also linear, but with the steeper slope of 2 (i.e.
G0 w u2), which reects progressively greater storage of energy by
contortion of individual polymer coils as the frequency of
Intermolecular association of polymer molecules in solution

results initially in formation of small, soluble clusters of chains. As
the extent of association increases these clusters grow, until ultimately one becomes large enough to span the entire volume of the
solution and form a continuous crosslinked network: this is the
critical 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 over
many 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. In
some, 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 d z 1. For
materials that gel on cooling (such as carrageenans and gellan) the
temperature at the critical gel point (Tc) can be obtained by plotting
curves of tan d versus temperature (T) for a range of different
frequencies of oscillation. The curves cross one another at a single
point of intersection where T 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 & RossMurphy, 2003) and include n 0.5 from the WintereChambon
model (Chambon & Winter, 1985; Winter & Chambon, 1986;
Winter & Mours, 1997) and n 0.67 (2/3) from percolation theory,
assuming Rouse-like dynamics (Martin, Adolf, & Wilcoxon, 1989).
Experimental values for gelling polysaccharides include n 0.7 for
calcium-induced gelation of pectin (Lopes da Silva, Gonalves,
Doublier, & Axelos, 1996) and n 0.42 for gelation of 1 wt % iota
carrageenan on cooling, decreasing monotonically as the carrageenan concentration was increased (Hossain, Nemoto, & Nishinari,
1997). In a study of commercial gellan (Gelrite) in water, Dai, Liu,
Liu, and Tong (2008) obtained values of n 0.77, 0.53, 0.43 and
0.38 at polymer concentrations of 1.0, 1.5, 2.0 and 2.5 wt %,
respectively. Variation of n with concentration appears to be
conned to polymers that form physical gels by co-operative noncovalent association, and may reect departure from the random
growth of network structure that occurs on chemical (covalent)
crosslinking and is assumed in theoretical models that predict
universal values of n.
382
The onset of intermolecular association into soluble clusters is

accompanied by a sharp increase in G00 , reecting increased resistance to movement through the solvent. The temperature at which
this occurs can be taken as an index of the coilehelix transition
temperature (Tch). After initial formation of a continuous network
at the critical gel point, G0 increases steeply with increasing intermolecular association on further cooling, until stabilising when the
network is fully formed. The point at which the curves of G0 and G00
versus temperature cross one another (i.e. going from G0 < G00 to
G0 > G00 ) is often taken as the solegel transition temperature (Tsg).
Although less rigorous than nding the temperature at which tan d
becomes independent of frequency, use of the G0 , G00 crossover
criterion is far simpler and less time consuming. Its validity
depends on the frequency at which the measurements are made.
As discussed above, the response of semi-dilute solutions to
high-frequency oscillatory deformation (Fig. 10b) may be dominated by elastic distortion of the entangled network, and further
increase in G0 during cooling may simply reect greater entanglement between individual growing clusters of crosslinked chains,
rather than formation of a continuous network. However, at low
frequencies, where entanglement makes little contribution to
solid-like (elastic) response, the crossover of G0 and G00 gives
a reasonably valid index of Tsg.
3.3. Weak gels
Passing through the critical gel point does not necessarily imply
formation of a cohesive gel. A number of polysaccharides give
solutions that ow freely but have mechanical spectra qualitatively
similar to that shown in Fig. 10a for a typical gel network. Systems
of this type, which show predominantly elastic (gel-like) response
to small perturbations but which cannot support their own weight
and can be stirred and poured like normal solutions, are commonly
known 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 that
are weak only in the sense of having low moduli. Conventional
gels, which are often described as self-supporting or demouldable, or as true gels (the term used in this review), respond to
high stress by fracturing, whereas weak gels ow. To avoid such
confusion, other descriptions such as pourable gels (Morris, 1991)
or uid gels (Sworn, Sanderson, & Gibson, 1995) have been used,
and indeed Professor S.B. Ross-Murphy, who was one of the rst to
adopt the term weak gel, has suggested more recently that
structured liquid might be better (Ross-Murphy, 2008). However,
the term weak gel seems rmly established in the literature, and
we will continue to use it in this review. Mechanical spectra of
weak gels normally differ from those of true gels in having greater
frequency-dependence of G0 and G00 and smaller separation
between the two moduli, but the main distinction is the difference
in response to unidirectional stress.
The most extensively-studied polysaccharide with weak gel
properties is xanthan. At high temperature and low ionic strength
xanthan is disordered in solution and shows normal solution
properties. On cooling and/or addition of salt, however, it
undergoes a (thermally-reversible) transition to a stiff, ordered
structure (Norton, Goodall, Frangou, Morris, & Rees, 1984). At
concentrations above w0.3 wt %, solutions of ordered xanthan give
gel-like mechanical spectra, and are capable of holding small
particles in suspension over long periods of time (which is central
to many of the industrial and food applications of xanthan).
Weak gel properties have also been reported for solutions of
other ordered polysaccharides, including welan and rhamsan
(Morris et al., 1996) which are branched variants of gellan, and for
dispersions (Haque, Richardson, Morris, & Dea, 1993) of ispaghula
(milled seed husk of Platago ovata Forsk). The common feature of

these systems is that the polysaccharide is in a rigid form, either at
the molecular level or in large, supramolecular assemblies.
Formation of true gels by association of disordered coils into
ordered junctions involves substantial loss of conformational
entropy, and will therefore occur only if the enthalpic advantage of
association is correspondingly large (i.e. if the non-covalent
bonding within the intermolecular junctions is strong). Rupture
of the resulting networks therefore requires considerable force.
Association of rigid structures, by contrast, involves little loss of
entropy, and can therefore occur even if the bonding is weak, giving
rise to tenuous weak gel networks that come apart at low stress.
Polysaccharides that form true gels on cooling under quiescent
conditions can also give dispersions of microscopic gel particles
with weak gel properties if they are subjected to shear on cooling
through the temperature-range of the solegel transition (Harris &
Pointer, 1986; Norton, Jarvis, & Foster, 1999). By applying this
approach to gellan, Sworn et al. (1995) obtained weak gel
networks capable of suspending small particles at very low polymer concentration (0.125 wt %) where ordered xanthan shows only
the predominantly liquid-like properties of a normal polysaccharide solution.
The internal rheology of the gellan microgel particles in such
preparations was compared with the macroscopic weak gel
properties in an investigation by Caggioni, Spicer, Blair, Lindberg,
and Weitz (2007). Mixed solutions of commercial gellan (Kelcogel) and NaCl were prepared at 80 C and cooled (at 0.5 C/min) to
25 C, either quiescently or under shear (at a constant shear rate of
100 s1). As would be expected, the true gels formed on quiescent
cooling had higher moduli (G0 and G00 ) than the weak gels formed
under shear. The microrheology of both was probed on a micrometre length-scale by video imaging of the Brownian motion of
tracer particles (1 mm diameter polystyrene beads) incorporated in
the gellan solutions prior to cooling. The results obtained for the
sheared and non-sheared preparations were indistinguishable,
demonstrating that the microgel particles formed by cooling under
shear have the same internal structure as the continuous gel
networks obtained on quiescent cooling. The weak gel networks
were capable of holding the polystyrene tracer particles in
suspension over long periods of time: at a gellan concentration of
0.05 wt % (in 100 mM NaCl) only slight sedimentation was observed
after storage for 2 months.
On application of small stresses (steady or oscillatory) the
resulting deformation of the weak gels increased in direct
proportion to the stress (giving constant elastic modulus, as would
be observed for a true gel). Above a critical stress, however, the
strain increased steeply, showing failure of the weak gel network,
with elastic deformation being replaced by viscous ow. The stress
at the point of failure increased in direct proportion to gellan
concentration (over the range 0.05e0.40 wt % in 100, 200 or
300 mM NaCl), and the strain at failure decreased inversely (i.e.
with the onset of ow occurring at progressively smaller
deformation as the concentration of gellan was raised). Yielding
of the weak gel network of microgel particles was tentatively
attributed to a shear-induced transition from a jammed to an
un-jammed state.
Failure of weak gel networks formed by gelation of gellan
under shear was investigated further by Garcia, Alfaro, Calero, and
Muoz (2011). A xed concentration of NaCl (0.22 M) was incorporated in solutions of Kelcogel (0.025e0.25 wt %) at 80 C. The
onset temperature for gelation on cooling, as determined by lowamplitude oscillatory measurements of G0 and G00 , was w41 C.
Weak gels were prepared by shearing at this temperature for
1 min at 700 rpm, and were then stored for at least 2 days at 4.5 C
before characterisation at 20 C.
The sheared preparations were visually cloudy, and confocal

laser scanning microscopy (CSLM), with uorescent labelling of
gellan by uoresceinamine, showed irregularly-shaped particles
with 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) were
similar to the typical true gel spectrum shown in Fig. 10a, with G0
about an order of magnitude greater than G00 , and with both moduli
showing only a slight decrease with decreasing frequency (u).
Equilibrium values of G0 (Ge), obtained by extrapolation to u 0,
varied with gellan concentration in the same way as the elastic
moduli of conventional (un-sheared) biopolymer gels (Section 3.1),
with Ge w C2 for C 0.25e0.05 wt %, and a steeper decrease on
further reduction to C 0.025 wt %, the lowest concentration
studied.
Failure of the weak gel networks of microgel particles was
characterised by measurement of transient changes in rheology in
response to shear. After loading onto the rheometer, the samples
were left to equilibrate (at 20 C) until they had reached constant
values of G0 , which occurred within 10 min. They were then subjected to a constant shear rate g_ of 10 s1 for 5 min. When the
shear was applied, resistance (stress, s) rose almost instantaneously
to an abrupt maximum, and then decreased progressively over time
towards a constant value (i.e. towards constant steady-shear
viscosity, h s=g_ ). Recovery of weak gel rheology on cessation
of shear became progressively slower with increasing concentration of gellan.
The values of complex dynamic viscosity jh j for the initial
weak gel networks were much (w20 times) higher than the
values of h observed at the end of the 5 min period of steady shear
at 10 s1. Thus the gellan weak gels violate the CoxeMerz rule
(Section 3.1): jh j of the intact weak gel network is substantially
greater than the viscosity (h) characterising ow after network
failure. Similar violation of the CoxeMerz rule, and stress overshoot 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 corresponding to the stress needed to initiate ow of weak gels is an
important consideration in engineering operations such as pumping the sheared networks from the tanks in which they were
prepared, with the power required being substantially greater than
would 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 curves
from investigation (Manning, 1992; Robinson, Manning, & Morris,
1991; Robinson, Manning, Morris, & Dea, 1988) of 1 wt % Na gellan (CP 15 mM) in the presence of NaCl at concentrations ranging
from 0 to 130 mM (i.e. CT 15e145 mM; Eq. (1)). Throughout this
range, the DSC cooling scans showed only a single exotherm which,
as expected from previous studies by other techniques (Section
2.3), moved to progressively higher temperature as the concentration of Na was raised. Heating at low CT gave single endotherms
(e.g. trace A in Fig. 11) which were essentially equal and opposite to
the exotherms observed for the same samples on cooling. At a salt
concentration of w50 mM NaCl (CT z 65 mM), however, a second
thermal process became evident in the DSC heating traces. On
further increase in salt concentration, the rst endotherm in the
heating curves became progressively smaller, but remained in
approximately the same position as the exotherm in the corresponding cooling curves. The second endotherm, by contrast,
moved to much higher temperature and increased in size, until at
the highest concentration of NaCl studied (130 mM; CT 145 mM)
383
Fig. 11. Representative DSC cooling and heating traces for 1 wt % (15 mN) Na gellan at
total concentrations (mM) of Na (counterions to the polymer added NaCl) of: A: 25;
B: 67; C: 115 and D: 145 (Manning, 1992; Robinson et al., 1991). Baselines have been
subtracted from the cooling curves; the heating curves are displaced vertically by
arbitrary amounts to avoid overlap.
it became the only detectable process. Closely similar behaviour for

the same concentration of Na gellan was observed by Mazen,
Milas, and Rinaudo (1999).
The proposed interpretation (Robinson et al., 1991), which now
seems 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 the
second thermal process on heating comes from melting of double
helices stabilised by aggregation, and the rst from residual
unaggregated helices which melt at the same temperature as they
formed on cooling, with the proportion and stability of aggregated
helices increasing with increase in CT above a critical threshold
value (CT*). An alternative interpretation (Miyoshi, Takaya, &
Nishinari, 1996; Robinson et al., 1988) in which the rst DSC
endotherm on heating was attributed to dissociation of aggregates
and the second to melting of unaggregated helices has now been
retracted by the senior coauthors of the papers in which it was
proposed (E.R. Morris and K. Nishinari).
The progressive increase in thermal hysteresis between formation of individual helices on cooling and melting of helixehelix
aggregates on heating as CT is raised above CT* is shown directly
in Fig. 12. At low concentrations of Na, up to CT z 25 mM, the
solutions 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 gave
mechanical spectra similar to those of gels (Fig. 10a) but remained
uid (i.e. showing the weak gel properties described in Section
3.3). True gels were formed only when CT became greater than CT*
(Fig. 12), with moduli then increasing steeply as the concentration
of Na was raised. The accompanying increase in extent of
384
Fig. 12. Effect of total concentration of Na on peak-maximum temperatures (Tmax) for

the single DSC exotherm observed on cooling (B) and the rst (C) and second (-)
endotherms on heating (see Fig. 11) for 1 wt % (15 mN) Na gellan with varying
concentrations of NaCl (Manning, 1992). The three types of rheological response
observed (Morris, E.R. et al., 1999) for the ordered form at low temperature over
different ranges of Na concentration are indicated at the foot of the gure (solution,
weak gel and true gel).
aggregation, characterised by the magnitude of the second endotherm in DSC (Fig. 11), and stability of the aggregates, characterised
by the degree of thermal hysteresis between formation of double
helices on cooling and melting of helixehelix aggregates on heating, indicates strongly that true gels are formed by association of
gellan double helices into stable aggregates.
Measurements of optical rotation (Milas & Rinaudo, 1996) also
showed hysteresis between values of transition temperature (Tm)
obtained on cooling and on heating at cation concentrations above
a minimum threshold (Fig. 13). For Na ions, the value of CT*
derived by OR was w45 mM, which is somewhat lower than the
corresponding value of w65 mM from DSC (Fig. 12). In both studies,
however, the values of CT* were estimated by extrapolation from
higher cation concentrations, and the comparatively small
discrepancy between them probably reects the experimental
error inherent in the extrapolation, rather than any genuine
difference between the processes characterised by the two
different techniques.
Despite this experimental imprecision, it is clear that the value
CT* z 20 mM (Fig. 13) derived by Milas and Rinaudo (1996) from
optical rotation of K gellan with added KCl is appreciably lower
than the corresponding value for the Na salt form. Thus, in
contrast to the lack of selectivity between different monovalent
cations in promoting formation of gellan double helices (Section
2.3), association of the helices into aggregates does appear to
depend on which cation is used.
As shown in Fig. 14, the concentration-dependence of modulus
(Milas & Rinaudo, 1996) for Na and K gellan (in the presence of
the corresponding chloride salt at a xed concentration of 100 mM)
is close to the C2 relationship commonly found (Section 3.1) for
gelling biopolymers at concentrations well above the minimum
value (Co) required to form a continuous network, giving a slope of
w2 on a double logarithmic plot. At each concentration of gellan,
the moduli for the K salt form are about twice those observed for
the Na form, and the single value obtained for the Li salt form at
Fig. 13. Variation of transition temperature (Tm) from optical rotation on cooling (open
symbols) and heating (lled symbols) with activity of Na (circles) and K (squares) in
solutions of gellan (Milas & Rinaudo, 1996). The cooling curve is identical to the line
shown for monovalent cations in Fig. 9, but some points have been omitted for clarity
of presentation.
the highest gellan concentration studied (1.3 wt %) is about 30

times lower, which is consistent with the order of effectiveness
reported by Grasdalen and Smidsrd (1987) for gelation of gellan
with Group I cations: Li < Na < K < Cs.
Further evidence of differences in the effectiveness of different
Group I cations in promoting aggregation of gellan double helices
has come from investigation (Yuguchi, Urakawa, Kitamura,
Wataoka, & Kajiwara, 1999) of 1.5 wt % Na gellan (NaGG-3,
Table 1) in the presence and absence of 50 mM LiCl, NaCl, KCl or
CsCl by small-angle X-ray scattering. Measured values of cross-
Fig. 14. Variation of Youngs modulus (E) with polymer concentration (C) for K, Na
and Li gellan in 0.1 M KCl, NaCl or LiCl, respectively (Milas & Rinaudo, 1996).
sectional radius of gyration for the ordered form (at 10 C) were

0.30 nm with no added salt; 0.44 nm with LiCl; 0.50 nm with NaCl;
1.01 nm with KCl; and 1.37 nm with CsCl. The increase in aggregate
size with increasing size of the cations from the added salt parallels
the order of effectiveness in promoting gelation of gellan and is
consistent with the proposal (Robinson et al., 1991) that association
of gellan double helices into stable aggregates is responsible for
formation of true gels.
In the disordered state, the coil dimensions of polyelectrolytes
such as gellan are expanded by intramolecular electrostatic repulsion. On addition of increasing concentrations of salt the repulsions
are progressively screened by the simple anions and cations, with
consequent decrease in the dimensions of the polyelectrolyte coils,
which in turn leads to reduction in viscosity and in dynamic
moduli, G0 and G00 . Indeed, the extent to which intrinsic viscosity
decreases with increasing concentration of salt has been used to
characterise the stiffness of the polyelectrolyte (Smidsrd & Haug,
1971). However, when the polymer is in an ordered conformation, resistant to contraction, charge screening by added salt has the
opposite effect of enhancing rheology, by suppressing repulsion
between 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 % Na gellan
(NaGG-2, Table 1) in the disordered and ordered states.
Non-specic charge screening, however, is determined simply
by ionic strength (Smidsrd & Haug, 1971), and cannot therefore
account for the observed selectivity between different monovalent
cations in promoting aggregation and gelation of gellan. The most
likely interpretation (Morris et al., 1996) is that Group I cations
attach directly to the gellan double helices by site binding (which
can alternatively be described as formation of stable ion pairs
between the metal cations and the carboxylate groups of gellan),
with consequent reduction (or elimination) of repulsion between
the helices. In addition to electrostatic attraction, site binding of
cations to conformationally-ordered anionic polysaccharides
involves coordination (chelation) of the cation by appropriatelyspaced oxygen atoms around the charged groups of the polysaccharide, as postulated in the widely-accepted egg box model
(Grant, Morris, Rees, Smith, & Thom, 1973) for cation-induced
gelation of alginate (Morris, Rees, Thom, & Boyd, 1978) or pectin
(Morris, Powell, Gidley, & Rees, 1982). The order of effectiveness of
Group I cations in promoting aggregation of gellan double helices
385
would imply that Cs ions give the best geometric t to the binding
site, with progressively less efcient coordination as the size of the
cation decreases.
Site binding of K ions to the gellan double helix in the solid state
has been demonstrated by X-ray bre diffraction (Chandrasekaran,
Puigjaner, Joyce, & Arnott, 1988). Each cation is coordinated
(bound) to ve oxygen atoms of the double helix: the two atoms
from the carboxylate group of the glucuronate residue (Fig. 1) and
O(2) of the glucose residue adjacent to it in the non-reducing
direction of one strand in the helix, and, from the second strand,
O(2) of glucuronate and O(6) of the adjacent glucose in the reducing
direction. The separation of the K ion from one of the oxygen atoms
of the carboxylate group is greater than the distance required for
optimum coordination, suggesting that, as postulated above, larger
Group I cations (Rb and Cs) would be bound more efciently. It
was proposed from the X-ray analysis that helixehelix aggregates
are formed by carboxylateeKewatereKecarboxylate interactions. However, although present in the solid state, it seems unlikely
that such water bridges would survive in the presence of the large
excess of water in gellan gels.
Binding of Group I cations in gellan gels has been demonstrated
by Annaka, Honda, Nakahira, Seki, and Tokita (1999) using multinuclear NMR. The samples studied contained 1.5 wt % Na gellan
(NaGG-3, Table 1) with 20 mM NaCl, KCl or RbCl, and spectra for the
23
Na, 39K and 87Rb isotopes were recorded at temperatures spanning
the range of the coilehelix and helixecoil transitions of gellan. As
shown in Fig. 15, conformational ordering of gellan on cooling was
accompanied by loss of detectable 39K and 87Rb resonances, which
implies 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 highresolution NMR signal from gellan itself. No such changes were
seen (Fig. 15) for the 23Na signal from solutions of 1.5 wt % (22.5 mM)
Na gellan with 20 mM NaCl. However, the total concentration of
cations present (CT CP CS 22.5 20 42.5 mM) is below the
critical concentration (CT*) of Na at which cation-mediated aggregation can be detected (Figs. 12 and 13), but above the corresponding
critical value for K (Fig. 13), which could explain the difference in
mobility (Fig. 15) of these two cations in the presence of ordered
gellan. Immobilisation of Rb is then consistent with the evidence
from other techniques, discussed above, that binding afnity of
Group I cations to ordered gellan increases with increasing size.
Fig. 15. Effect of temperature on 23Na, 39K and 87Rb NMR spectra for 20 mM NaCl, KCl or RbCl in the presence of 1.5 wt % Na gellan (NaGG-3; Table 1). Almost no thermal hysteresis
was observed between the changes seen as temperature was raised or lowered (Annaka et al., 1999).
386
3.5. Gelation of gellan with Me4N cations

Fig. 16 shows mechanical spectra recorded (L.E. Whittaker, R.K.
Richardson & E.R. Morris, unpublished) for 1.0 wt % (13.9 mM)
Me4N gellan in water, and in 0.25, 0.4, and 0.7 M Me4NCl, after
cooling to the ordered state at 10 C. The spectrum for ordered
Me4N gellan in water (Fig. 16a) is broadly similar to those observed
for semi-dilute solutions of disordered polysaccharide coils
(Fig. 10b). With 0.7 M Me4NCl present in the sample, the
mechanical spectrum (Fig. 16d) has the form typical of a normal
polysaccharide gel (Fig. 10a). Formation of true gels by Me4N
100
10
0.1
100
10
0.1
100
gellan at high concentrations of Me4NCl was reported almost 25

years ago by Grasdalen and Smidsrd (1987), although Me4N
cations have often been described subsequently as non-gelling. At
lower concentration of Me4NCl (0.4 M), the rheological response
(Fig. 16c) is still predominantly gel-like, but the moduli now show
appreciable frequency-dependence and the separation between G0
and G00 is smaller. At 0.25 M Me4NCl (CT z 264 mM), log G0 and log
G00 vary linearly with log u over the entire range of frequency (u) at
which measurements could be made, with the same slope (w0.54)
for both. As described in Section 3.2, such behaviour is typical of
a gelling system where the degree of crosslinking is just sufcient
to give a continuous network, and pinpoints the onset of gelation.
Conformational ordering of Me4N gellan on cooling under both
gelling and non-gelling conditions could be seen as a sharp increase
in G0 , with an accompanying sharp increase in G00 . The effect of
increasing concentration of Me4NCl on the temperature (To) at the
onset of this steep increase on cooling and on completion of the
corresponding decrease on heating is shown in Fig. 17a. The two
values are coincident up to the critical gel point (Fig. 16b) at 0.25 M
Me4NCl, but then diverge progressively, with the extent of thermal
hysteresis (i.e. the difference in To between heating and cooling
scans) showing an approximately linear increase with increasing
concentration of Me4NCl.
At Me4NCl concentrations of 0.7 M and above, the gels became
strong enough to be removed from cylindrical moulds and characterised by compression testing (Section 3.1). Fig. 17b shows the
variation of break stress (sb) for 1.0 wt % Me4N gellan with varying
concentrations of Me4NCl, in direct comparison with the corresponding values of log G0 from small-deformation oscillatory
measurements. At Me4NCl concentrations below w0.9 M, sb
decreased linearly with decreasing salt, extrapolating to zero at
w0.6 M Me4NCl. The mechanical spectra, however, remained gellike at salt concentrations down to the critical gel point at 0.25 M
Me4NCl. There is thus a window of salt concentrations, extending
from w0.25 to w0.6 M Me4NCl, where 1.0 wt % Me4N gellan shows
weak gel properties (Section 3.3).
3.6. Gelation of gellan with Group II (alkaline earth) cations
10
c
0.1
10000
1000
100
10
1
0.1
10
100
Frequency (rad/s)
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) Me4N gellan in (a) water, (b) 0.25 M
Me4NCl, (c) 0.4 M Me4NCl and (d) 0.7 M Me4NCl (L.E. Whittaker, R.K. Richardson & E.R.
Morris, unpublished).
In a continuation of the work described in the previous section, the

effect of divalent cations was explored by incorporation of CaCl2 in
non-gelling solutions of 2.0 wt % (27.8 mN) Me4N gellan. As shown in
Fig. 18a, low concentrations of CaCl2 caused massive increases in G0 ,
for example by about a factor of 10,000 at 5 mM (10 mN) Ca2. The
accompanying increases in G00 , though still massive, were smaller
than those of G0 , and there is a sharp minimum in tan d at w9 mN Ca2,
which is about 1/3 of the stoichiometric requirement of the carboxylate groups of the polymer (Fig. 1).
The effect of polymer concentration at xed concentration of
CaCl2 (10 mM) was investigated by Rodriguez-Hernndez, Durand,
Garnier, Tecante, and Doublier (2003) using Na gellan prepared
from Kelcogel by cation exchange. The variation of log G0 with
concentration (C) had the form typical (Section 3.1) of a gelling
biopolymer, with a progressive increase in slope as C was reduced
towards the minimum critical gelling concentration (Co), which
was extremely low (slightly below 0.005 wt %), and G0 w C2 at
C >> Co. Confocal laser scanning microscopy (CSLM) with uorescent labelling of gellan by uoresceinamine showed the expected
progressive increase in network connectivity as the polymer
concentration was raised.
In contrast to the behaviour observed with monovalent cations
(Sections 3.4 and 3.5), where thermal hysteresis (Figs. 12, 13 and
17a) occurs only when the cation concentration exceeds
a minimum critical value (CT*), there was immediate thermal
hysteresis on addition of CaCl2 to Me4N gellan, with melting
387
100
Hysteresis (C)
To (C)
80
60
40
20
0
0.0
0.2
0.4
0.6
0.8
1.0
[Me4NCl] (M)
1.2
1.4
50
40
30
20
10
(kPa)
log (G'/Pa)
0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0
1.4
[Me4NCl] (M)
Fig. 17. (a) Thermal hysteresis (:) between the temperature (To) at the onset of
gelation on cooling (C) and completion of gel melting on heating (-) and (b) breaking
stress, sb (C) and G0 (-; 1 rad/s, 1% strain), measured at 5 C, for 1.0 wt % (13.9 mM)
Me4N gellan with varying concentrations of Me4NCl (L.E. Whittaker, R.K. Richardson &
E.R. Morris, unpublished).
temperature (Fig. 18b) increasing linearly with Ca2 concentration,

and reaching 100 C at w10 mN (5 mM). Formation of gels stable to
above 100 C has been observed previously (Gibson & Sanderson,
1997; Sanderson, 1990) on addition of low concentrations of Ca2
to commercial gellan. Comparative studies with MgCl2 (L.E. Whittaker, R.K. Richardson & E.R. Morris, unpublished) showed no
signicant or systematic difference between gellan gels formed
with Ca2 or Mg2, which is consistent with the study by Grasdalen
and Smidsrd (1987) which showed no difference in gel strength
for Group II cations (Mg2, Ca2, Sr2 and Ba2), although divalent
cations of transition metals (Zn2, Cu2 and Pb2) were found to
give stronger gels. Tang, Tung, and Zeng (1996), however, have
reported that Ca2 gives gels that are 1.1e1.4 times stronger than
those formed with Mg2.
It is well established (e.g. Gibson & Sanderson, 1997; Grasdalen
& Smidsrd, 1987; Milas & Rinaudo, 1996; Miyoshi, Takaya, &
Fig. 18. (a) G0 (-), G00 (C) and tan d (:) at 7 C and (b) the temperature (To) at the
onset of gelation on cooling (C) and completion of gel melting on heating (-) for
2.0 wt % (27.8 mM) Me4N gellan on progressive addition of CaCl2. Measurements were
made at 1 rad/s and 1% strain. (L.E. Whittaker, M.W.N. Hember & E.R. Morris,
unpublished).
Nishinari, 1994a, 1995, 1996; Sanderson, 1990) that the concentration of divalent cations required to induce gelation of gellan is far
lower than for monovalent cations, and that the resulting gels have
greater thermal stability. Indeed the content of divalent cations in
commercial gellan is usually sufcient to give strong, stable gels
without addition of extraneous salt. However, when divalent and
monovalent cations are present together, as would occur if salts
such as CaCl2 or MgCl2 are added to solutions of gellan in a monovalent, or mixed, salt form, the gelation and melting processes, and
the properties of the gels, can be complex and difcult to interpret.
The purpose of the investigation leading to the results shown in
388
Fig. 18 was to avoid such complexity by using solutions in which the

only cation present initially was Me4N at a concentration
(27.8 mM) about an order of magnitude lower than the critical
gelling concentration (Fig. 16b) of w264 mM, thus allowing the
effect of subsequent incorporation of divalent cations to be studied
in 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 initial
increase in gel strength on progressive addition of salt is followed
by a decrease at higher salt concentrations. This effect is illustrated
in Fig. 19 for mixtures of 0.8 wt % (w12 mM) K gellan with KCl and
of 0.3 wt % (w4.5 mM) Na gellan with MgCl2 (Milas & Rinaudo,
1996). Both show maxima in Youngs modulus (E) and force at
break (proportional to breaking stress, sb), but the molar concentrations at which these occur are w30 times higher for K than for
Mg2 (i.e. w15 times higher on the basis of normality). For both
salts, the maximum in break stress comes at substantially lower salt
concentration than the maximum in modulus. Similar behaviour
was observed (Morris, E.R. et al., 1999) for Na gellan with added
NaCl when the salt-dependence of sb was compared with that of G0 .
In the investigation by Milas and Rinaudo (1996) three different
patterns of fracture under compression were observed as the
concentration of added KCl or MgCl2 was increased. These are
shown schematically in Fig. 19, and the regions of salt concentration
over which they occurred are identied as a, b and c. In region a,
which corresponds to salt concentrations below the maximum in
break stress, the gels were weak and broke sharply along a single
fracture plane. The stiffer gels formed in region b, which corresponds roughly to salt concentrations between the maximum in
break stress and the maximum in modulus, formed multiple small
fractures during break. In region c, at salt concentrations above the
maximum in modulus, the gels were softer and their disintegration

resembled a coagulation or phase-separation process.
Gelation of gellan can also be induced by reduction in pH, and in
their study of the effect of monovalent and divalent cations
Grasdalen and Smidsrd (1987) described HCl as the most potent
gel-former. However, the variation in gel strength with increasing
concentration of acid is not monotonic. Initial acidication from
neutral 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 the
glucuronate residues of gellan (Fig. 1), at wpH 3.4 (Haug, 1964),
break stress decreases (Norton, Cox, & Spyropoulos, 2011) and by
pH 2 the gels are extremely weak and turbid, and show phase
separation of polymer and solvent (Moritaka, Nishinari, Taki, &
Fukuba, 1995). Indeed, precipitation by acid can be used as
a method for isolation and purication of gellan (Sanderson, 1990).
Initial increase and subsequent decrease in Youngs modulus,
giving curves similar to those shown in Fig. 19, has also been
observed on progressive addition of alkali metal salts to kappa
carrageenan (Watase & Nishinari, 1982), and maxima in gel
strength on varying salt concentration and/or pH are commonly
observed in thermogelation of globular proteins (Foegeding,
Bowland, & Hardin, 1995). Indeed, such maxima seem to be
universal, and are perhaps to be expected. Biopolymer gels form
part of a continuum from solutions of individual molecules at one
extreme to close-packed solids at the other as the extent of intermolecular association is increased (by varying conditions such as
pH and ionic environment). Optimum crosslinking will occur
somewhere along the continuum; less association will give
a weaker network; greater association will give larger aggregates,
with consequent reduction in the effective number of individual
junctions, until ultimately the network collapses into a solid
precipitate. This interpretation is consistent with the apparent
phase separation observed for gellan gels at very low pH or with
a large excess of salt (region c in Fig. 19), and with the observation
(Ohtsuka & Watanabe, 1996) that the gels become turbid at the
point where their strength begins to decrease.
3.8. Summary and interpretation
Fig. 19. Changes in Youngs modulus, E (circles) and maximum force (Fm) at break
(squares), from compression testing (25 mm/min) of cylindrical samples (17 mm
height; 17 mm diameter) at 25 C, on addition of MgCl2 to 0.3 wt % Na gellan (lled
symbols) or KCl to 0.8 wt % K gellan (open symbols). The different patterns of failure
observed in regions a, b and c for each salt are shown schematically between the traces
for MgCl2 (from Milas & Rinaudo, 1996).
The rst step in gelation of gellan is conversion of the polymer

from 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 into
stable aggregates. Aggregation is inhibited by electrostatic repulsion between the helices. One way in which the repulsion can be
suppressed, allowing gels to form, is reduction in pH, which
reduces the charge on the helices by conversion of glucuronate
carboxyl groups (Fig. 1) from the negatively-charged COO form to
the uncharged COOH form.
Aggregation can also be promoted by salt. The simple anions and
cations from dissolved salt screen electrostatic repulsion between
the gellan helices. Cations cause further reduction in repulsion by
clustering around the helices and thus lowering their effective
negative charge. Both of these mechanisms are non-specic: charge
screening depends solely on ionic strength, determined by the
concentration and charge of both anions and cations, and the
extent to which the (negatively-charged) helices are surrounded
preferentially by (positively-charged) cations is determined by the
concentration and charge of the cations. Group I (alkali metal)
cations, however, can cause further reduction in the effective
negative charge of the gellan double helices by attaching to them in
specic binding sites. Site binding is triggered initially by electrostatic attraction of cations to the carboxylate groups of the polymer,
but is augmented and stabilised by formation of a coordination
complex with appropriately-spaced oxygen atoms from both
strands of the double helix. The equilibrium between bound and

unbound cations is determined by the efciency of coordination,
which, as discussed in Section 3.4, seems to decrease with
decreasing ionic size (Cs > Rb > K > Na > Li).
Stabilisation of gellan double helices by association into
aggregates leads to thermal hysteresis between the temperatures
at which helices form on cooling and aggregates melt on heating.
On gelation with alkali metal (Group I) cations, the onset of
hysteresis is accompanied by the onset of formation of true gels,
and occurs at progressively lower cation concentration as ionic
size increases.
At low concentrations of monovalent cations, solutions of
ordered gellan give mechanical spectra similar to those of solutions
of disordered coils (Fig. 10b or c). On further increase in salt
concentration there is a region of weak gel response (Section 3.3)
before reaching the threshold concentration for formation of true
gels. This progression, which can be attributed to progressive
suppression of electrostatic repulsion between the gellan double
helices, is observed for both alkali metal and M4N cations. The salt
concentrations over which the three types of rheology (solution,
weak gel and true gel) occur are, however, much higher for M4N
than for Group I cations. This can be readily explained by the
inability of organic cations such as M4N to form coordination
complexes, which makes them incapable of reducing the effective
negative charge of the gellan double helices by the site binding that
occurs with Group I cations.
Gelation with divalent cations (Ca2 and Mg2) is qualitatively
different. The thermal hysteresis associated with stable aggregation
begins immediately (Fig. 18) on progressive incorporation of CaCl2
(or MgCl2) in solutions of M4N gellan, and is accompanied by
immediate massive increase in G0 and G00 . Maximum gel strength is
attained (Tang et al., 1996) when the concentration of divalent
cations reaches w100% of stoichiometric equivalence to the
carboxyl groups of the polymer (for gellan concentrations ranging
from 0.6 to 2.2 wt %), which argues against salting out as the
cause of reduction in gel strength after the initial increase (Fig. 19).
A proposed interpretation (Morris et al., 1996; Tang et al., 1996) is
that divalent metal ions promote aggregation by site binding
between pairs of carboxylate groups on neighbouring helices,
rather than by suppressing electrostatic repulsion by binding to
individual helices. Computer modelling (Chandrasekaran &
Thailambal, 1990) has shown that direct bridging between gellan
double helices by Ca2 is sterically feasible.
In Ca2-gelation of alginate and pectin, the poly-L-guluronate
sequences of alginate (Morris et al., 1978) and poly-D-galacturonate
sequences of pectin (Morris et al., 1982) both form highly-stable
dimeric junctions in which the participating chains adopt
a buckled, 2-fold conformation with site-bound Ca2 cations
bridging between them. Larger assemblies, involving more chains
and more layers of site-bound cations can be built up in the same
way, but the dimer structure is particularly stable. This can be
explained by simple electrostatic considerations: incorporation of
the rst layer of divalent cations causes a gross reduction in
negative charge density, so that binding between dimers is far
weaker than initial binding within individual dimers.
Since the participating chains have 2-fold symmetry and only
the inner faces are involved in chelation (coordination) of cations,
the Ca2 content of polyguluronate and polygalacturonate dimers is
half the full stoichiometric requirement of the polymer carboxyl
groups. Formation of analogous Ca2-mediated dimeric junctions
between gellan double helices, which have 3-fold symmetry
(Fig. 2b), would therefore reach completion when the content of
site-bound cations is 1/3 of stoichiometric equivalence. The sharp
minimum in tan d at this point (Fig. 18a) can then be explained by
transition from a primary mechanism of association through
389
highly-stable helixehelix dimers to a secondary process involving

much weaker association between dimers.
Finally, as described in Section 3.6, excess salt or low pH can
weaken gellan networks by promoting excessive aggregation,
leading to collapse of gel structure and, ultimately, precipitation of
the polymer.
4. Gelation of gellan in water, with no added salt

Fig. 20 shows DSC traces recorded on cooling and heating at
0.5 C/min for essentially pure Na gellan (NaGG-3, Table 1) at
concentrations ranging from 1.0 to 5.5 wt % in water, with no added
salt (Miyoshi & Nishinari, 1999a). As would be expected, the
magnitude of the thermal transitions increases with increasing
polymer concentration. The cooling traces show a single exotherm
which moves to progressively higher temperature as the concentration of gellan is raised. At gellan concentrations up to 4 wt %, the
heating traces show a single endotherm which is essentially equal
and opposite to the corresponding exotherm on cooling. At 4.5 wt
%, however, a small second endotherm can be detected. On further
increase in gellan concentration, the second endotherm increases
in magnitude relative to the rst, and the separation between them
increases.
The accompanying rheological changes, characterised (Miyoshi
& Nishinari, 1999a) by low-amplitude oscillatory measurements
of G0 and G00 at 0.1 rad/s during cooling and heating at 0.5 C/min are
shown in Fig. 21. The initial sharp increase in G00 during cooling
coincides 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; the
temperature at which it occurs can therefore be regarded (Section
3.2) as the coilehelix transition temperature (Tch). G0 also increases
Fig. 20. DSC traces recorded (Miyoshi & Nishinari, 1999a) on cooling (left) and heating
(right) at 0.5 C/min for Na gellan (NaGG-3; Table 1) in water at the concentrations
shown to the right of the heating curves (1.0e5.5 wt %).
390
sharply below Tch, but at gellan concentrations less than w2 wt % it

remains lower than G00 (Fig. 21a and b). At higher concentrations,
however, G0 rises above G00 , and, at the low frequency of oscillation
used (0.1 rad/s), the temperature at which the two curves cross one
another can be taken (Section 3.2) as the solegel transition
temperature (Tsg). Both Tch and Tsg increased (Table 2) with
increasing concentration of gellan, paralleling the increase of
transition temperatures in DSC (Fig. 20).
Over the concentration-range where the DSC heating curves
showed a single endotherm at the same temperature as the single
exotherm on cooling (up to w4 wt %) the rheological changes on
heating followed the same temperature-course as those observed
on cooling (Fig. 21aef). However, at 4.5 wt %, the concentration at
which a second, higher temperature, endotherm was rst observed
in DSC, the nal steep decrease in G0 and G00 on heating also
occurred at higher temperature (Fig. 21g) than the initial steep
increase on cooling.
Mechanical spectra recorded (Miyoshi & Nishinari, 1999a) at
selected temperatures during cooling are shown in Fig. 22 for
NaGG-3 in water at concentrations of 1.0, 2.0, 2.5, 3.0 and 3.5 wt %.
As described in the gure legend, the spectra are shifted along the
horizontal axis (frequency) and/or along the vertical axis (moduli)
to avoid overlap and allow the relative magnitudes of G0 and G00 , and
their response to frequency (u), to be compared at each concentration and temperature.
For the 1% solution (Fig. 22a) in the disordered coil form at 30 C,
0
G was too low to measure, and log G00 increased linearly as log u
was increased, with the slope of 1 typical (Section 3.1) of a dilute
polymer solution. At 25 C, which is just below the onset of
conformational ordering at Tch 26.5 C (Table 2), the slope of log
G00 versus log u remains the same (1) and G0 is now detectable,
with a slope of 2 on the double logarithmic plot. As described in
Section 3.1, this is again the behaviour typical of a dilute solution of
polymeric species free to move independently. On further reduction in temperature, to 15, 5 and 0 C, however, there is a progressive decrease in the frequency-dependence of both moduli towards
the lower slopes typical of semi-dilute solutions (Fig. 10b), which
can be explained by progressive entanglement between growing
Table 2
Variation of coilehelix and solegel transition temperatures, Tch and Tsg (Fig. 21),
with concentration (C) of Na 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)
Tsg ( C)
20
Nonea
26.5
Nonea
34
9.4
37.7
26
38.2
30.5
42
42
43
43
a
G0 remained below G00 to the lowest temperature at which measurements were
made (5 C).
clusters of chains crosslinked through double helices. Formation of

a continuous network, with gel-like mechanical response (Fig. 10a),
was not, however, observed for this concentration of gellan in the
absence of salt, even at 0 C.
A similar progression towards increasing entanglement as
temperature was decreased below Tch (Table 2) is evident at 2 wt %
(Fig. 22b), but at this concentration a solegel transition was
observed (at 9.4 C), and the mechanical spectrum recorded at
lower temperature (5 C) has some gel-like character (G0 > G00 ),
although still showing pronounced frequency-dependence of both
moduli.
The solegel transition temperatures (Table 2), by the criterion of
G0 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 temperatures, 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-crosslinked
network (Section 3.2): log G0 and log G00 both increase linearly
with log u over the entire range of accessible frequencies and have
the same slope of w0.5. On progressive reduction in temperature
below the critical gel point, there is a progressive decrease in
frequency-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 at w42 C and was accompanied by a simultaneous
sharp 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
Fig. 21. Temperature-dependence of G0 (circles) and G00 (triangles), measured at 0.1 rad/s during cooling (open symbols) and heating (lled symbols) at 0.5 C/min, for Na gellan
(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, (f) 3.5 and (g) 4.5. The coilehelix transition temperature (Tch in frame c) is taken as the
start of the steep increase in G00 on cooling, and the solegel transition temperature (Tsg) as the point where the traces for G0 and G00 cross (Miyoshi & Nishinari, 1999a).
391
Fig. 22. Frequency-dependence of G0 and G00 for Na 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 temperatures
shown within the frames. At each concentration and temperature the symbols used for the two moduli are the same, but are open for G0 and lled 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 are
shown to the left of each pair of curves (a rst and b below it). Temperatures of the coilehelix and solegel transitions (Tch and Tsg) from Fig. 21 are shown within the frames (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-dilute
solution of entangled coils (Fig. 10b), suggesting that some
limited association of chains into entangled clusters has already
occurred. The spectrum recorded at 42 C, by contrast, has the form
typical of a polymer gel (Fig. 10a), with G0 about an order of
magnitude higher than G00 and little frequency-dependence of
either modulus. It is evident, therefore, that the critical gel point, by
the criterion (Section 3.2) of constant tan d, must lie within the
narrow window of temperature between 43 and 42 C but, because
of the very large changes in moduli within this range, determination of the precise temperature at the gel point was not experimentally practicable.
Fig. 23 shows the variation of Tch and Tsg with concentration of
NaGG-3 during cooling in water, with no added salt (as in
Figs. 20e22). Below w2 wt % gellan, Tch increased steeply with
increasing 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 increased
steeply with increasing concentration of gellan, until becoming
approximately equal to Tch at w3.5 wt %. At higher concentrations,
Tch and Tsg remained approximately equal to one another, and
showed only slight further increase with increasing concentration
of gellan (Miyoshi & Nishinari, 1999a).
Three different physical states of gellan can be distinguished in
Fig. 23. The region above Tch corresponds to solutions of disordered
coils, the region between Tch and Tsg to solutions of ordered gellan,
and the region below Tsg to continuous gel networks. In the
Fig. 23. Variation of coilehelix (B) and solegel (C) transition temperatures (Tch and
Tsg) on cooling (see Fig. 21) with concentration (C) of Na gellan (NaGG-3; Table 1) in
water; the region labelled Sol-I corresponds to solutions of disordered coils, Sol-II to
ordered (helical) structures in solution, and Gel to continuous networks (Miyoshi &
Nishinari, 1999a).
392
mechanical spectra of the networks formed at concentrations

below 3.5 wt % (Fig. 22bed) there is little separation between G0
and G00 and both show large variations with frequency. As
mentioned in Section 3.3, spectra of this type are commonly seen
for weak gels. The mechanical spectra observed (Fig. 22e) for the
networks formed at 3.5 wt % gellan, by contrast, have the form
typical (Fig. 10a) of true gels (G0 >> G00 ; little variation with
frequency). The Gel region in Fig. 23 can perhaps therefore be
subdivided, tentatively, into true gels at gellan concentrations of
3.5 wt % and above, and weak gels at lower concentrations, down
to 2 wt %, below which there is no solegel transition.
The concentrations of NaGG-3 used in the rheological studies
described above ranged (Fig. 23) from 0.20 to 4.75 wt %. Since the
solutions were prepared in water with no added salt, and 1 wt %
Na gellan corresponds (Section 2.3) to 15 mM, the total concentration of Na cations therefore ranged from 3.0 to 71.25 mM.
Fig. 24 shows the variation of coilehelix transition temperature
(Fig. 23) with Na concentration plotted according to Eq. (3), in
direct comparison with the corresponding values for solutions in
which the concentration of Na gellan was held xed at 0.05 wt %
(Milas & Rinaudo, 1996; Fig. 9) or 1.0 wt % (Manning, 1992; Fig. 12)
and the total concentration of Na was varied by incorporation of
NaCl. Despite the widely different gellan concentrations, the three
families of points lie convincingly on the same straight line,
demonstrating that, as would be expected from Eqs. (1)e(3), the
temperature at which gellan converts from coils to double helices
on cooling is determined by the total cation concentration, with
counterions to the polymer having the same effect as the same
cations introduced by addition of salt, and is unaffected by the
concentration of the polymer chains themselves.
The changes in DSC heating traces (Fig. 20) on varying concentration of Na gellan with no added salt bear a striking qualitative
resemblance to the changes observed (Fig. 11) on progressive
addition of NaCl to a xed concentration of Na gellan (1 wt %).
Quantitatively, the gellan concentration of 4.5 wt % at which
splitting of the DSC heating curve becomes detectable (Fig. 20) is
equivalent (Section 2.3) to a counterion concentration of
Fig. 24. Dependence of Tch on total concentration of Na (counterions to the polymer

plus added NaCl, where present) for 0.20e4.75 wt % Na gellan with no added salt (C;
Fig. 23) and for 0.05 wt % (B; Fig. 9) and 1.0 wt % (6; Fig. 12) Na gellan with or
without NaCl.
15 4.5 67.5 mM Na, which is in close agreement with the

corresponding values of CT 67 mM or 65 mM observed by,
respectively, Robinson et al. (1991) and Mazen et al. (1999) for 1 wt
% Na gellan with added NaCl. It seems likely, therefore, that the
orderedisorder transition temperatures observed for gellan in
water, like the temperature of the disordereorder transition
(Fig. 24), are determined by the concentration of counterions to the
carboxylate groups of the polymer, rather than being affected
directly by the concentration of polymer itself.
A similar investigation, using the same procedures and experimental conditions, was carried out by Miyoshi, Takaya, and
Nishinari (1996) for aqueous solutions of the less pure Na gellan
sample NaGG-2 (Table 1). At gellan concentrations of 1 and 2 wt %
the changes in G0 and G00 on cooling and heating were broadly
similar to those observed (Fig. 21b and c) for the same concentrations of the purer sodium salt form, NaGG-3. At 3 wt %, however, the
initial steep increase in G0 on cooling was followed by a second
wave of increase at lower temperature, which was not seen for
NaGG-3 (Fig. 21e). At concentrations up to 3 wt %, the heating
curves for NaGG-2 superimposed closely on the cooling curves,
with no detectable thermal hysteresis, as was also observed for
NaGG-3. On slight increase in the concentration of NaGG-2 to
3.2 wt %, however, reduction in both G0 and G00 occurred in two
steps, the rst coincident with the increases in moduli observed on
cooling, and the second at higher temperature. On further slight
increase in gellan concentration to 3.3 wt % the second melting
process was displaced to even higher temperature, and at 3.5 wt %
loss of gel structure had not gone to completion by the highest
temperature at which measurements were made (60 C). The onset
of (slight) thermal hysteresis for NaGG-3, by contrast, was not
observed until the polymer concentration had reached 4.5 wt %
(Fig. 21g). The obvious interpretation of the greater thermal
stability of the gels formed by NaGG-2 is that the polymer has
a signicant content of K and, particularly, Ca2 and Mg2 cations
which are present only in much smaller amounts in NaGG-3
(Table 1). The ability of these cations to promote formation of
more stable junctions than those formed with Na is discussed in
Sections 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 of
cations other than Na on thermal transitions of NaGG-2. The DSC
cooling curves (Fig. 25) are broadly similar to those observed
(Fig. 20) for NaGG-3, but are displaced systematically to slightly
higher temperature, which may reect the ability of divalent
cations to induce conformational ordering of gellan at higher
temperatures (Fig. 9) than equivalent concentrations of monovalent cations.
The main difference in DSC between NaGG-2 and NaGG-3,
however, is in the heating curves. Detectable splitting of the DSC
endotherm for NaGG-3 did not occur until the polymer concentration had reached 4.5 wt % (Fig. 20) and only two endothermic
processes were then observed up to the highest concentration
studied (5.5 wt %). For NaGG-2, by contrast, there is obvious splitting at 3.2 wt % (Fig. 25). As the concentration of NaGG-2 is
increased to 3.5 and 3.6 wt % the separation between the two
endothermic processes increases, and on further increase in polymer concentration up to the highest studied (4.2 wt %) multiple
endothermic processes are observed, strongly indicating that the
various cations present in NaGG-2 form aggregated junctions of
different thermal stabilities. As might therefore be expected,
addition of extraneous salts increases the complexity of the heating
traces in DSC (Miyoshi, Takaya, & Nishinari, 1994b). The ability of
the comparatively small amounts of divalent cations in NaGG-2
(Table 1) to cause large enhancements in thermal stability
emphasises the importance of the higher concentrations normally
Fig. 25. DSC traces recorded (Miyoshi, Takaya, & Nishinari, 1996) on cooling (left) and
heating (right) at 0.5 C/min for Na gellan (NaGG-2; Table 1) in water at the
concentrations shown to the right of the heating curves (1.0e4.2 wt %).
present in commercial gellan in yielding thermally-stable gels at

low 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 of
gellan gels are shown in Fig. 26. The rst (Fig. 26a) was based on
393
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 M4N gellan by light scattering (Gunning & Morris, 1990).
Although rather different visually, the two models have many
features in common. Both envisage formation of true gels by
association of double helices in the presence of gel promoting
cations (i.e. metal ions). In the paper by Robinson et al. (1991) the
associated helices are termed cation-mediated aggregates and in
Gunning and Morris (1990) they are described as crystalline
junction zones, but the underlying concept of ordered assemblies
of double helices incorporating metal cations to balance the
negative charge of the polysaccharide appears to be the same in
both.
The schematic network shown on the right-hand side at the top
of Fig. 26a includes both cation-mediated aggregates and unaggregated stretches of double helix, with the unaggregated helices
melting rst on heating, leaving a network crosslinked solely by
helixehelix aggregates. This is an essential feature of the model,
invoked to rationalise the two thermal transitions observed in DSC
heating scans (Fig. 11). The simultaneous presence of aggregated
and unaggregated double helices in gelled lms of K gellan
(KGG-1, Table 1) has been observed directly (Nakajima et al., 1996)
by scanning tunnelling microscopy (STM). Coexisting stretches
of aggregated and unaggregated double helices are also present in
the network shown bottom-left in Fig. 26b, although the double
helices are represented by solid black bars, whereas in Fig. 26a they
are shown as two strands wound round one another. Two-stage
melting does not form part of the second model, but is not
precluded by it.
A central feature of the second model, shown schematically on
the right-hand side of Fig. 26b, is formation of long laments on
cooling under non-gelling conditions (including specically M4N
gellan with or without low concentrations of added M4N). These
are described by Morris, V.J. (1995) as lamentous aggregates.
However, to avoid confusion with use of the term aggregates to
refer to structures formed by lateral (side-by-side) association of
Fig. 26. Models for gelation of gellan proposed by (a) Robinson et al. (1991) and (b) Gunning and Morris (1990). In both models, lled circles denote cations that promote
aggregation of gellan double helices.
394
double helices, we will continue to use the term clusters to

describe assemblies of chains that are crosslinked solely by individual double helices and do not form a continuous network (as in
the discussion of critical gel point in Section 3.2). Transient association of soluble clusters (foot of Fig. 26a) was incorporated in the
rst model to explain weak gel properties. Although linear clusters were not envisaged specically when this model was proposed,
there was certainly no intention (or experimental evidence) to
preclude them. Conversely, the second model (Fig. 26b) includes
some branching of the linear laments. Thus, although the
emphasis of the two models is different, there is no fundamental
clash between them.
One genuine difference, however, is that the schematic
networks shown in Fig. 26a include some stretches of disordered
polymer chains between the gellan double helices (aggregated or
non-aggregated), while there are no such disordered regions in the
second 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 ordered
form, when a non-gelling solution of Na gellan was cooled
through the temperature-range of the coilehelix transition
observed by other techniques. In a more recent investigation,
Hossain and Nishinari (2009) recorded high-resolution 1H NMR
spectra for 1 wt % K gellan in the solution state at 50 C and in the
gel state at 20 C. The spectrum at 50 C showed the comparatively
sharp resonances characteristic (Section 2.3) of disordered polymer
coils. The same resonances were still detectable at 20 C, although
their intensity was decreased to w10% of that at 50 C. DSC cooling
traces recorded (Tanaka & Nishinari, 2007) for the same concentration of the same gellan sample showed a well-dened exotherm
with a peak-maximum temperature of w30 C, and no evidence of
further conformational ordering on continued cooling after the
temperature had reached 20 C. A possible explanation of the
visible 1H NMR resonances detected (Hossain & Nishinari, 2009) at
this temperature is that they arose from disordered sequences
trapped by topological constraints in formation of the crosslinked
network, which would not occur in the non-gelling solution
studied by Milas and Rinaudo (1996), and that the ratio of order to
disorder in the resulting gel was about 9:1. This conclusion is
consistent with the schematic model in Fig. 26a, although the
presence of disordered sequences was not necessary to explain the
experimental evidence on which the model was based, and could
not be inferred from that evidence.
The presence or absence of disordered sequences is important in
understanding the mechanical properties of gels (Nishinari, Koide,
& Ogino, 1985). For networks with an appreciable content of
disordered sequences the main response to applied stress is
stretching of these exible regions, and the elastic resistance to
deformation comes predominantly from the consequent reduction
in conformational entropy. Since change in entropy (DS) becomes
increasingly signicant as temperature (T) is raised (DG DHTDS),
the elastic modulus of entropic networks increases with
increasing temperature, as explored for gellan gels by Watase and
Nishinari (1993). The elasticity of networks formed by lateral
association of brillar strands (Fig. 26b), by contrast, would come
predominantly from the increase in enthalpy (DH) on distortion of
the aggregated laments, or the entire brillar network (Morris, V.J.
et al., 1999).
5.2. Dimensions of strands in gellan networks
Branched brillar strands have been visualised directly
(Gunning et al., 1996; Gunning, Kirby, Ridout, Brownsey, & Morris,
1997) by atomic force microscopy (AFM) for networks formed by
drying very dilute solutions (3 or 10 mg/L) of K or M4N gellan on
freshly-cleaved mica. The length of the linear regions between

branch points in the micrographs varies considerably, but is typically around 150 nm. As discussed in Section 2.3, the tetrasaccharide repeating unit of (Na) gellan has mass 668 D, giving
almost exactly 2 kD (3 0.668) for a full turn (pitch 5.64 nm) of
each strand in the double helix (Fig. 2). The mass per unit length of
the double helix is therefore 0.71 kD/nm (2 2/5.64). Thus the
typical length of w150 nm for the linear regions visualised by AFM
corresponds to a mass of 106 kD, which is in perfect agreement
with the value of Mw (Section 2.4) obtained from light-scattering
studies of well-claried solutions of ordered gellan by the same
group (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 adjacent
chains over its entire length.
Although technically challenging, it has also proved possible to
use AFM to obtain images of the surface of fully-formed gellan gels
at molecular resolution. This was rst achieved (Gunning et al.,
1996, 1997) for very strong gels of 1.2 wt % gellan obtained by
controlled reduction in pH using D-glucono-d-lactone (GDL),
yielding images of a network of long brillar strands. Surface
images of gels formed by 2 wt % gellan in the presence of 100 mM
KCl or CsCl were obtained in a subsequent study by Ikeda, Nitta,
Temsiripong, Pongsawatmanit, and Nishinari (2004), again
showing networks of brillar strands.
In the same investigation, images were recorded for structures
formed by deposition of very dilute solutions onto freshly-cleaved
mica, as in the study by Gunning et al. (1996) described above.
Solutions of gellan (KGG-1, Table 1) were prepared at a concentration 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 with
the same concentration of M4N gellan. The images obtained for
the M4N salt form showed branched clusters of strands with fairly
uniform height of around 0.5 nm, consistent with crosslinking
solely through unaggregated double helices. Isolated clusters were
also observed for the sample incorporating K, but the individual
strands were longer and their heights extended to w1.2 nm, indicating side-by-side association of double helices. For samples
incorporating Cs, which, as discussed in Section 3.4, is more
effective than K in promoting gelation of gellan, a continuous
network structure was observed, with the height of the strands
again corresponding to aggregates formed by lateral association of
a few double helices.
Strand dimensions in gellan gels have been explored (Yuguchi
et al., 1993, 1996, 1999) by small-angle X-ray scattering (SAXS) for
each of the three samples (Table 1) studied in the Japanese
collaborative research initiative (Section 1). In the rst of these
investigations (Yuguchi et al., 1993) it was concluded that gels
formed by 1.5 wt % KGG-1 (with no added salt) had (at least) three
different 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 relative
mass-fractions of 63, 7 and 30%.
In the second investigation (Yuguchi et al., 1996) NaGG-2, again
with no added salt, was studied at four concentrations: 1.0, 1.5, 2.9
and 5.7 wt %. At the two lower concentrations the increase in crosssectional radius of gyration observed on cooling from the disordered state (at 60 C) to the ordered state (at 10 C) was consistent
with conversion of single chains to unaggregated double helices.
Rheological studies (Miyoshi, Takaya, & Nishinari, 1996) of the same
batch 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 two
populations 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%; the
corresponding 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-1
can reasonably be attributed to its high content of K and divalent cations.
In the third investigation (Yuguchi et al., 1999) 1.5 wt % solutions
of 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-sectional
radii 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 SAXS
scattering proles were tted by combining different proportions
(Table 3) of calculated proles for individual double helices and for
aggregated strands consisting of 2, 4, 8 or 12 double helices. As
emphasised by the authors, these specic models were used for
computational convenience and do not imply that there are no
structures consisting of intermediate (or larger) numbers of helices,
but they give a useful general indication of the size of the brillar
strands in gellan gels, and the way in which they vary with ionic
environment. At the specic concentration of polymer (1.5 wt %)
used by Yuguchi et al. (1999) the strands varied (Table 3) from
individual double helices in the absence of added salt to aggregates
consisting predominantly of around 12 double helices in the
presence of 50 mM Cs.
In a more recent investigation by TEM, Kasapis, Abeysekara,
Atkin, Deszczynski, and Mitchell (2002) observed strands with
diameters up to w40 nm and lengths of several hundred nanometres for Na gellan (0.7 wt %) in the presence of Ca2 at
a concentration (10 mN) around stoichiometric equivalence to the
carboxyl groups of the polymer, demonstrating that divalent
cations give longer, thicker strands than monovalent (Group I)
metal ions.
Finally, measurements of the diffusion coefcient of a probe
molecule (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 into
a 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 by
cooling 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 of
gellan at ambient temperature, or by the internal set procedure
(Sime, 1990) developed for alginate, in which Ca2 ions are released
into solution at a controlled rate by dissociation of an insoluble
calcium salt on reduction in pH by GDL.
Introduction of cations at ambient temperature can also be used
to reinforce existing gels. This was demonstrated in a study by
Nitta, Ikeda, and Nishinari (2006) in which gels of K gellan (KGG-1,
Table 1) were immersed in 1 M solutions of LiCl, NaCl, KCl or CsCl
at 25 C. Large, progressive increases in E0 were observed as
Table 3
Relative proportions (%) of model structures used (Yuguchi et al., 1999) to t SAXS
scattering proles for ordered gellan (1.5 wt % NaGG-3) with no added salt, and with
50 mM LiCl, NaCl, KCl or CsCl.
Double helices per strand
No salt
LiCl
NaCl
KCl
CsCl
100
81
68
17
12
19
32
17
66
27
73
395
immersion time increased, with moduli after w2 h immersion

following the same order of cation-dependence as observed
(Section 3.4) for gels formed by cooling hot solutions
(Li < Na < K < Cs). The rheological changes were accompanied
by large changes in circular dichroism, indicating formation of
double helices in addition to cation-induced association of existing
helices.
5.4. Conformational freedom and release of polymer chains

The ability of gellan double helices to form within the
constraints of an existing gel network was also demonstrated in an
earlier study by Nitta et al. (2001). The gellan used was again
KGG-1, at a concentration of 1.6 wt % in water, with no added salt.
The gels formed under these conditions remained intact up to the
highest temperature at which rheological measurements could be
made, which is consistent (Fig. 18b) with the high content of
divalent cations in the gellan sample (Table 1). However, DSC
heating scans showed a sharp endotherm at w28 C, indicating
melting of unaggregated double helices, to give disordered chain
sequences crosslinked solely by cation-mediated aggregates (as
illustrated schematically at the top of Fig. 26a).
On subsequent cooling (Fig. 27), there was a corresponding DSC
exotherm, essentially equal and opposite to the endotherm
observed on heating. This was accompanied by a sigmoidal increase
in E0 and E00 (Fig. 27a), showing reinforcement of the network
structure remaining after initial heating, and by a sigmoidal
decrease in circular dichroism at 202 nm (Fig. 27b), as seen (Fig. 4)
on conversion of gellan chains from disordered coils to double
helices. It is evident, therefore, that disordered chain sequences
formed during heating of the KGG-1 gel have sufcient freedom of
movement within the surviving crosslinked network to revert to
the double helix state on cooling.
Two recent investigations (Hossain & Nishinari, 2009; Tanaka &
Nishinari, 2007) have shown release of polymer chains from gellan
gels immersed in excess water. Both studies used the same sample
of K gellan. Cylindrical gels of height 1.5 cm and diameter 1.0 cm
(volume z 1.18 mL) were prepared by cooling hot solutions of
gellan in distilled water with no added salt, and immersed in 50 mL
distilled water at 10 C. The concentration of gellan released into
the surrounding liquid was quantied by phenol-sulphuric acid
assay.
In one series of experiments (Hossain & Nishinari, 2009) release
of K ions was also monitored (by ICP). The gellan concentration
used was 2 wt %; the polymer content of the gel (volume 1.18 mL)
was therefore 23.6 mg, which for K gellan (formula weight per
tetrasaccharide repeat unit 684 D) equates to 34.4 mmol.
Measurements were made after immersion of the gel for 1, 2, 4, 6
and 8 h. At the end of this period, the concentration of K cations in
the surrounding solution (volume 50 mL) was 8.19 ppm, which
corresponds to release of 10.5 mmol K. The accompanying
concentration of gellan in the solution was 0.0146 wt %, which
corresponds to release of 10.7 mmol of tetrasaccharide units, in
close agreement with the value of 10.5 mmol for K (as would be
expected from the requirement to preserve electrical neutrality in
both the gel and the surrounding liquid). Studies by size-exclusion
chromatography (Hossain & Nishinari, 2009) showed that the
distribution of molecular mass for the released chains was displaced to somewhat lower values than in the corresponding
chromatograph 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, demonstrating
that the chains released from the gellan network were capable of
forming double helices.
396
progressive slower release on longer immersion, although, since

the volumes and surface areas of the gels were different, the
absolute concentrations of gellan in the surrounding solution were,
of course, also different.
Measurements of E0 throughout the 8 h immersion period
showed an initial increase in modulus over about the rst 3 h of
immersion, and a steep decrease after w5 h. These were attributed
(Hossain & Nishinari, 2009) to, respectively, swelling and erosion of
the gel. Swelling of chemically-crosslinked (entropic) networks
leads to a decrease in moduli (Flory, 1953) in response to the
decrease in polymer concentration. For gellan gels, which are
crosslinked (Section 5.1) by extended regions of aggregated double
helices, the observed increase in modulus may arise from enthalpic
resistance to distortion of the polymer network during swelling.
Similar initial increase in modulus was observed (Hossain &
Nishinari, 2009) when the gellan concentration was raised from
2.0 wt % to 2.5 and 3.0 wt %, but the subsequent decrease at longer
times was much smaller, indicating that the gel networks were
more stable and resistant to dissociation. As found (Section 5.3) by
Nitta et al. (2006) and conrmed by Hossain and Nishinari (2009),
immersion of gellan gels in salt solutions gives a progressive
increase in modulus towards constant values, abolishing the
downturn observed on immersion in water. It seems likely, therefore, that increase in stability of gellan networks with increasing
concentration comes, at least in part, from the accompanying
increase (Section 4) in the concentration of counterions to the
polymer chains.
5.5. Texture of gellan gels
Fig. 27. Rheological, conformational and thermal changes observed (Nitta et al., 2001)
for K gellan gels (sample KGG-1, Table 1; 1.6 wt % in water) on cooling, after heating
from 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 min
at each temperature. (b) Specic ellipticity (202 nm) from circular dichroism (A) and
DSC exotherm (solid line with no symbols), both recorded at a cooling rate of
0.5 C/min.
After immersion for 8 h, the volume of the gel had increased by

w19% and w31% (100 10.7/34.4) of the polymer had been
released into the surrounding solution. Movement of water into the
gel and of polymer into the solution is analogous to the processes
that occur during dissolution of solid particles, with equilibrium
being reached only when the polymer is distributed homogeneously throughout the solvent. Rapid release of gellan (over 10% of
the total polymer content of the gel) occurred within the rst 1 h of
immersion, with slower, progressive release at longer times.
According to classic theory of polymer gelation (Flory, 1953) there is
a residual sol fraction of polymer chains and/or crosslinked
clusters of chains that do not form part of the continuous network,
even when the extent of intermolecular association is much greater
than at the critical gel point (Section 3.2). It seems likely that the
initial rapid increase in the concentration of gellan in the solution
surrounding the gel comes predominantly from release of this sol
fraction, and the subsequent slower increase from progressive
dissociation (dissolution) of the continuous network.
Comparative studies (Hossain & Nishinari, 2009) using gel
samples with the same height (1.5 cm) but smaller diameters (0.5
or 0.3 cm) also showed initial rapid release of gellan and
The technique of texture prole analysis (TPA), developed

initially by General Foods in the 1960s, has been used extensively
by Kelco to characterise gellan gels and to compare their textural
properties 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 consecutive cycles of compression. In the specic procedure used by Kelco
(Sanderson, 1990) the sample is compressed to 30% of its initial
height at 2 inches per minute (w0.85 mm/s), the crosshead of the
instrument is then raised, and a second cycle of compression is
made under the same conditions as the rst.
Three parameters are obtained from the rst compression:
modulus, hardness and brittleness. The modulus is identical to
Youngs modulus (E), although usually expressed in different units
(pounds force per square inch). Hardness is dened as the
maximum force generated in resistance to the rst compression,
and is usually equivalent to break stress (sb), although again
expressed in different units. For some samples, however, maximum
resistance may occur during further compression after initial
failure. The brittleness value is identical to strain at break (3 b),
which means, confusingly, that very brittle materials (i.e. those that
break at low strain) have very low brittleness values, and very
elastic (non-brittle) materials have high brittleness.
Two further parameters can be obtained from the second cycle
of compression: elasticity and cohesiveness. Elasticity, which
characterises the ability of the sample to recover from compression,
is derived from the point at which the descending crosshead
touches the top of the sample and is dened as the height of the
sample at this point expressed as a percentage of the initial height
prior to compression. Cohesiveness, which is intended to indicate
toughness during eating, is measured as the area under the
forceedistance curve obtained in the second cycle of compression
expressed as a percentage of the corresponding area from the rst
compression.
Early studies by this technique (Sanderson & Clark, 1984)

showed that commercial gellan gum gave gels strong enough to be
characterised by compression testing at polymer concentrations as
low as 0.2 wt %, and that hardness values increased initially and
then decreased on progressive reduction in pH or progressive
addition of Na, K, Mg2 or Ca2, as conrmed (Section 3.7) in
later studies under better-dened ionic conditions. It was also
found that brittleness values decreased systematically with cation
concentration (i.e. that the gels became more brittle as more salt
was added).
Subsequently it was demonstrated (Sanderson, Bell, Clark et al.,
1988) that the modulus, hardness, brittleness, elasticity and cohesiveness of agar in traditional Japanese food products could be
matched well using gellan at much (2e3 times) lower concentrations, although agar itself is a very efcient gelling agent. In the
same study it was found that on progressive increase in concentration of gellan at xed concentration of added Ca2 there was
a progressive reduction in brittleness values (i.e. with the gels
becoming more brittle as the polymer concentration was increased)
and a progressive, large, increase in elasticity, as well as the expected increase in modulus and hardness.
TPA has been particularly useful in comparing gellan with other
gelling agents. These can give a wide spectrum of different textures
(Sworn, 2009) ranging from rm and brittle for agar and kappa
carrageenan to soft and exible for gelatin and the synergistic
gels (Morris, E.R., 1995b) formed by mixtures of xanthan with plant
polysaccharides such as locust bean gum (LBG) or konjac glucomannan, with alginate and pectin giving gels of intermediate
texture. The gels formed by commercial deacylated gellan (gellan
gum) typically have brittleness values of around 25e30%, placing
them at the rm and brittle extreme of this continuum.
However, as discussed further in Section 7.2, high acyl gellan
gives gels that lie at the opposite extreme (soft and exible).
5.6. Mobility of water in gellan networks
In an investigation of the dielectric properties of solutions and
gels of Na gellan (NaGG-2, Table 1) by the time domain reectometry 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). The
relaxation time for the high-frequency process agreed well with
that of pure water, and the magnitude of this process demonstrated
that, even in the gel state at low temperature, most of the water had
the same mobility as bulk water. The conclusion that most of the
water in hydrated biopolymer networks is present as free water has
also been reached for other gelling biopolymers and, indeed, for
high-moisture food products (Ablett & Lillford, 1991; Ablett,
Lillford, Baghdadi, & Derbyshire, 1978; Davies et al., 2010). The
process at lower frequency was attributed to bound water. On
cooling through the temperature-range of the disordereorder
transition, there was a large (w8-fold) increase in the relaxation
strength of this process, indicating that water molecules bind more
tightly to the ordered structure of gellan in the gel state than to the
disordered coils in solution. The same conclusion has been reported
for agarose (Watase, Nishinari, & Hatakeyama, 1988) and also for
KGG-1 (Hatakeyama, Quinn, & Hatakeyama, 1996).
The mobility of water in gellan networks was also investigated
by Ohtsuka and Watanabe (1996) using the pulsed eld gradient
stimulated echo NMR method. Samples were prepared by cooling
hot solutions of 2 wt % Na gellan (NaGG-2, Table 1) incorporating
a wide range of concentrations of either KCl or CaCl2. Experimental
values of the diffusion coefcient of water were analysed by
a simple model in which the polymer is regarded as a series of
permeable barriers running parallel to one another and separated
397
by a xed distance, a. One immediate conclusion was that the

diffusional mobility of water in the channels between the barriers
was essentially the same as that of bulk water, consistent with the
dielectric studies (Mashimo et al., 1996) summarised above.
On progressive increase in K concentration, the separation (a)
of the hypothetical barriers increased sharply, passed through
a shallow maximum, decreased sharply, and then levelled out
towards a constant value; permeability of the barriers (p) showed
converse changes over the same range of K concentration (i.e.
decreasing initially, passing through a shallow minimum,
increasing again, then becoming essentially constant). The initial
increase in a, with accompanying decrease in p, was attributed to
conversion from a homogeneous solution to a network structure
with larger void spaces and decreased permeability, and began at
[K] z 20 mM. As shown in Fig. 13, thermal hysteresis of K gellan,
attributed to aggregation of double helices, also became detectable
(Milas & Rinaudo, 1996) at 20 mM K.
The steep decrease in a, with accompanying increase in p,
occurred at [K] z 90 mM, which coincides with the point at which
the force required to fracture K gellan gels (Fig. 19) was found to
decrease (Milas & Rinaudo, 1996). The corresponding changes in
a and p for the gels formed with CaCl2 occurred at [Ca2] z 3.3 mM,
which again agrees well with the salt concentration (Fig. 19) at
which Milas and Rinaudo (1996) observed reduction in gel strength
(force at break) on addition of MgCl2 to Na gellan (bearing in mind
that, as discussed in Section 3.6, Ca2 and Mg2 are essentially
indistinguishable in their interaction with gellan). Thus, although
the physical reality of the simple model used by Ohtsuka and
Watanabe (1996) may be debatable, it seems clear that the
changes in restricted diffusion of water derived by NMR correlate
convincingly with changes in the macromolecular organisation of
gellan observed by other techniques.
K concentrations around the shallow maximum in a and
shallow minimum in p (from w50 to w80 mM) gave transparent,
elastic gels. Only this region of high a and low p, with little
change in either parameter, and the subsequent steep decrease in
a and increase in p, were observed for gels formed with CaCl2,
which is consistent with the observation (Fig. 18) of immediate
gelation at very low concentrations of Ca2, without the preceding
regions of solution and weak gel response (Fig. 12) that occur on
progressive addition of monovalent cations.
Finally, Ohtsuka and Watanabe (1996) found that the plots of
a and p versus concentration of Ca2 superimposed closely on the
corresponding plots for K when moved horizontally along the
concentration axis, indicating that the networks formed by gellan
in the presence of monovalent or divalent cations have the same
overall structure, and the same effect on mobility of water, and
change in the same way when cation concentration is varied,
although the concentration-range over which the changes occur is
much lower for divalent cations than for monovalent (as can also be
seen for the changes in rheology shown in Fig. 19).
5.7. Syneresis
Gellan gels are comparatively stable when stored quiescently at
ambient temperature or under refrigeration, but there may be
some release of uid (syneresis), particularly at polymer concentrations below w0.2 wt % (Gibson & Sanderson, 1997). Syneresis is
undesirable in most food products, and it may be necessary to add
thickeners to prevent it.
Syneresis can also cause problems of slippage when gellan
networks are investigated by rheological measurements under
shear. This was demonstrated (Morris, E.R. et al., 1999) in an
investigation of the effect of NaCl on gelation of Na gellan.
Measurements of G0 and G00 were made using either smooth (cone-
398
and-plate) geometry, or a novel perforated-cylinder arrangement

in which the sample surrounds and penetrates both the moving
and stationary elements, making slippage impossible (Richardson &
Goycoolea, 1994). At low concentrations of added salt, the coneand-plate geometry gave plots of G0 and G00 versus temperature
similar to those shown in Fig. 21. At higher salt concentrations,
where stiffer gels are formed, there was again an initial sharp
increase in G0 and G00 on cooling, but G0 then decreased slightly, and
both moduli uctuated erratically. On switching to the perforatedcylinder geometry, however, these unusual features were no longer
observed, demonstrating that they were artefacts caused by slippage of the sample on the smooth surfaces of the cone and plate.
Release of uid when gellan gels are squeezed can be observed
visually (Gibson & Sanderson, 1997), and was demonstrated
dramatically 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 polymer
concentration of 1.33 wt % in water, with no added salt, and were
compressed at rates ranging from 1000 to 0.005 mm/min. Three
different patterns of response were observed over different ranges
of compression rate: w1 mm/min and higher; w0.02 mm/min and
lower; and intermediate rates. Fig. 28 shows photographs of the
gels taken before, during and immediately after compression at
representative rates within the three ranges (100, 0.1 and
0.005 mm/min), and after the compressed samples had been
soaked 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 returned
almost fully to their original height when applied stress was
removed. On reduction of compression rate to 0.1 mm/min, the
pattern became similar to those shown in regions b and c in Fig. 19:
vertical cracks 3e5 mm in height formed at the surface of the
cylindrical sample and water owed from them. There was still
substantial recovery towards the initial height of the sample when
the crosshead of the instrument was raised, but less than occurred
after more rapid compression (as would be expected from loss of
uid at the slower compression rate). At the slowest rate of
compression studied (0.005 mm/min) no fracture was observed.
Instead, there was continuous, progressive expulsion of water from

the gel until it had been compressed to a at disc 1 mm thick (i.e.
10% of the original height), which showed little recovery when
stress was removed. There was no change in diameter during
compression, so the apparent value of Poissons ratio is zero!
These observations indicate that gellan gels respond to
compression by two different mechanisms: (i) elastic deformation
up to the point of fracture, and (ii) rearrangement of the polymer
network to accommodate the imposed strain, with consequent
expulsion of uid. As the rate of compression is decreased, allowing
more time for rearrangement to occur, the second of these becomes
dominant. Time-dependent reduction in the resistance of gellan
gels to imposed deformation has been observed directly by
measurements of stress relaxation (Morris, V.J. & Brownsey, 1995).
When the compressed gels (Fig. 28) were soaked in water for
two 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 phenomena
demonstrating their dynamic nature.
5.8. Flavour release
It is well known in the food industry that perceived intensities of
avour and taste are lower for gelled products than for uid products (such as sauces and drinks) incorporating the same objective
concentrations of avour compounds or tastants such as sugar or
salt. Gellan causes less suppression than most other gelling agents,
or, as more commonly expressed, it has outstanding avour
release properties. One possible explanation (Gibson & Sanderson,
1997), suggested by release of uid when gellan gels are compressed
(Section 5.7), is that water is released from the gels during mastication, carrying with it avour and taste compounds. An alternative,
though possibly related, interpretation was suggested (Gothard,
1994; Morris, E.R., 1994, 1995a) from sensory and rheological
comparisons of gellan with other gelling polysaccharides spanning
a wide range of different textures (Section 5.5).
The materials studied were xanthan in combination with LBG
(giving exible, elastic gels), commercial gellan gum (giving stiff,
Fig. 28. Compression of K gellan gels (sample KGG-1, Table 1; 1.33 wt % in water at 22.5 1 C) at (a) 100, (b) 0.1 or (c) 0.005 mm/min (Nakamura et al., 2001). Photographs show
the gels (1) before compression (height 10 mm; diameter 11.5 mm), (2) during compression, (3) after release of applied stress by raising the crosshead of the instrument to its
original position, and (4) after soaking in water for 2 days. Fluid released from the gels during compression was removed before the photographs in column 3 were taken; the
compressed gel in 3(c) can be seen as a thin sheet at the bottom of the photograph.
brittle gels) and two others (calcium alginate and the potassium
salt form of kappa carrageenan) that gave gels of intermediate
texture. Polysaccharide concentrations were varied across the full
range at which measurable gels could be obtained and xed
concentrations of sucrose and avouring were included in all
samples. The gels were rated for perceived rmness, sweetness and
intensity of avour by a sensory panel, and characterised by
compression testing to give Youngs modulus (E), breaking stress
(sb) and breaking strain (3 b). As found in earlier studies of thickened
systems (Baines & Morris, 1989), sweetness and avour were suppressed to the same extent. The avour/taste intensities for
samples of equivalent perceived rmness differed widely between
the different gel systems, with highest intensities for gellan and
lowest for xanthaneLBG.
No correlation was found between panel scores for sweetness
and avour and instrumental measurements of modulus and break
stress. However, when avour/taste intensity was plotted doublelogarithmically against breaking strain a good linear relationship
was observed, with progressive reduction in perceived intensity as
the deformation required to break the network increased, and no
systematic discrepancies between the results obtained for the
different gelling agents. It was therefore concluded that avour
release is determined by the extent of deformation required to
fracture the gel and create new surfaces from which release can
occur. The same conclusion was reached in a later investigation
(Bayarri, Rivas, Izquierdo, & Costell, 2007) in which gellan was
compared with only one other gelling agent (kappa carrageenan)
but two different sweeteners were studied and panellists assessed
changes in perceived sweetness over time. There is no necessary
conict between this interpretation and the proposal (Gibson &
Sanderson, 1997) that avour release comes from expulsion of
uid under compression, since release of water could provide
a mechanism for transport of avour/taste compounds across the
fresh surfaces created by fragmentation of the gel network.
5.9. Gellan liquid crystals
In a recent investigation by Nitta, Takahashi, and Nishinari
(2010), Na gellan was partially depolymerised by alkaline hydrolysis (10e45 mM NaOH; 12 h at 50 C). Three samples, denoted as
G-10, G-15 and G-30 in order of increasing molecular weight (i.e.
decreasing concentration of NaOH used in their preparation), were
selected for investigation. When a concentrated (8 wt %) solution of
the sample of lowest molecular weight (G-10) was held quiescently
in a vial at low temperature, it gradually resolved into two layers.
Examination by polarised light microscopy showed that the upper
layer was an anisotropic liquid crystalline phase. The concentration
at which birefringence, diagnostic of anisotropy, became detectable
decreased with increasing molecular weight, as has also been
observed for xanthan (Inatomi, Jinbo, Sato, & Teramoto, 1992; Lee &
Brant, 2002) and for kappa carrageenan in solutions with sodium
iodide (Borgstrm, Egermayer, Sparrman, Quist, & Piculell, 1998).
When solutions of G-10 were cooled through the temperaturerange of the disordereorder transition, G00 increased steeply, as
expected, with an accompanying increase in G0 . At concentrations
above w2.5 wt %, however, the initial increase was followed by
a large decrease on further cooling. Similar behaviour was observed
for G-15 and G-30, but reduction in moduli occurred at progressively lower concentration as molecular weight increased, being
clearly evident at 2 wt % G-30. The highest moduli recorded were at
least an order of magnitude lower than those required to trigger the
slippage artefacts described in Section 5.7, strongly indicating that
the observed reductions were caused by formation of an anisotropic phase, rather than by slippage. Similar changes in moduli
have been reported for other polysaccharides known to form
399
anisotropic liquid crystals, including xanthan (Lee & Brant, 2002;

Maret, Milas, & Rinaudo, 1981; Milas & Rinaudo, 1983), schizophyllan (Fang, Takemasa, Katsuta, & Nishinari, 2004) and methylcellulose (Yin, Nishinari, Zhang, & Funami, 2006). Like these
systems, the solutions studied by Nitta et al. (2010) were all nongelling, which is an essential requirement for formation of liquid
crystals.
6. Effect of sugars
The ability of high concentrations of sucrose, or other sugars, to
induce gelation of high-methoxy pectin under acidic conditions in
production of jams and jellies is well documented (e.g. Rolin, 1993).
Incorporation of sugars has also been shown to enhance the
strength and thermal stability of networks formed by gelling
biopolymers 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 Na gellan was studied in
investigations by Miyoshi, Takaya, and Nishinari (1998) and
Miyoshi and Nishinari (1999b). In both of these the concentration of
gellan was held xed at 1 wt %, and sugar concentrations were
varied within the range 0e72 wt %. The experimental procedures
used were the same as those described in Section 4, where gellan
concentration was varied in the absence of sugar. Concentrations of
sugar were expressed as molarities. Since the molecular weight of
monosaccharides such as glucose and fructose is 180, a 1 M solution
corresponds to 18.0 wt %; for disaccharides such as sucrose
(molecular weight 342), 1 M 34.2 wt %.
In the rst of these investigations (Miyoshi et al., 1998) the
gellan used was NaGG-2 (Table 1) and the sugars studied were
glucose and mannose. When 1 wt % NaGG-2 was cooled in the
absence 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 to
be measured, but became detectable on incorporation of a low
concentration of glucose (0.01 M 0.18 wt %). Addition of 1.6 M
(28.8 wt %) glucose to 1 wt % NaGG-2 gave cooling and heating
curves of G0 and G00 virtually identical to those observed for 2 wt %
NaGG-2 in water, with increase in Tch to w37 C and G0 rising above
G00 at Tsg z 7 C. On further increase in glucose concentration, Tch
continued to increase, with an accompanying steeper increase in
Tsg, as seen (Fig. 23) when gellan concentration was varied in the
absence of sugar, until conformational ordering and gelation
occurred simultaneously at Tch Tsg 41 C when the concentration of glucose had reached 3.5 M (63 wt %). Thermal hysteresis
between cooling and heating scans became apparent at a glucose
concentration of 1.8 M (32.4 wt %) and increased in magnitude as
the concentration of glucose was raised further.
Qualitatively similar changes in Tch and Tsg were observed with
mannose as cosolute, but the sugar concentrations required were
systematically higher than for glucose, and no thermal hysteresis
was observed even at the highest concentration of mannose
studied (63 wt %). Two central conclusions from this investigation
are therefore (i) incorporation of sugars promotes conformational
ordering and gelation of gellan, and (ii) glucose is more effective
than mannose.
In the second investigation (Miyoshi & Nishinari, 1999b) the
gellan used was NaGG-3 (Table 1) and the sugars studied were
glucose, fructose, sucrose and trehalose. The changes in Tch with
increasing concentration of glucose were virtually identical to those
seen (Miyoshi et al., 1998) for NaGG-2, but the accompanying
increase in Tsg was less steep: at 3.3 M (63 wt %) glucose, where
conformational ordering and gelation of NaGG-2 occurred simultaneously, gelation of NaGG-3 (crossover of G0 and G00 ) was not
400
observed until w9 C below Tch, and even at the highest concentration of glucose studied (4 M 72 wt %) Tsg was still slightly lower
than Tch. The most likely explanation for the earlier gelation of
NaGG-2 is that it arises from the higher content of divalent cations
in NaGG-2 than in NaGG-3 (Table 1).
DSC scans showed a systematic increase in peak-maximum
temperatures as concentration of sugars was increased, with no
thermal hysteresis between cooling and heating. The disaccharides
studied gave greater increases than the monosaccharides. The
increases were slightly greater with sucrose than with trehalose, and
much greater with glucose than with fructose. Taken together with
the comparison between glucose and mannose by Miyoshi et al.
(1998), the order of effectiveness in promoting ordered association
of gellan is: sucrose > trehalose > glucose > mannose >> fructose.
One obvious way in which high concentrations of sugars, or
other cosolutes, can promote association of polymer chains is by
replacing most of the solvent. Miyoshi and Nishinari (1999b)
explored this effect by expressing concentrations of gellan relative to the mass of residual water, rather than total mass, and
making comparisons of Tch and Tsg with those observed for the
same concentrations of gellan in water (with no sugars present).
Much of the increase in transition temperatures with increasing
concentration of sugar could be explained in this way, but the
values of Tch and Tsg for mixtures of gellan with the disaccharides
(sucrose and trehalose) were still substantially higher than for
equivalent concentrations of gellan alone, those for mixtures with
glucose were slightly higher, and at high concentrations of fructose
they were lower.
The enhancements observed with sucrose, trehalose and
glucose were ascribed to sugarewater associations in competition
with interactions between water and gellan, and the order of
effectiveness was correlated with dynamic hydration number,
which is determined by the number of equatorial hydroxyl groups
in the sugar (Katsuta et al., 1992; Nishinari & Watase, 1992;
Nishinari et al., 1990). It was suggested that the inhibitory effect of
fructose might be due to attachment of fructose molecules to gellan
by hydrogen bonding, with consequent inhibition of selfassociation of the polymer. Direct attachment of fructose to polymer chains has also been proposed for mixtures with highmethoxy pectin (Tsoga, Richardson, & Morris, 2004b), on the
basis of an intense exothermic process on heating that was not
observed for mixtures with other sugars or polyols (Tsoga,
Richardson, & Morris, 2004a).
Other investigations have focussed on the effect of sugars on
commercial gellan (Kelcogel) at concentrations where gels are
formed in the absence of sugar. Bayarri, Costell, and Durn (2002)
used compression testing to study the effect of sucrose at concentrations in the range 0e25 wt % on three gelling concentrations of
Kelcogel: 0.30, 0.75 and 1.2 wt %. In all cases there was a systematic
increase in Youngs modulus (E), break stress (sb), and strain at
break (3 b) with increasing concentration of sucrose (i.e. with the
gels becoming stronger and less brittle). At the lowest concentration of gellan studied (0.3 wt %) the changes were very small, but
they became clearly evident at the higher concentrations. For the
maximum concentrations of gellan and sucrose studied (1.2 and
25 wt %, respectively), E was w38% higher than in the absence of
sugar, sb was w72% higher and 3 b increased from the very low value
of w17% strain at break to w23%.
Small increases in gel strength with accompanying small
increases in breaking strain continue (Sworn & Kasapis, 1998) as the
concentration of sucrose is raised to 40 wt %, where the strain at
break is w32%. On further increase in sucrose concentration to
60 wt %, however, there is a massive increase in 3 b, with the gels
remaining intact up to w65% strain. Fig. 29 shows illustrative
compression curves for gellan with no added sugar (Fig. 29a) and in
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 show
experimental data; the solid lines are ts calculated using a modied reel-chain model.
Stretch ratio, l d/do, where d is the diameter of the sample during compression and
do is the initial diameter prior to compression (Kawai et al., 2008).
the presence of 60 wt % sucrose (Fig. 29b). The extent of

compression is characterised by the accompanying increase in the
width of the sample (Section 3.1). The curves were tted (Kawai,
Nitta, & Nishinari, 2008) by a reelechain model proposed previously to explain the temperature-dependence of the elastic
modulus of thermoreversible gels (Nishinari et al., 1985). The ts
are so precise that the calculated curves, shown as solid lines in
Fig. 29, are almost entirely obscured by the experimental points
(shown as symbols). The reduction in brittleness on incorporation
of 60 wt % sucrose (Fig. 29b) is evident from the much greater
deformation at break (stretch ratio z 1.7) in comparison with the
sample (Fig. 29a) with no added sugar (stretch ratio z 1.16). The
concentration of gellan (0.8 wt %) in the mixture with sucrose is
somewhat lower than in the sample without sucrose (1.2 wt %) but
the break stress is substantially higher (w32 kPa in comparison
with w7 kPa), demonstrating the effectiveness of sucrose in reinforcing gellan networks.
Partial replacement of sucrose by either glucose or fructose at
a total sugar concentration of 60 wt % gives stronger gels than at
60 wt % sucrose alone (Gibson & Sanderson, 1997). This was
detected in an experiment intended to test for possible degradation
of 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 on
cooling was found to be stronger than before exposure to high
temperature and low pH. This unexpected behaviour was traced to
partial hydrolysis (inversion) of sucrose to glucose and fructose. It
was also found (Gibson & Sanderson, 1997) that values of modulus
at break (i.e. sb/3 b) were higher for gellan gels incorporating fructose than for those incorporating equivalent concentrations of
glucose. The apparent order of effectiveness of sucrose and its
hydrolysis products in strengthening gellan gels is therefore
fructose > glucose > sucrose, which is the reverse of the order
found by Miyoshi and Nishinari (1999b).
It seems likely that the difference arises from the divalent
cations that are present in much larger amounts in normal
commercial gellan gum than in the NaGG-2 and NaGG-3 samples
studied by, respectively, Miyoshi et al. (1998) and Miyoshi and
Nishinari (1999b). Sworn and Kasapis (1998) explored the effect
of small additions of CaCl2 on the modulus (E) of gels formed by
0.5 wt % Kelcogel in the absence of sugar and in the presence of
sucrose, glucose or fructose at concentrations of 20, 40 or 60 wt %.
As discussed in Section 3.7 and illustrated in Fig. 19, the moduli
showed an initial increase and subsequent decrease with increasing
concentration of salt. The maximum modulus was displaced to
progressively lower concentration of Ca2 as the concentration of
sugar was increased. The Ca2 concentration at which the moduli
began to fall were lowest for the gels incorporating sucrose and
highest for those incorporating fructose.
The apparent clash of experimental evidence mentioned above
could perhaps, therefore, be explained as follows. Sugars promote
association of gellan in the order sucrose > glucose > fructose. When
the extent of association in the absence of sugar is well below that
required for optimum gelation, as in the Na gellan systems studied
by Miyoshi and coworkers, gel strength increases in the same order.
However, commercial gellan contains sufcient divalent cations to
give gels that are already at, or close to, the optimum degree of
crosslinking, so that further association in response to the presence
of sugar can cause excessive aggregation (Section 3.7) and thus
weaken the network rather than strengthening it. Gel strength in the
presence of different sugars would then decrease with increasing
extent of further association, giving the reverse order of effectiveness: fructose > glucose > sucrose, as observed.
Mixtures of commercial gellan with low or moderate concentrations (up to w40 wt %) of sucrose or other cosolutes give cooling
and heating curves similar in form to those observed for NaGG-2 at
concentrations above w3.2 wt % in the absence of cosolute: G0 and
G00 are extremely low in the solutions state at high temperature but
increase sharply at the coilehelix transition temperature (Tch) on
cooling, and show substantial thermal hysteresis on heating.
Entirely different behaviour, however, has been reported (Kasapis
et al., 2002; Papageorgiou, Kasapis, & Richardson, 1994) for
mixtures of Kelcogel (0.5 wt %) with very high concentrations
(80e85 wt %) of cosolute. Experimentally, these concentrations
were attained by using 50 wt % sucrose and adding the required
further amount of cosolute as corn syrup of dextrose equivalent
(DE) 42, which corresponds to a number-average degree of polymerisation of w2.4.
At high temperature (90 C) these mixtures gave mechanical
spectra similar to those of polysaccharide gels (Fig. 10a). On cooling
to 5 C there was no indication of the sharp increase in moduli
observed in the absence of cosolute, and no detectable thermal
hysteresis on heating. Instead, there was a smooth, progressive
increase in moduli during cooling, with G00 rising above G0 . Such
behaviour is typical (Haward & Young, 1997; Sworn & Kasapis,
1999) of a material that undergoes vitrication, transforming
from a rubbery solid to a high-viscosity liquid (glass). In mechanical
spectra recorded on completion of cooling to 5 C (Papageorgiou,
Kasapis et al., 1994), G00 remained higher than G0 at all values of
frequency (u) studied (0.01e10 Hz). At the upper end of this
frequency range, log G00 versus log u had the slope of 1 characteristic (Section 3.1) of a liquid, with log G0 versus log u running
roughly parallel. At lower frequencies, however, the slope of both
plots decreased and G0 rose towards G00 , indicating survival of
a crosslinked network within the vitried sample.
Crystalline materials do not undergo vitrication. For vitrication to occur, the sample must be largely amorphous. This led to the
401
proposal (Sworn & Kasapis, 1999) that the networks formed by

gellan in the presence of high concentrations of cosolute consist
predominantly of exible, disordered chains, with only occasional
junctions between them. As discussed in Section 5.1, the resistance
to deformation of such networks would be predominantly entropic
in origin, contrasting with the networks of aggregated strands
(Fig. 26b) formed in the absence of cosolute, where resistance
would be dominated by enthalpy.
Changes in G0 and G00 during cooling suggest that both types of
network are formed at cosolute concentrations between w40 and
w80 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 initial
cooling, both moduli show the smooth, progressive increase
observed at higher concentrations of cosolute and attributed to
vitrication. On reaching the coilehelix transition temperature,
however, the moduli show the steep, sigmoidal increase observed
in the absence of cosolute, demonstrating formation of an enthalpic
network. As the concentration of cosolute is increased, the
magnitude of the sigmoidal transition decreases (Sworn & Kasapis,
1999), until eventually, at w80 wt % cosolute, it becomes undetectable, leaving only the smooth increases from vitrication of the
entropic network.
In an attempt to rationalise the effect of a wide range of sugars and
polyols on gelation of high-methoxy pectin, Tsoga et al. (2004a,b)
proposed that the effect of replacement of water by cosolute might
be partially offset by cosolute molecules clustering around the polymer chains and thus hampering association of chains into a gel
network (i.e. by enthalpically-favourable polymerecosolute interactions competing with polymerepolymer interactions). Although
speculative, this proposal is consistent with the observed behaviour
of gellan in the presence of sugars and other cosolutes such as corn
syrup, as summarised below.
(i) Inhibition of formation and aggregation of gellan double helices
by condensation of cosolute molecules around the polymer
chains would explain the progressive loss of the enthalpic
network as concentration of cosolute is increased, and the
accompanying large decrease in transition enthalpy (DH)
observed by DSC (Al-Marhoobi & Kasapis, 2005; Kasapis, 2006).
(ii) Similarly, it is consistent with only very limited residual
association of gellan chains in the surviving entropic networks
at very high concentrations of cosolute.
(iii) In the presence of soluble solids at concentrations above
w15 wt % commercial gellan gives crystal clear gels
(Sanderson, 1993), without the slight haze that can sometimes
be observed in the absence of cosolute. Increased clarity with
increasing concentration of cosolute (0e35 wt % sucrose or
fructose) has been demonstrated objectively (Tang, Mao, Tung,
& Swanson, 2001) by instrumental measurements of turbidity,
and can again be explained by decreased aggregation from
polymerecosolute interactions competing with interactions
between the gellan double helices.
Finally, Nickerson, Paulson, and Speers (2004) carried out an
investigation under experimental conditions essentially identical to
those used by Papageorgiou, Kasapis et al. (1994) and obtained
broadly similar results. They proposed, however, that gellan in the
presence of high concentrations of cosolute forms gel particles or
gel islands embedded within the cosolute matrix, rather than
a continuous network. This interpretation was strongly challenged
in a spirited response by Kasapis (2006). One of the most direct
lines of rebuttal was that TEM micrographs (Kasapis et al., 2002) for
gellan (0.7 wt %) in the presence of 80 wt % cosolute showed no
features that could be attributed to gel particles, although, as
402
mentioned in Section 5.2, extended brillar strands were successfully visualised in the same investigation for 0.7 wt % gellan in the
presence of 5 mM CaCl2 with no cosolute.
7. Effect of acyl substituents
post-fermentation processing (and, perhaps, in the culture broth

during continued fermentation), which seems more likely than
initial biosynthesis with varying contents of acyl substituents.
Consistent with this interpretation, Jay et al. (1998) detected
substantial amounts of free glycerate in preparations with unusually low values of fg.
7.1. Acyl groups in native gellan

7.2. High acyl gellan
The helical structure of gellan with attached acyl groups (Fig. 1)
was rst explored by using computer modelling to extrapolate from
the known solid-state geometry (Fig. 2) of the deacylated polymer
(Chandrasekaran & Thailambal, 1990). The main conclusion was
that acetyl substituents would lie on the periphery of the duplex
(Fig. 2b), with no modication of the underlying helix geometry,
but that the glyceryl group would lie in the interior of the helix and
force the carboxylate group of the adjacent glucuronate residue to
swing round through an angle of w30 about the C(5)eC(6) bond,
with consequent major changes in the pattern of hydrogen bonding
within and between the participating strands. When diffraction
patterns of the quality needed for detailed analysis were subsequently obtained (Chandrasekaran, Radha, & Thailambal, 1992) it
was found that the actual rotation of the carboxylate group is less
than predicted (w14 ), but that the required separation from the
glyceryl substituent is achieved by the glucuronate residue itself
also undergoing a signicant rotation (17 3 ) about the glycosidic
bonds linking it to the two adjacent glucose residues (Fig. 1).
In normal commercial production of gellan gum, acyl groups are
removed by brief exposure to alkali (KOH) at high temperature
(w95 C). The reaction can be expressed as:
ReCOOeR0 KOH/ReCOOK HOR0

where ReCOO and R0 denote, respectively, the substituent and the
polymer chain. Partial removal can be achieved by limiting the
amount of alkali used. When hydrolysis is carried out in this way, at
elevated temperature with the polymer in the disordered form,
glyceryl substituents are liberated somewhat more rapidly than
acetyl groups. An alternative procedure is to hydrolyse for much
longer times at lower temperature, where the polymer is conformationally ordered. Under these conditions, release of acetyl
groups from the periphery of the double helix occurs far more
rapidly than removal of glyceryl substituents embedded within the
helix. Thus by appropriate manipulation of temperature, alkali
concentration, and hydrolysis time (Baird, Talashek, & Chang, 1992),
it is possible to obtain samples differing widely in the proportion of
repeat units carrying residual acetyl or glyceryl substituents
(denoted here as, respectively, fa and fg). To take account of possible
unintended loss of acyl groups, it is more correct to refer to gellan
prepared without deliberate deacylation as high acyl rather than
native.
In the investigation where the presence of L-glyceryl substituents in high acyl gellan was rst detected (Kuo et al., 1986) it was
reported that every repeat unit carried a glyceryl group, but that
only half the repeating units were acetylated (i.e. fg 1.0; fa 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 0.72e0.87 and fa z 0.52.
In an investigation of mutant strains cultured in their laboratory,
Jay et al. (1998) reported fg 0.49 and fa 0.60 for gellan from
normal (wild type) bacteria, and Mazen et al. (1999) reported
fg 0.80 and fa 0.80 for high acyl gellan that was also produced
and puried in their own laboratory.
A possible explanation for the variability is that the polymer is
expressed initially with stoichiometric content of both substituents, but that some loss of acyl groups occurs during extraction and
Comparison (Sanderson, Bell, Clark et al., 1988) by TPA (Section

5.5) has shown that high acyl gellan gives gels with lower modulus
and 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%, in
comparison with w30%). In contrast to the ease with which gellan
gum gels release uid (Section 5.7), the gels formed by high acyl
gellan show no syneresis (Sworn, 2009). As described below, the
high acyl and deacylated forms also differ substantially in their
response to changes in temperature and ionic environment.
As shown in Fig. 30a, the conformational transitions (monitored
by optical rotation) that accompany formation and melting of high
acyl gellan gels (1.0 wt %) show no detectable thermal hysteresis,
either in water or in 100 mM NaCl (Morris et al., 1996), and there
are only small variations (Fig. 30b) in melting temperature of the
gels and in transition midpoint temperature from DSC heating
scans (Mazen et al., 1999) on varying concentration of NaCl
between 10 and 100 mM, in contrast to the massive changes in
melting temperature observed (Fig. 12) for deacylated gellan over
the same range of NaCl concentrations.
The comparative insensitivity of high acyl gellan to changes in
ionic environment was also observed by Huang, Singh, Tang, and
Swanson (2004) who studied the effect of monovalent cations
(10e200 mM Na or K) and divalent cations (2e80 mM Mg2 or
Ca2) on the temperature of the solegel transition (Tsg). There was
no signicant difference between Na and K, or between Mg2
and Ca2, but the divalent cations gave somewhat higher transition
temperatures 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, but
the plots for the high acyl form were less steep and the transitions
occurred at much higher temperature than for the deacylated
polymer.
Higher transition temperatures are also evident in the DSC
cooling scans (Morris et al., 1996) shown in Fig. 31a for high acyl
gellan (Na salt form; 1.0 wt % in water) in comparison with
deacylated gellan under the same conditions. The other noticeable
difference is that the DSC peak is substantially wider for the high
acyl form, indicating that the transition is less co-operative. Heating
curves for the same samples, recorded in the same investigation but
reported separately (Baird et al., 1992), were essentially equal and
opposite to the cooling curves in Fig. 31a (i.e. with again no indication of thermal hysteresis).
7.3. Blends of high acyl and deacylated gellan
As discussed further in Section 8, mixtures of high acyl and
deacylated gellan give gels with textures that lie between the
extreme brittleness of the deacylated form and the extreme
extensibility of the high acyl form (Morrison et al., 1999). On
cooling from high temperature the mixtures show two regions of
steep increase in G0 (Kasapis et al., 1999), the rst coincident with
the solegel transition of high acyl gellan at high temperature and
the second with the corresponding transition of the deacylated
polymer at much lower temperature.
403
-540
-1
Molar rotation (deg cm dmol )
-560
-580
-600
-620
-640
-660
-680
0
20
40
60
Temperature (C)
80
100
100
Temperature (C)
90
80
70
60
50
40
0
20
40
60
80
100
[NaCl] (mM)
Fig. 30. (a) Variation of optical rotation (436 nm) with temperature (Morris et al.,
1996) for high acyl gellan (1.0 wt %) on heating (lled symbols) and cooling (open
symbols) 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 characterised 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 % high
acyl gellan in the Na salt form (Mazen et al., 1999).
Two separate transitions are also observed by DSC (Kasapis

et al., 1999; Morris et al., 1996), with peaks in the same positions
as those shown in Fig. 31a for the individual constituents, and by
the temperature-course of reduction in intensity of detectable 1H
NMR resonances on cooling and accompanying changes in circular
dichroism spectra (Matsukawa & Watanabe, 2007). It seems clear,
therefore, that high acyl and deacylated gellan do not form double
helices incorporating strands of both types.
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 Na salt form. (b) DSC cooling scans
(0.7 C/min) for gellan samples devoid of acetyl substituents, but with L-glyceryl
groups 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 for
samples A and B are identied by the corresponding lower-case letters (Morris et al.,
1996).
%) of fully deacylated gellan. AFM micrographs for dilute aqueous

solutions (1 g/L) dried on mica showed brillar strands, but no
continuous 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 progressively lower temperatures as acyl content decreased (Fig. 32), but
reduction in modulus on heating moved in the opposite direction,
giving progressively greater thermal hysteresis, which was
accompanied by progressive development of continuous network
structure in the micrographs from AFM. Thus the ability of fully
deacylated gellan to form brillar networks by cation-mediated
association of double helices is progressively restored as acyl
groups are removed from the native polymer.
7.5. Individual roles of glyceryl and acetyl groups
7.4. Partially deacylated gellan

In an investigation by Noda et al. (2008) four samples of gellan
with different degrees of deacylation were studied by oscillatory
rheological measurements and AFM. The content of glyceryl and
acetyl groups in these samples is shown in Table 4, expressed both
on a weight basis and as the fraction of repeat units carrying
substituents (i.e. as fg and fa). Solutions (1.0 wt %) of these materials
in water, with no added salt, showed a sharp, sigmoidal increase in
G0 on cooling, with no thermal hysteresis on heating. The temperature at the onset of the rise in G0 decreased progressively with
decreasing content of acyl substituents, towards the value of
Tch z 26.5 C observed (Table 2) for the same concentration (1.0 wt
Fig. 33 shows changes in G0 observed (Morris et al., 1996) on

cooling and heating for two gellan samples with, respectively,
fg 0.43, fa 0.03, and fg 0.52, fa 0.50. Although both have
a similar, substantial, content of residual glycerate, the rst sample,
which is virtually devoid of acetate, shows pronounced thermal
hysteresis (Fig. 33a), but no hysteresis is detectable (Fig. 33b) for the
second sample, which has a high content of acetate. It would
therefore appear that the presence of acetyl groups on the
periphery of the double helix is the dominant factor in blocking
helixehelix aggregation. However, the temperature-course of
gelation is closely similar for both samples, indicating that acetate
groups have little inuence on the formation of individual double
404
Table 4
Content of acyl groups in gellan samples studied (Fig. 32) by Noda et al. (2008).
Glyceryl (wt %)
Acetyl (wt %)
fg
fa
GG1
GG2
GG4
GG6
10.0
3.0
0.72
0.39
9.1
2.8
0.65
0.36
7.2
2.7
0.50
0.34
5.6
2.5
0.38
0.31
Data are presented as means of duplicate determinations. All samples were in the
Na salt form, with negligible content of other cations.
helices, and that the enhanced stability of the helix structure in

high acyl gellan is due predominantly, or solely, to glycerate.
The effect of glyceryl substituents on the thermal stability of
the gellan double helix was explored in greater detail (Morris
et al., 1996) by using DSC to monitor conformational ordering
of ve gellan samples (identied as A, B, C, D and E) which were
prepared by Kelco from the same fermentation broth by
progressively longer exposure to alkali (25 mM KOH) at 40 C,
where the polymer is in the ordered form. The shortest hydrolysis time used (4 h) was sufcient to give almost complete
removal of acetyl groups from the periphery of the helix. The
glycerate level, however, decreased systematically over time,
giving fg values of 0.615, 0.434, 0.194, 0.083 and 0.038 for
samples A, B, C, D and E, respectively.
Fig. 31b shows the DSC cooling scans recorded for these materials. At glycerate levels above w40% stoichiometric (samples A and
B) the traces show a large exotherm at high temperature, widely
separated from a much smaller peak centred close to 25 C. On
further reduction of glycerate content to below w20% stoichiometric (samples C and D) the two peaks overlap, but with obvious
increase in the relative contribution of the second transition to
overall thermal change. Finally, at low glycerate content (<4%
stoichiometric; sample E) the peaks merge, but the position and
width of the exotherm suggest that it is still a composite of two
(heavily overlapping) transitions.
As shown in Fig. 31b, the rst, major, transition moves to
progressively lower temperature with decreasing content of
glycerate, whereas the temperature of the second, smaller, transition remains essentially constant. The interpretation proposed
by Morris et al. (1996) was that the rst process corresponds to
formation of the high acyl structure, with progressive reduction
in stability as the proportion of missing glyceryl groups increases,
and that the second comes from ordering of chain sequences
totally devoid of glycerate. If the fraction of tetrasaccharide units
lacking glyceryl substituents (1fg) is denoted as f, then the
probability of nding n such units adjacent to one another in the
polymer chain is f n. This was compared (Morris et al., 1996) with
the relative contribution of the second transition to overall
thermal change to give an approximate value of the minimum
Fig. 32. Temperature-dependence of G0 (bold lines; left-hand axis) and tan d (faint lines; right-hand axis) for Na gellan samples (a) GG1, (b) GG2, (c) GG4 and (d) GG6 (Table 4) at
1.0 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 at the same rate; the direction of temperature change is indicated by arrows within the
frames (Noda et al., 2008).
remain conformationally mobile on the periphery of the double

helix (Fig. 2b), introducing a steric and entropic barrier to
aggregation.
100
8. Mixtures and applications
G' (Pa)
10
a
0
20
40
60
80
100
80
100
Temperature (C)
100
G' (Pa)
10
b
0
405
20
40
60
Temperature (C)
Fig. 33. Variation of G0 (10 rad/s; 2% strain) on cooling (B) and heating (:) at
1 C/min for partially deacylated gellan preparations (0.5 wt % in deionised water) with
acyl 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-deacylated

helix structure.
The two DSC peaks (Fig. 31b) for sample C (fg 0.194; f 0.806)
are particularly well resolved, and could be quantied with good
precision. The lower temperature transition contributes w26% of
the total heat change (i.e. f n z 0.26), which is in close agreement
with the calculated value for n 6 (0.8066 0.27). A consecutive
run of six glycerate-free repeating units on each of the participating
strands would correspond to two full turns of the deacylated
double-helix structure (Fig. 2), which seems a physically realistic
length for stable association.
In summary, L-glyceryl groups in high acyl gellan increase the
stability of the double helix by forming additional hydrogen
bonds within and between the participating strands, but destroy
the binding site for metal cations by changing the orientation of
the adjacent carboxyl group (Fig. 2b). The consequent loss of
cation-mediated aggregation (Sections 3.4 and 3.6) reduces gel
strength and eliminates thermal hysteresis. In mixtures of high
acyl and deacylated gellan, each forms its own type of double
helix, with no intermediate structures involving both acylated
and non-acylated chains. Sequences of six or more repeat units
devoid of glycerate terminate the high acyl structure, but are
sufcient 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
In many practical applications, gelling agents are present in

mixtures with other biopolymers, present as natural constituents of
food materials such as fruit, vegetables, meat, sh and eggs, or
added as industrial hydrocolloids to modify texture, create
composite structures, or control syneresis. Applications of gellan
and its behaviour in combination with other biopolymers are
therefore discussed together in this section.
Interactions between two different polymers can be classied as
associative if they are thermodynamically more favourable than
interactions between the individual polymers of each type and
segregative if they are less favourable. The most common
mechanism of association is electrostatic attraction between polyanions, such as negatively-charged polysaccharides, and polycations, such as proteins below their isoelectric point. Less
commonly, associative junctions can be formed between polymers
with the same chain geometry, as is believed to occur in mixtures of
alginate and high-methoxy pectin at low pH, and in synergistic
gelation of xanthan with konjac glucomannan (KGM) or galactomannans such as LBG which, like xanthan, have backbones consisting of (1 / 4)-diequatorially-linked residues. Segregative
interactions can cause separation into a dispersed phase containing
most of one polymer surrounded by a continuous phase containing
most of the second polymer. The two phases may, however, run
through one another, giving a bicontinuous structure like a jelly set
in the pores of a sponge. Alternatively, the two polymers may
remain intimately mixed (without being conned to only part of
the total volumes as in phase-separated bicontinuous networks)
and form two separate gels permeating through one another, to
give an interpenetrating network (IPN) structure (Morris, E.R.,
1990; Morris, V.J., 1986).
Mixtures of Na gellan (NaGG-2, Table 1) with KGM (MW 950 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) after
cooling to low temperature (0 C). Mechanical spectra recorded
under these conditions had the form typical (Fig. 10a) of a gel
network, although the individual constituents of the mixture showed
only solution-like response. On addition of monovalent cations (Na
or K) G0 and G00 of the mixtures increased and the solegel transition
moved to higher temperatures. Low concentrations of divalent
cations (Mg2 or Ca2) also promoted gelation of the gellaneKGM
mixtures, but higher concentrations decreased the moduli, as found
(Fig. 19) for gellan alone. The proposed interpretation was that KGM
enhances the strength and thermal stability of gellan networks by
binding to the surface of aggregated assemblies of double helices. A
similar interpretation has been proposed for mixtures of KGM (or
galactomannans) with agarose, furcellaran or kappa carrageenan (as
reviewed by, for example, Morris, E.R., 1990, 1995b).
Dried lms cast from mixtures of commercial gellan (Kelcogel)
with KGM (MW 1320 kD) were studied by Xu, Li, Kennedy, Xie, and
Huang (2007). Maximum tensile strength was observed when the
KGM content of the lms was around 70%, which is in reasonable
agreement with the mixing 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-angle
X-ray diffraction analysis of the dried lms (Xu et al., 2007) indicated that the crystalline packing found for gellan or KGM alone
was inhibited by hydrogen bonding between the two polymers, and
406
Fourier-transform infrared (FT-IR) spectra suggested that the

bonding was predominantly between the carboxyl groups of gellan
and the acetyl and hydroxyl groups of KGM. It was suggested (Xu
et al., 2007) that gellaneKGM lms incorporating an antimicrobial agent (nisin) could be used in active packaging of
food products to extend their shelf-life.
Tamarind xyloglucan has the same (1 / 4)-diequatorial backbone geometry as KGM, but is solubilised by monosaccharide and
disaccharide sidechains rather than by acetyl substituents.
Mixtures of this material with Na gellan were examined (Nitta
et al., 2003; Nitta & Nishinari, 2005) at a total polymer concentration of 1.0 wt %, where the individual polysaccharides are nongelling. The mixtures converted from solutions to gels on cooling.
The temperature at the critical gel point (Section 3.2), where tan d is
independent of frequency (u), increased with increasing content of
xyloglucan, from 21 C at 0.6 wt % gellan plus 0.4 wt % xyloglucan to
25 C at 0.3 wt % gellan plus 0.7 wt % xyloglucan. Values of the
common slope of log G0 and log G00 versus log u within this range of
compositions varied between 0.31 and 0.42, closely similar to the
values of 0.32e0.43 observed (Hossain et al., 1997) for iota
carrageenan.
The solegel transition of the gellanexyloglucan mixtures was
accompanied by an exothermic transition in DSC, at higher
temperature than the exotherm from the coilehelix transition of
gellan alone. As the concentration of xyloglucan in the mixtures
was increased, the magnitude of the higher-temperature transition
also increased, until ultimately it became the only thermal process
detectable by DSC. The same progressive replacement of the
conformational transition of gellan alone by a new transition at
higher temperature was also seen in DSC heating scans. Circular
dichroism spectra showed an increase in ellipticity at 202 nm on
heating through the temperature-range of the rst transition in
DSC, as observed (Fig. 4) for gellan alone, but on further heating
through the range of the second transition the ellipticity decreased,
indicating dissociation of a different type of intermolecular
structure.
It would appear, therefore, that both KGM and tamarind xyloglucan associate with gellan to give coupled networks (Morris,
V.J., 1986) in which the participating polysaccharides are directly
attached to one another. In the mixtures with KGM, gellan retains
its double-helix structure, whereas in the mixtures with tamarind
xyloglucan self-association of gellan through double helices
appears to be replaced by a new type of association between gellan
and xyloglucan chains.
Direct association between gellan and gelatin by electrostatic
attraction (complex coacervation) was explored by Chilvers and
Morris (1987), using commercial gellan (Gelrite). The gelatin was
obtained from pig skin by extraction with acid, giving an isoionic
point of pH 8. Mixtures were prepared at a temperature (60 C) well
above the solegel transition temperatures of the individual polymers. Coacervate droplets (1e100 mm diameter) were formed at pH
values in the range 3.5e5.0, where the gelatin had a net positive
charge, and the gellan a negative charge. The droplets gelled on
cooling.
The potential use of gellanegelatin coacervates to encapsulate
oils or solid particles was demonstrated by adding model substrates
(parafn oil, sunower oil, aluminium powder or beads of ion
exchange resin) to the biopolymer mixtures in the solution state,
reducing pH into the range required for coacervation, and cooling to
gel the coacervate layer that had formed around the core material.
The resulting beads were then xed with glutaraldehyde, washed,
and dehydrated with isopropanol to yield concentrated slurries or
free-owing powders.
Mixtures of K gellan with kappa carrageenan, also in the K salt
form, were found (Nishinari, Watase, Rinaudo, & Milas, 1996) to
give two separate transitions in DSC, one at about the same

temperature as for carrageenan alone and the other at higher
temperature, about the same as for gellan alone, which argues
against any association between the two polymers. However,
values of jh j obtained for solutions prepared at a total polymer
concentration of 0.25 wt % with varying proportions of gellan and
kappa carrageenan were substantially higher than those of the
individual polymers, reaching a maximum at w2:8 gellan:carrageenan. This was attributed (Nishinari, Watase et al., 1996) to phase
separation, with consequent increase in the effective concentration
of each polymer when conned to only part of the total volume. At
higher total concentrations of polymer (2e5 wt %), where gels were
formed, E0 dropped below the values observed for the individual
polysaccharides, reaching minimum values at w1:1 gellan:carrageenan. This can again be explained by phase separation, with the
weaker network forming the continuous phase and the stronger gel
conned to dispersed particles.
Phase separation has also been reported (Papageorgiou, Gothard
et al., 1994) for mixed gels of commercial gellan (Kelcogel) with
calcium alginate. Incorporation of moderate concentrations of
gellan (0.1e0.3 wt %) increased the strength (break stress) of the
gels formed by 2.0 wt % alginate, but the breaking strain was
unaffected, indicating that the brittle gellan network was distributed as dispersed particles embedded in a more elastic calcium
alginate matrix. At higher concentrations of gellan (above w0.7 wt
%), however, the rheological response to addition of Mg2 cations
suggested a bicontinuous (phase-separated) network structure,
although the possibility of phase inversion to a continuous gellan
network surrounding dispersed particles of calcium alginate gel
could not be eliminated conclusively.
Mixtures of K 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 kappa
carrageenegellan mixtures, and a phase-separated network was
again proposed. A subsequent study by Amici, Clark, Normand, and
Johnson (2000), however, found no evidence of the large increase
in turbidity expected to occur on phase separation, or of biphasic
structures in images obtained by confocal microscopy or TEM.
Instead, the TEM micrographs showed a homogeneous distribution
of both polymers, and rheological analysis was entirely consistent
with interpenetrating networks of gellan and agarose.
Initial formation of an interpenetrating network structure was
also proposed (Clark, Eyre, & Ferdinando, & Lagarrique, 1999) for
mixtures of commercial gellan (Kelcogel) with gelling potato maltodextrin (Paselli SA2), although interpretation was complicated by
subsequent self-association of the maltodextrin component into
large aggregates.
Mixtures of high acyl and deacylated gellan show a smooth
progression of modulus and brittleness values from one extreme to
the other as the relative proportions of the two components is
varied from high acyl alone to deacylated alone (Morrison et al.,
1999). There is no indication of the discontinuity in properties
that would be expected from phase separation, which again
strongly indicates an interpenetrating network structure.
Mixtures of Na gellan (NaGG-3, Table 1) with sodium and
calcium salts of hyaluronic acid were studied by Mo, Kubota, &
Nishinari (2000). Incorporation of Na hyaluronate raised the
values of both G0 and G00 observed (Fig. 21a) for 0.5 wt % NaGG-3, but
had little effect on the changes that occurred on cooling through
the temperature-range of the disordereorder transition, and the
mixtures did not gel. Gelation was, however, observed for mixtures
of NaGG-3 (0.1e0.3 wt %) with Ca2 hyaluronate. At pH 7, there was
a slow, progressive increase in G0 and G00 when the mixtures were
held for 2 h at 25 C, and mechanical spectra recorded at the end of
this holding period had the form typical (Fig. 10a) of true gels. On
reduction in pH to 2.5, the moduli increased more rapidly, reaching

constant values within w30 min, and the resulting gels were much
(w20 times) stronger. They were also more transparent than those
formed at pH 7, although gellan alone gives turbid gels at low pH
(Section 3.7). Gelation of mixtures of Na gellan with Ca2 hyaluronate can, of course, be explained, at least in part, by interaction
between the gellan component and Ca2 cations from the hyaluronate, but substantial further research would be required to
unravel the complexity of other possible macromolecular and ionic
interactions in this system.
Mixtures of commercial gellan (Gelrite) with a wide range of
starches (native and chemically-modied) were studied by
Sanderson, Bell, Burgum, Clark, and Ortega (1988), using a standard
Brabender Amylograph procedure in which samples are heated
from 50 to 95 C over 30 min, held at 95 C for 30 min, and cooled to
50 C over a further 30 min period, with measurement of viscosity
throughout. Gellan (0.5 wt %) had little effect on the maximum
viscosity generated by 4.5 wt % starch during heating, in contrast to
other hydrocolloids such as xanthan and carboxymethylcellulose
which can give undesirably high values of peak viscosity. The starch
pastes formed in the Amylograph were cooled overnight, and then
sheared. The viscosities of the pastes containing gellan were
reduced signicantly by shearing, but remained higher than those
of the corresponding pastes of starch alone, suggesting that gellan
could be useful in preparation of a variety of starch-based llings,
which are generally prepared by cooking, stored in bulk, sheared
during lling into their nal containers, and allowed to recover
viscosity on standing.
Most gelling agents had well-established areas of application
long before there was any proper molecular understanding of their
mechanisms of gelation. Fundamental research on gellan, by
contrast, has proceeded alongside development of practical applications (Sanderson, 1990; Sanderson & Clark, 1984).
The rst applications of commercial gellan (Gelrite) were in
growth media for microbial cultures, as a replacement for agar
(Harris, 1985; Lin & Cassida, 1984; Morris, V.J., 1995). Gellan
substrates have the advantages of clarity, purity, and the ability to
withstand prolonged incubation at high temperature. Gellan was
also identied (Shimomura & Kamada, 1986) as a promising
substitute for agar in plant tissue culture, because of the absence of
impurities found in agar, the lower polysaccharide concentrations
needed, and the greater clarity of the gels, allowing clearer observation 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-line
at the web address shown at the end of Section 1. Examples given
below are from the same source, unless other references are cited. A
list of potential food applications broadly similar to that in Table 5
was published by Sanderson and Clark (1984) before gellan was
cleared for use in food (Section 1).
Preparation of gellan solutions at factory scale is more difcult
than in the laboratory. The following concise description of the
problem and the way in which it can be addressed is quoted
verbatim from Sanderson (1993). In low solid systems, calcium
ions need to be removed to allow gellan gum to hydrate. This is
easily done using a sequestrant such as sodium citrate. Unlike low
levels of calcium, low levels of sodium ions from the sequestrant
do not inhibit hydration. When gelation subsequently takes place,
usually brought about by cooling, it is sometimes but not always
necessary to add back ions to obtain optimal gelation. For acidic
products such as jams, jellies and confectionary, acidication of
the hot solution liberates calcium that is bound to the citrate to
boost gel strength. Simply stated, the essence of Kelcogel gellan
gum technology is calcium ion control through the use of
sequestrants.
407
Table 5
Typical food applications of gellan.
Major food area
Typical products
Confectionery
Jams and jellies
Starch jellies, pectin jellies, llings, marshmallow

Reduced calorie jams, imitation jams, bakery
llings, jellies
Fabricated fruits, vegetables, meats
Dessert gels, aspics
Instant desserts, canned puddings, pre-cooked
puddings, pie llings
Bakery icings, canned frostings
Ice cream, gelled milk, yogurt, milkshakes,
low-fat spreads, dips
Fruit, milk-based, soy and carbonated drinks
Batters, breadings, coatings, adhesion systems
Fabricated foods
Water-based gels
Pie llings and puddings
Icings and frostings
Dairy products
Beverages
Films/coatings
The same difculty of hydration/dissolution does not, however,

occur in production of confectionary, where high concentrations
of sugar and/or other soluble solids are present and gellan forms
only a sparingly-crosslinked network (Section 6). Gellan can be
used as a replacement for gelatin in marshmallows and sweets
(candies) such as Gummi bears, giving products acceptable to
vegetarians and followers of religions that forbid materials from
pigs or cattle. Another advantage is heat-stability. Marshmallows
prepared from gellan and starch do not melt when added to hot
cocoa or when baked in cake mixes (Sanderson, 1993). Sweets
structured by gellan, alone or in combination with other gelling
agents such as xanthan/LBG, do not have the undesirable tendency
to stick to one another when exposed to high ambient temperature, as often happens when gelatin is used. Sworn (2009) has
reported detailed formulations for jelly sweets incorporating
gellan 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 baked
products.
Gellan can also be used, in complete or partial replacement of
gelatin, to improve the characteristics of savoury gels or aspics in
meat, sh or vegetable products, and to raise the settingtemperature of gelatin desserts, removing the need for refrigeration. Incorporation of gellan in starch-based puddings gives additional structure and stability, while still retaining the characteristic
thick and pasty consistency. Gellan can also be used in production
of 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 were mitsumame jelly cubes, hard red bean
jelly, soft red bean jelly, and tokoroten noodles, and the concentrations of gellan required were, 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 uid weak gel dispersions of ordered
gellan (Section 3.3) have been described by Oomoto, Uno, & Asai
(1999) and by Sworn (2009), who also reported uid-gel formulations for beverages prepared with and without incorporation of
fruit juice. Low concentrations (w0.12 wt %) of ordered gellan are
also effective in stabilising soy milk and soy beverages.
Novel beverages can be obtained by incorporating beads of
gellan gel. Beads can be prepared easily (Section 5.3) by dripping
a solution of gellan, with appropriate avouring and colour, into
a solution containing Ca2. A similar procedure can be used to form
carbonated beads, by dispersing calcium carbonate in a gellan
solution and dripping the dispersion into a solution of citric acid, or
into fruit juice. The consequent reduction in pH triggers gelation of
408
the gellan, and converts calcium carbonate into carbon dioxide,

which is trapped within the gelled beads, and released on storage in
a stoppered bottle or other sealed container to give a carbonated
drink. Gellan beads can also be used as carriers of savoury ingredients 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 sequestrant, and the network that forms on cooling can suspend cocoa
particles in chocolate milk drinks, and stabilise products such as
milkshakes, ice cream, sour cream and yogurt. Low concentrations
of gellan (w0.05 wt %) can extend the shelf-life of acid milk drinks,
probably by electrostatic association of acid-casein fragments to the
gellan weak gel network, restricting sedimentation of the casein
(Kiani, Mousavi, Razavi, & Morris, 2010).
Gellan can also be used to form a protective lm around materials such as chicken, sh, cheese, potatoes and other vegetables,
and dough-coated products such as egg rolls, to reduce absorption
of oil during frying. Breaded products can be prepared in a similar
way, by dipping into, or spraying with, a solution of gellan, followed
by breading, then freezing or partial baking. Subsequent heating in
a conventional or microwave oven gives a product similar to those
obtained by frying, but with lower fat content. Surface coating with
gellan has also been suggested (Nussinovitch & Hershko, 1996) as
a way of extending the shelf-life of vegetables, including specically
garlic.
A related application of gellan is as an adhesion system for
surface coating of products such as crackers (biscuits), cookies
(buns), potato chips (crisps), corn chips, pretzels and rice cakes. A
dilute solution of gellan is sprayed, as a ne mist, onto the surface of
the product. Spices, avouring or sweeteners can be incorporated
in the solution, or applied to the surface of the product immediately
after spraying. The adhesion system can also be used to attach other
materials, such as fruit pieces, herbs, or dairy-based powders.
Gellan has recently (January 2011) been listed as an allowed
ingredient for use in organic foods and beverages by the USDA
National Organic Program (http://www.foodingredientsrst.com/
news/KELCOGEL-Gellan-Gum-Approved-for-Use-in-Organic-Foodsand-Beverages.html), which should add to its existing use by the
food industry.
Gellan also has a wide range of established or potential nonfood uses, in addition to the applications in microbiology and
tissue culture mentioned above. The weak gel properties of gellan, coupled with clarity and preservation of network structure at
high temperature, can be exploited in creams and lotions, including
suntan lotions and sunscreens, hair-care products such as shampoos and conditioners, toothpaste, and gelled air-fresheners. Gellan
can also be used in paper, to increase its strength and improve its
performance in ink-jet printing, and in pharmaceutical formulations for sustained release of drugs (Alhaique et al., 1996; Kubo,
Miyazaki, & Attwood, 2003).
It has also been suggested (Norton et al., 2011) that acid-induced
gelation of gellan at gastric pH could induce satiety by structuring
the contents of the stomach, and thus be useful in combating
obesity.
In view of its short history relative to that of other gelling agents,
it seems likely that many further advances will be made before
gellan comes close to realising its full potential. These will
undoubtedly include identication of new areas of application, and
formulation of novel or improved products. An additional
approach, however, might be informed manipulation of acyl
content, by variant cultures or controlled deacylation (Section 7.1),
to tailor the properties of gellan to the requirements of specic
applications. Although the feasibility of this approach has already
been demonstrated (Sections 7.4 and 7.5), it has not yet been
exploited on a commercial scale.
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Food Hydrocolloids 28 (2012) 373e411

Contents lists available at SciVerse ScienceDirect

Gelation of gellan e A review

* Corresponding author. Tel.: 81 6 6605 2818; fax: 81 6 6605 3086.

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

(Jansson, Lindberg, & Sandford, 1983; ONeill, Selvendran, & Morris,

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

Throughout this review, however, we will use the term gellan to

In the rst phase of the Japanese collaborative research initiative,

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

International Hydrocolloids Conference. This included an informal

Fig. 2. Double helix structure of deacylated gellan (Chandrasekaran, Millane et al.,

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

counterions. These three parameters are not normally identical to

where CP is the concentration of charged groups on the polymer,

d1=Tm =dlog aT Rfc fh =DH

The slope of 1/Tm versus log aT is determined by two factors: the

2.3. Conformational transitions of gellan in solution

Fig. 3. Temperature-dependence of optical rotation (302 nm) for 1.2 mM (0.086 wt %)

approximately linear, increase in (negative) optical rotation with

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

exotherms on cooling and corresponding endotherms on heating

even at the lowest temperature studied (10 C). DSC exotherms

Fig. 6. Temperature-dependence of reduced specic viscosity on heating (:) and

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

Fig. 7. Temperature-dependence of intrinsic viscosity, [h], for Na gellan in 25 mM

salts of the same cations, and measuring midpoint temperature of

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

In an early investigation, Brownsey, Chilvers, IAnson, and

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

Fig. 9. Inverse of transition midpoint temperature (Tm) on cooling as a function of the

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

claried using a 0.45 mm lter, but Okamoto et al. (1993), using

(stress, s) as the extent of compression (strain, 3 ) is increased are

Dividing jG j by the frequency of oscillation (u) gives the

jh j jG j=u

tan d G00 =G0

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

oscillation is increased. As polymer concentration is raised, a point

3.2. Critical gel point

Intermolecular association of polymer molecules in solution

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

The onset of intermolecular association into soluble clusters is

(milled seed husk of Platago ovata Forsk). The common feature of

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

The sheared preparations were visually cloudy, and confocal

it became the only detectable process. Closely similar behaviour for

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

Fig. 12. Effect of total concentration of Na on peak-maximum temperatures (Tmax) for

the highest gellan concentration studied (1.3 wt %) is about 30

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

sectional radius of gyration for the ordered form (at 10 C) were

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

3.5. Gelation of gellan with Me4N cations

gellan at high concentrations of Me4NCl was reported almost 25

In a continuation of the work described in the previous section, the

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

temperature (Fig. 18b) increasing linearly with Ca2 concentration,

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

Fig. 18 was to avoid such complexity by using solutions in which the

maximum in modulus, the gels were softer and their disintegration

The rst step in gelation of gellan is conversion of the polymer

Dividing jG j by the frequency of oscillation (u) gives the

jh j jG j=u

15 4.5 67.5 mM Na, which is in close agreement with the