Role of Substituents on the Properties of Some Polysaccharides

Biomacromolecules 2004, 5, 1155-1165

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Role of Substituents on the Properties of Some Polysaccharides M. Rinaudo* Centre de Recherche sur les Macromole´ cules Ve´ ge´ tales, CNRS, associated with Joseph Fourier University, BP 53, 38041 Grenoble Cedex 9, France Received November 17, 2003; Revised Manuscript Received February 23, 2004

This paper concerns the influence of the chemical structure on the physical properties of some polysaccharides. Especially, we proposed to discuss the role of the substituents on these properties. In some cases, noncarbohydrate substituents play a minor role on rheological properties in the presence of a salt excess as shown on xanthan and succinoglycan. The rheology of aqueous solution of these stereoregular polysaccharides is controlled by the conformation (helical conformation) whose stability is not largely influenced by these substituents. On the other hand, the interaction between galactomannan and xanthan depends on the presence of acetyl substituents on xanthan but also on the xanthan conformation. However, for polymers such as gellan, XM-6 or BEC 1615, complete deacetylation induces the ability to form physical gels in given thermodynamic conditions. The presence of carbohydrate substituents or short side chains was also examined. Especially in the gellan family, the role of position of substitution (position 3 on the glucose unit C or position 6 on the A glucose) was presented. It is concluded that the substituents giving the higher stability for the helical conformation (higher ∆H and Tm values) also cause a lower salt sensitivity for the helical stability. The role of the substituents on the properties is also described for natural polymers and their chemically or enzymatically modified derivatives. 1. Introduction In this paper, the case of some known bacterial exopolysaccharides will be mainly examined; these polymers (usually water soluble) are mainly produced by bacteria (or some fungi) and often excreted in the fermentation medium. After purification, they can be characterized using the usual techniques of polymer science. An advantage of these polymers is that they have a regular chemical structure based on 2 up to 8 sugar repeat units. In addition, they are stereoregular and they may adopt an ordered conformation (helical conformation formed by one up to three chains). The stiffness of the molecules depends strongly upon this conformation. A few examples examined in our laboratory after similar purification treatments and using the same experimental techniques will be described in which the role of the substituents, independently of the backbone, is clearly demonstrated. As shown, the substituents will play a role on interchain and/or intrachain interactions. Some evidences were described in the literature. Gellan, as produced by bacteria and in its native structure, contains two different substituents per repeat unit: L-glyceryl groups stabilize a double helix whereas acetyl groups inhibit gelation.1,2 Absence of these substituents gives a strong and clear gel, able to compete with agarose or gelatine. Xanthan molecule contains also two types of substituents: pyruvyl and acetyl groups.3 The substituents play a very moderate role on conformational stability and rheology * To whom correspondence should be addressed. Phone: 33 476037627. Fax: 33 476547203. E-mail: [email protected].

of xanthan in aqueous solutions, but acetyl groups are shown to control the interactions with other polysaccharides such as galactomannans.4 A new polysaccharide was recently studied in our group: the name is YAS 34 and the trade name Soligel (Soliance Cy, France).5,6 The structure is based on an eight-sugar repeat unit and it contains acetyl substituents; in the native structure, it stabilizes under a double helix conformation. It is a thickener in aqueous solution, but after a controlled thermal treatment causing a partial modification of the acetyl groups, it turns to a thermoreversible physical gel. After complete deacetylation, no more helical conformation exists nor gel. Many other examples can be discussed for bacterial polysaccharides. Atkins et al. have mainly investigated these systems from solid state by X-ray diffraction7-9 and Chandrasekaran et al. also developed molecular models.10-13 Some interesting examples of the role of substituents can also be found in the field of chemically modified natural polysaccharides such as chitin and cellulose: deacetylation of insoluble chitin turns to a water soluble irregular derivative in acidic conditions (chitosan);14 introduction of hydrophobic substituents (-CH3) on cellulose (rich in hydrophilic -OH groups) allows to transform cellulose to water soluble or organo-soluble derivatives depending on the degree of substitution (DS).15 In these cases, not only the DS but also the distribution of the substituents along the chain are very important. This paper will demonstrate the role of substituents and their location on the physical properties for some examples of polysaccharides.

10.1021/bm030077q CCC: $27.50 © 2004 American Chemical Society Published on Web 04/13/2004

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Figure 1. Some characteristics of polysaccharides.

Figure 2. Classifcation of polysaccharides.

Figure 3. Main physical properties of some water soluble polysaccharides.

Figure 4. Origins of some water soluble polysaccharides.

2. Origin and Main Characteristics of Polysaccharides Polysaccharides form a large series of polymers with different molecular structures and very different physical properties (Figures 1 and 2). Consequently, they can find applications in different domains such as cosmetics, food, paints, and water clarification. Among the water soluble polysaccharides, some have thickening properties, i.e., they increase the viscosity of the solvent (xanthan or succinoglycan) or gelling properties depending on the thermodynamic characteristics of the medium (deacylated gellan,

alginates, agarose, curdlan) or suspending and stabilizing characteristics for dispersed solids or emulsions (xanthan)16 (Figure 3). Bacterial polysaccharides are very important polysaccharides. They are produced by fermentation in reproducible conditions (Figure 4), and based on stereoregular structure, they adopt an ordered conformation usually assumed to be a helical conformation as found in the solid state. This characteristic exists in solution in given thermodynamic conditions, and a conformational transition (helixcoil transition) is often observed when the temperature

Role of Substituents on Polysaccharide Properties

Figure 5. Chemical structure of the different polysaccharides in the gellan family.

increases (Tm is defined as the temperature of half conformational transition in given thermodynamic conditions) or when the ionic concentration decreases due to electrostatic repulsions increase between neighbor ionic sites on the chain. The helical chain conformation gives a higher local stiffness than the coiled conformation. The stiffness also in some conditions induces the packing of the chains going to physical gel formation. In addition, polysaccharides are rich in -OH groups able to form intra or intermolecular H-bond interactions. This organization controls the solubility of the polymers then also the rheological behavior (as we will show with galactomannan), the stability of the helical conformation and that of the physical gels obtained as example with gellan or agarose. 3. Influence of Carbohydrate Substituents Carbohydrate side groups modify the physical properties of the polysaccharides as it can be demonstrated with the gellan family consisting of polysaccharides obtained from different strains.17-19 The chemical structure of the repeating units is given in Figure 5 comparing five bacterial polysaccharides. Gellan is produced by the bacteria Sphingomonas elodea; the structure is linear based on a four sugars repeating unit (sugars A-D). In addition, it contains in the native state two different non-carbohydrate substituents; their role will be discussed later.1 In the absence of these substituents, the deacylated gellan structure can be compared with that of deacetylated welan and S-657, which have a one sugar and two sugar side chain respectively grafted on the position 3 of the C glucose unit.20-22 A new polysaccharide, RMDP17 also named I-886, also belongs to this usual series of polysaccharides. It was demonstrated that its structure is very similar to that of rhamsan but containing a 2-deoxy unit on the B unit.23 Rhamsan and RMDP 17 have a two sugars side chains grafted on the 6 position of the A unit of gellan. All these polysaccharides are acetylated in the native state and can be easily deacetylated by alkali.17 The later four polysaccharides, whatever the type of side chain substituents, are non gelling systems, contrary to deacetylated gellan. The

Biomacromolecules, Vol. 5, No. 4, 2004 1157

stability of the conformations for the acetylated and deacetylated polysaccharides was examined and compared using the same approaches (namely optical rotation, microcalorimetry, circular dichroism, and NMR).17,24 Some results are compared in Table 1. From this table, it is clear that wellan and S-657 conformations are much more stable than gellan due to the presence of a side chain on the C unit; from molecular structure discussed by Chandrasekaran and al.,13,25 the side chains fold back on the helix and shield the carboxylate groups from the surrounding molecules, in agreement with the higher stability of the double helix. The lower influence of the ionic concentration is predicted. On the contrary, rhamsan and RMDP 17 are destabilized just like gellan by deacetylation.24 The linkage of the side chain in the 6 position of the A glucose unit is more flexible with no shielding of the carboxylate groups, but it stabilizes the double helix at low temperature by H bond formation as described by Chandrasekaran et al.13 It was shown from X-ray diffraction that rhamsan and RMDP 17 adopt a gellanlike double helix structure. The behaviors of deacylated gellan and deacetylated rhamsan and RMDP17 are compared in the following; the small structural difference between rhamsan and RMDP 17 will be discussed later. The salt sensitivity is larger for gellan than that for rhamsan which is just a little larger than that for RMDP17 (Figure 6). It is seen that, for a given ionic concentration (CT), Tm is higher for deacetylated RMDP 17 than deacetylated rhamsan and confirms the larger values of enthalpy found for RMDP17 (Table 1). In Table 2, the change in Tm for an increase of salt concentration by a factor 10 indicates also that the more stable conformation stabilized by intrachain interactions gives the lower salt sensitivity. The larger interhelical separation due to the side chain prevents the aggregation of double helices as well as the gelation. It explains the difference of behavior with that of gellan. This is demonstrated by the comparison of rheological curves for 10 g/L solution prepared in 0.1M NaCl at 25 °C. Gellan gives a strong gel (G′ ) 10 000 Pa in all of the range of frequencies with G′ . G′′) when rhamsan and RMDP17 behave as viscoelastic fluids with much lower storage modulus (at 1 Hz, G′ ) 100 and 10 Pa respectively) The results are compared in Figure 7 for the three deacetylated polysaccharides.2,24 The interchain interaction is related to the side chain length, but it is also shown that RMDP 17 is less interacting (and the conformation is more stable) than rhamsan. Another interesting example on the role of carbohydrate side groups is found in glucomannan and galactomannan series. The last case was particularly examined and is discussed below. Galactomannans are natural polysaccharides that are extracted from plant seeds and are named tara gum, guar gum, carob gum (locus bean gum), etc. and differ in the mannose/galactose ratio (M/G). They are used as thickeners in aqueous systems for many applications (food, paints, and cosmetics). Their chemical structure is a linear (1 f 4)-β-D-mannan chain substituted with single Dgalactopyranosyl units as side chain by a (1 f 6) R-linkage. The various galactomannans differ from each other not only in the mannose/galactose ratio but also in the molar mass

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Table 1. Comparison of Conformational Stability of Some Polysaccharides in the Gellan Familly

Gellan

Rhamsan RMDP17 Welan S-657 a

conformation at 25°C in 0.1 M Nacl

Tm (°C)a on cooling

DH DH DH DH DH DH DH DH DH DH DH

78 ( 2 76 ( 2 44.6 stable 37 stable 44.2 stable stable stable stable

native deacetylated deacylated native deacetylated native deacetylated native deacetylated native deacetylated

∆H, J/g

∆H, kJ/repeating unit

refs

23.6 ( 1 20 ( 1 14.4

16.2 13.7 9.9

1 1 1 17,24 17,24 24 24 17 17 17 17

8

8.1

10.2

10.2

In 0.1 M NaCl DH double helix; Tm temperature for the helix-coil transition; ∆H in J/repeating unit (four sugars in the main chain).

Figure 6. Phase diagram relating the inverse of the temperature for conformational change Tm and the total ionic concentration of the solution including free counterions for different bacterial polysaccharides. Table 2. Variation of Tm (K) for One Decade of the Ionic Concentration Change (NaCl) on the Cooling Curves polysaccharides

∆Tm (°)

refs

native gellan deacylated gellan deacetylated rhamsan deacetylated RMDP17 xanthan succinoglycan BEC 1615

6 21 15 13 44 12 35

1 2 24 24 42 42 78

distribution and the distribution of galactose side groups along the main chain. All behave in solution as random coil conformation but their rheological behavior is much dependent on M/G due to loose interaction promoted by mannan block interactions (stabilized by interchain H bonds). The viscosity at low shear rate demonstrates these interactions. It has been shown that the specific viscosity is directly related, for perfectly soluble polymers, to the overlap parameter C[η] by the following relation:26 ηsp ) [η]C [1+ k1 [η]C + k2 ([η]C)2 + k3 ([η]C)3) with k1 ) 0.4, k2 ) k12/2!, k3 ) k13/ 3!.

Figure 7. Comparison of the rheological behavior of deacetylated RMDP 17 and rhamsan with that of deacylated gellan. Polymer concentration 10 g/L, ionic concentration 0.1 M NaCl.

As soon as the experimental values deviate from this curve, it means that secondary loose interchain interactions exist in solution;27 the slope of the curve in the semidilute regime is usually in the range of 3.4-4; it increases up to 6.5 for galactomannan with low G/M ratios.28-32 It must be recalled that the intrinsic viscosity of a polymer is related to the molar mass and to the persistence length of the molecule, and then the values in terms of overlap parameter must fit with the prediction. It is not the case with galactomannan having a low G/M ratio. To show the role of the galactose units on the backbone stiffness, the persistence length of galactomannan with different M/G ratios and distributions of galactose along the mannan chain was determined from molecular modeling.33,34 From this study, the values given in Table 3 show that the stiffness of the chain only changes by maximum by a factor 2. Thus, this is not the parameter which implies the large increase of the specific viscosity observed. As will be mentioned later, interesting behavior is obtained when galactomannans are complexed with xanthan depending on the role of the galactose unit distribution but also on the degree of acetylation of the xanthan. This point will be discussed later. An other example comes from the comparison of curdlan produced by Alcaligenes faecalis Var.myxogenes, a neutral

Role of Substituents on Polysaccharide Properties Table 3. Lp Values for Different Galactomannans [ref 33] galactomannan composition M/G ratios

Lp values (Å)

1:1 2:1 3:1 4:1 5:1 Mannan

96 85 91 100 113 145

β (1 f 3) glucan forming triple helices in aqueous medium35,36 and scleroglucan having side glucose groups with different distribution (usually one β (1 f 6) glucose as side chain per 3 glucoses in the main chain).37-39 Curdlan gives stiff and stable gels formed at high temperature when scleroglucan is water soluble. Nevertheless, scleroglucan forms a gel in water at a temperature lower than 8 °C and the chains slightly interact in solution at higher temperatures. It forms also a gel when treated in alkaline conditions.40 These neutral β (1 f 3)/ β (1 f 6) glucans were recognized for their thickening properties but also for their immune modulating activity.41


4. Influence of Minor Carbohydrate Modification The chemical structure of deacetylated RMDP17 and rhamsan is given in Figure 8. The only difference is the presence of a deoxy-sugar in RMDP17.13,23 It has some influence on the thermal stability of the double helix conformation and on the salt sensitivity as shown in Figure 6 and Tables 1 and 2. These results are not in perfect agreement with the conclusions of Chandrasekaran et al.13 It may be necessary to review the 3-D structure of these two polymers. When xanthan and succinoglycan are compared (Figure 9), the backbone is a β (1 f 4) glucan for xanthan but β (1 f 4) glucan interrupted by a β (1 f 3) linkage in the repeat unit for succinoglycan.42 This difference changes the stiffness of the chain in the coil conformation, succinoglycan being more flexible than xanthan. 5. Influence of Non-Carbohydrate Substituents in Bacterial Polysaccharides 5.1. Low Influence of Substituents. On Figure 9, the chemical structure of xanthan and succinoglycan are recalled;

Figure 8. Comparative chemical structure for RMDP 17 and rhamsan, two bacterial polysaccharides.

Figure 9. Chemical structure of (A) succinoglycan and (B) xanthan.

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Table 4. Influence of Substituents on the Melting Temperature Tm for Xanthan [ref 3]a

a

samples

Tm (°C)

partially hydrolyzed xanthan acetyl free xanthan pyrute free xanthan pyruvate and acetyl free xanthan

76 65 82 72.5

Polymer concentration 1 g/L; solvent 4 × 10-2 M NaCl.

Figure 11. DSC scanning calorimetry on heating at 0.2°/min. for xanthan: galactomannan (M/G ) 3) mixture of 4:2 (g/L) in water, 5 and 10 mM NaCl. Tg indicates the gel-sol transition of the complex and Tm is the conformational transition of xanthan at the corresponding ionic concentration.55 Reprinted with permission from Rinaudo, M.; Milas, M., Bresolin, T.; Ganter, J. Macromol. Chem. Phys., Macromol. Symp. 1999, 140, 115-124. Copyright 2003 Wiley-VCH.

Figure 10. Role of the substituents on the Mark-Houwink dependency for xanthan and modified xanthan.3 Reprinted with permission from Callet, F.; Milas, M.; Rinaudo, M. Int. J. Biol. Macromol. 1987, 9, 291-293. Copyright 2003 Elsevier.

they are both charged due to the presence of an uronic acid in addition to pyruvic acid for xanthan and pyruvic acid and succinic acid in succinoglycan. They also have variable acetyl contents. Xanthan is based on a five sugar repeating unit forming a single helical conformation in excess salt for the native sample;43-47 after thermal treatment, it forms aggregates which increase the apparent viscosity of solution. The persistence length in the native state is around 40 nm in the single chain helical conformation and becomes 5 nm in the coiled conformation.48-51 Interaction between the three sugar side chain and the backbone based on H bonds47 stabilizes a pseudo double helical conformation. Nevertheless, the transition has a low cooperativity and is very sensitive to the ionic concentration and temperature (due to low intrachain secondary interactions):42 at a given ionic concentration, the helix-coil transition needs more than 15 °C to be achieved and Tm increases of 44° when the salt concentration is multiplied by 10 (Table 6). Xanthan has pyruvate and acetyl substituents on the mannose units in the side chain. Their yields depend on the conditions of fermentation and post-fermentation processes. The substituents were hydrolyzed progressively and their role on Tm was determined. The values are given in Table 4. The role of the substituents on Tm is low at a constant ionic concentration. The intrinsic viscosity as a function of the molar mass was plotted in Figure 10. It is clear that the rheology of xanthan in the dilute regime is not perturbed by elimination of the substituents. They have no fundamental role the hydrodynamic behavior in the helical conformation, but it was shown that a noticeable influence exists on the specific interaction with galactomannans. A complex is formed between these two polysaccha-

rides giving a physical gel. Two types of gels can be recognized: the usual one formed around 60 °C for galactomannan having a low G/M ratio.52 We have shown from calorimetry that a complex is also formed at low temperature (Tm ∼ 20-25 °C) with a galactomannan having M/G ) 3 ratio (Figure 11); this complex is stronger with the deacetylated xanthan53-59 and when xanthan is in the coiled conformation.54 Deacetylation is also important for interaction with konjac mannan.60 It can be also mentioned that deacetylation of acetan, another bacterial polysaccharide,61,62 allows synergistic interaction with locust bean gum and konjac mannan.63 Succinoglycan is based on an eight sugar repeating unit; depending on the strain, it also contains acetyl, succinyl, and pyruvyl substituents introducing an ionic character. Our study was performed on a succinoglycan produced by Pseudomonas sp NCIB 11592.64-68 The pyruvyl and succinyl substituents were progressively hydrolyzed, and the intrinsic vicosity was determined as a function of the molar masses at a constant ionic concentration for which the helical conformation is stable at ambient temperature. It was shown that again there is a minor role of the substitutents on the hydrodynamic characteristics of the polymers so long as the solubility of the samples remains good.66 These results are in disagreement with some data given in the literature.69 The stability of the single chain helical structure was also examined as a function of the substituent yield;64,67 the results are given in Table 5. In addition, it was shown that, after heating over Tm, this polymer forms aggregates in welldefined conditions.67 It can be concluded that the substituents in succinoglycan have even a lower role on the helix stability than with xanthan; as mentioned before, the cooperativity and stability of this helix is much larger (Table 6). The role of the ionic concentration on Tm was determined and represented in Figure 6 and in Table 6. From all these data, it is clear that the succinoglycan forms a very stable


Role of Substituents on Polysaccharide Properties Table 5. Role of Substituents on the Conformational Stability (Tm) for Succinoglycan [ref 66]a

a

samples

Tm (°C)

native succinoglycan succinate free succinoglycan pyruvate free succinoglycan succinate and pyruvate free succinoglycan

70 68 66 63

Polymer concentration 1 g/L; solvent 0.1 M NaCl.

Table 6. Comparison Between the Conformation of Xanthan and Succinoglycan

∆H, J/mol xanthan succinoglycan

13.9 7.1 30

thermal transition width (°C)

∆Tm increase per decade

∼15°

44

2°

12

ref

Figure 13. Structure of the repeat unit of the polysaccharide produced by Klebsiella ATCC12657 strain.

54 93 67

Figure 12. Chemical structure of native gellan.

helical conformation with low ionic concentration sensitivity; this single chain helix was also observed by AFM.70 5.2. Strong Influence of Substituents. The structures of the native and deacetylated gellan are given in Figures 13 and 5, respectively. The two substituents were hydrolyzed successively to test their role on the gellan properties. In the native structure, the double helix conformation is stable and the helix-coil transition determined when the temperature increases or decreases is reversible and nearly independent of the ionic concentration.2 The rheology demonstrates a loose interacting system with G′ (the elastic modulus in dynamic experiments) slightly larger than G′′.1 When the acetyl substituents are removed, the DSC show that the stability of the double helix remains unchanged (Tm and ∆H do not change). However, when the L-glyceric substituents are removed, the stability of the helix decreases largely and the helix-coil thermal transition becomes sensitive to ionic concentration; the lower stability is reflected by the position of the thermal transition (Tm) at lower temperature as well as the lower value of the enthalpy for conformational change. In addition, with deacylated gellan, a physical gel is formed over a critical ionic concentration imposed by the cationic selectivity with a preference K + > Na + > Li+.71,72 This gellan presents a conformational helix-coil transition (without any ionic selectivity) associated with a sol-gel transition that is sensitive to the ionic concentration and the nature of the excess electrolyte producing a large hysteresis. The conclusions are that the L-glyceric substituents included inside the double helix19 stabilize the double helix and are shown to modify slightly the position of the atoms in the gellan conformational analysis;12 acetyl groups are located outside the double helix and prevent aggregation of the double helices and gelation.19 A very interesting result was obtained recently with the bacterial polysaccharide named BEC 1615 produced by the

Figure 14. Evolution of the relative viscosity of the native BEC 1615 polysaccharide (concentration 5 g/L) in 0.4M NaCl as a function of temperature on heating ([) and cooling (]). Temperature variation 0.4 °C/min., shear rate for measurements ) 0.02s-1.78 Reprinted with permission from Guetta, O.; Milas, M.; Rinaudo, M. Biomacromolecules 2003, 4, 1372-1379. Copyright 2003 American Chemical Society.

Klebsiella strain ATCC 12657 and developed by Solabia (France). In the native structure, the polymer contains acetyl substituents. It has the same structure as described earlier for polysaccharide K 54 produced by the same Kliebsiellea strain but for which the physical properties in solution were not studied before (Figure 13).73-78 In aqueous solution, it behaves as a thickener and increases the viscosity of the solvent in relation with some loose interactions manifested by the slow increase of the viscosity when ionic concentration increases and the temperature decreases. In its native structure, BEG 1615 was assumed to be in a coiled conformation.78 Whatever the experimental conditions (temperature, polymer, and external salt concentration), no conformational transition was observed (Figure 14). After complete deacetylation in mild conditions, the polymer becomes a gelling polymer in given thermodynamic conditions. In these conditions, the behavior of this new polysaccharide looks like that of XM-6 previously described in the literature and produced by an Enterobacter strain.79,80 XM-6 was claimed to adopt a double helix conformation with a similar X-ray pattern to K54,7 but K54 indicates a lower crystallinity. It was previously demonstrated that the solgel transition for XM-6 is reversible and very cooperative. We found exactly the same type of behavior in our work

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Figure 15. Influence of temperature on deactivated BEC1615 properties. Evolution of the relative 1H NMR signal of fucose on ] cooling and [ heating in 0.4 M NaCl and evolution of the elastic modulus G′ on heating. Cp ) 5 g/L.78 Reprinted with permission from Guetta, O.; Milas, M.; Rinaudo, M. Biomacromolecules 2003, 4, 1372-1379. Copyright 2003 American Chemical Society.

published recently.78 A reversible sharp sol-gel transition, associated with a partial helix-coil transition, is observed at 30 °C for a 3 g/L solution in 0.25 N NaCl without hysteresis. It is assumed that the physical gel is obtained by aggregation of double helices (Figure 15). The enthalpy of conformational change was found to be low and of the order of 4 kJ/repeating unit which can be directly compared with the values of Table 1. This is two times lower than for deacylated gellan, and Tm is also lower and varies more in dependence of salt content. Nevertheless, the gellan gel formation is less cooperative than this one. In addition, it was noticed that the G′ modulus of the gel formed in the presence of monovalent or divalent counterions (over 0.1 N) is independent of the amount of excess salt. In the case of gellan as well as for this new polymer, the role of substituents is essential for the physical properties. In the absence of molecular modeling, it is difficult to fully understand the exact mechanism of gelation of this Klebsiella polysaccharide. 6. Influence of Substituents in Polysaccharides Derivatives Cellulose, starch, and chitin are important natural polysaccharides, but because of their intrinsic characteristics, it is difficult to develop their applications. They are semicyistalline polymers insoluble in the majority of organic solvents. They are rich in H-bonds stabilizing their 3Dstructure, and they are also thermostable, and no melting for cellulose and chitin is observed before degradation limiting the methods available to process them. A good way to modify their performance is to derivatize them. This is usually performed in the solid state and gives very heterogeneous distribution of the substituents along the main chain. It is also the reasons why different initial materials with different age or origin will give different characteristics for the derivatives prepared. Few examples are given in the following.

Rinaudo

Cellulose in heterogeneous conditions can be transformed in methylcellulose with different degrees of substitution (DS). It is recognized that for DS > 1.3 methylcelluloses become water-soluble and for DS > 2.5 they become soluble in organic solvents. However, it was demonstrated that the distribution of methyl groups along the chain controls the properties in solution. For that purpose, polymers were prepared in the same range of DS in homogeneous conditions and tested for their properties.81-84 For a random distribution of the methyl groups, water solubility is obtained for DS > 0.9, with the methyl groups preventing the packing of cellulosic chains. The most interesting characteristic of methylcellulose, namely their gelling ability when temperature increases is characteristic of amphiphilic systems, disappeared for homogeneous polymers prepared with the same degree of substitution. The two step gelation was investigated on heterogeneous methylcelluloses and two steps were identified as, first, a cross-linking of the chains due to highly modified cellulosic zones forming the “clear gel” over 40 °C and, second, the phase separation of the rest of the heterogeneous material having a lower average DS producing the strong turbid gel over 60 °C. This is essentially related to the heterogeneity of the distribution of the methyl groups along the chain giving advantage for some applications especially in the food industry. Chitin is a polysaccharide extracted from many marine organisms such as crabs and shrimps. To find more applications, it is deacetylated under heterogeneous conditions by concentrated alkaline treatments. The polymer obtained is called chitosan and characterized by a certain degree of acetylation (DA) corresponding to the average fraction of N-acetyl group left along the chains.85 This polymer becomes soluble in acidic conditions for a DA < 0.5; nevertheless, this value is just indicative because the solubility is also controlled by the distribution of the functional groups along the chains. It was shown that the stiffness of the chain is not greatly modified by deacetylation86 and is not able to explain the modification obtained in rheological properties. The distribution of the hydrophobic N-acetyl group along the chain controls the solubility.8 Finaly, natural polymers after extraction may be found having a different structure from each other due to enzymic mechanism of hydrolysis; an example is given with pectin. Pectins as extracted are highly methyl-esterified on the galacturonic acid units and characterized by their degree of esterification (DE); they form gels in acidic conditions in the presence of highly concentrated sucrose. Pectins were hydrolyzed in alkaline conditions to get a random distribution of the carboxylic groups, and then, they form gels in the presence of calcium when DE < 50%. In other experiments, they were hydrolyzed by an enzyme giving a blockwise distribution of the carboxylic groups.88-91 For the same average degree of esterification, it was demonstrated that the distribution of the free carboxylic groups controls the ability to form a gel. It is shown in Figure 16, and the gel point for the blockwise distribution (where block of carboxylic groups are able to form a junction) is obtained for a lower amount of calcium added than for the random distribution.89



be different. It is one of the reason complete characterization of these polymers should be needed but is often too heavy and expensive. It makes the field of polysaccharides and their applications much more difficult to develop. It can also be suggested that the development of molecular modeling and solid state conformation studies on microbial polysaccharides will help in a better understanding of their properties as related with their conformation and the possible interactions. References and Notes

Figure 16. Changes in reduced viscosity and scattered light during the addition of calcium chloride on calcium pectinates having a degree of esterification DE ) 30% obtained (1) by enzymic hydrolysis (blockwise carboxylic groups distribution) and (2) by alkaline hydrolysis (random distribution of carboxylic groups). Polymer concentration: 0.2 g/L.89 Reprinted with permission from Thibault, J. F.; Rinaudo, M. Br. Polym. J. 1985, 17, 181-184. Copyright 2003 John Wiley & Sons.

In natural medium, different types of enzymes exist which can be hydrolyzed pectins randomly or blockwise. It can also be mentioned that enzymic attack by endoglycanase is related to the distribution of the substituents in cellulose derivatives used as the substrate for the enzyme.92 It shows that at least a sequence of three or more unsubstituted glucose are needed to get a hydrolysis of glucan backbone. 7. Conclusion The objective of this paper was to show how a small modification in the chemical structure of the polysaccharide molecules may have a large effect on the physical properties or at least some of them. For bacterial polysaccharides, the presence of carbohydrate and non-carbohydrate substituents on a polysaccharidic backbone was discussed. They play a role on the stability of the helical conformation with these stereoregular polymers, on the packing of the chains, and ability to form gels. They also play a role in interchain interactions for some mixed systems. It was also shown that very minor modifications of the structure (presence of an anhydro-sugar) are able to give modifications in the behavior of the polymer in solution. For chemically modified polysaccharides, it was mentioned that the distribution of the substituents along the chains is very important. It is the reason why the commercial samples obtained by heterogeneous reactions on the polysaccharides (being semicrystalline solids) often have irreproducible properties even if they have the same average degree of substitution and the same estimated molar mass. Natural products such as alginates, pectins, and galactomannans also have a chemical structure depending on the age of the material, on the source, or on the industrial processes to extract and purify. Respectively, for products having the same mannuronic/guluronic ratio (alginates), the same degree of esterification (pectins), or the same mannose/ galactose ratio (galactomannans), the physical properties may

(1) Mazen, F.; Milas, M.; Rinaudo, M. Conformational transition of native and modified gellan. Int. J. Biol. Macromol. 1999, 26, 109118. (2) Rinaudo, M.; Milas, M. Gellan gum, a bacterial gelling polymer. In NoVel Macromolecules in Food Systems; Doxastakis, G., Kiosseoglou, V., Eds., Elsevier: Amsterdam, 2000; pp 239-263. (3) Callet, F.; Milas, M.; Rinaudo, M. Influence of acetyl and pyruvate content on rheological properties of xanthan in dilute solution. Int. J. Biol. Macromol. 1987, 9, 291-293. (4) Goycoolea, F. M.; Milas, M.; Rinaudo, M. Associative phenomena in galactomannan-deacetylated xanthan system. Int. J. Biol. Macromol. 2001, 29, 181-192. (5) Villain, A.; Milas, M.; Rinaudo, M. A new bacterial exopolysaccharide (YAS34). I: Characterization of the conformations and conformation transition. Int. J. Biol. Macromol. 2000, 27, 65-75. (6) Villain-Simonnet, A.; Milas, M.; Rinaudo, M. A new bacterial exopolysaccharide (YAS34). II: Influence of thermal treatments on the conformation and structure. Relation with gelation ability. Int. J. Biol. Macromol. 2000, 27, 77-87. (7) Atkins, E. D. T.; P. Attwool, T.; Miles, M. J.; Morris, V. J.; O′Neill, M. A.; Sutherland, I. W. Effect of acetylation on the molecular interactions and gelling properties of a bacterial polysaccharide. Int. J. Biol. Macromol. 1987, 9, 115-117. (8) Atkins, E. Structure of microbial polysaccharides using X-ray diffraction, in NoVel Microbial Polymers; Dawes, E. A., Ed.; Kluwer Academic Publishers: Norwell, MA, 1990, 371-386. (9) Atkins, E. Effect of mono-o-acetyl groups on the conformation and interactions of microbial polysaccharides in Industrial Polysaccharides: Genetic Engineering, Structure/Properties Relations and Applications; Yalpani, M., Ed.; Elsevier Science Publishers: Amsterdam, 1987; pp 177-186. (10) Chandrasekaran, R.; Millane, R. P.; Arnott, S.; Atkins, E. D. T. The crystal structure of gellan. Carbohydr. Res. 1988, 175, 1-15. (11) Chandrasekaran, R.; Puigjaner, L. C.; Joyce, K. L.; Arnott, S. Cation interactions in gellan: an X-ray study of the potassium salt. Carbohydr. Res. 1988, 181, 23-40. (12) Chandrasekaran, R.; Radha, A.; Thailambal, V. G. Role of potassium, acetyl and L-glyceryl groups in native gellan double helix. Carbohydr. Res. 1992, 224, 1-17. (13) Bian, W.; Chandrasekaran, R.; Rinaudo, M. Molecular structure of the rhamsan-like exocellular polysaccharide RMPDP17 from Sphingomonas paucimobilis. Carbohydr. Res. 2002, 337, 45-56. (14) (a) Rinaudo, M.; Pavlov, G.; Desbrieres, J. Solubilization of chitosan in strong acid medium. Int. J. Polym. Anal. Charact. 1999, 5, 267276. (b) Rinaudo, M.; Pavlov, G.; Desbrie`res, J. Influence of acetic acid concentration on the solubilization of chitosan. Polymer 1999, 40, 7029-7032. (15) Hirrien, M.; Chevillard, C.; Desbrie`res, J.; Axelos, M. A.; Rinaudo, M. Thermogelation of methylcelluloses: new evidence for understanding the gelation mechanism. Polymer 1998, 39, 6251-6259. (16) Geremia, R.; Rinaudo, M. Biosynthesis, Structure and physical properties of some bacterial polysaccharides. In Polysaccharides: Structural DiVersity and Functional Versatility, 2nd ed.; Dumitriu, S., Ed.; Dekker: New York (in press). (17) Campana, S.; Ganter, J.; Milas, M.; Rinaudo, M. On the solution properties of the bacterial polysaccharides of gellan family. Carbohydr. Res. 1992, 231, 31-38. (18) (a) Moorhouse, R. Structure/Property relationships of a family of microbial polysaccharides, in Industrial Polysaccharides: Genetic Engineering, Structure/Properties Relations and Applications; Yalpani, M., Ed.; Elsevier Science Publishers: Amsterdam, 1987; pp 187-206. (b) Moorhouse, R.; Colegrove, G. T.; Sandford, P. A.; Baird, J. K.; Kang, K. S. PS-60: a new gel-forming polysaccharide. In Solution properties of polysaccharides; Brant, D., Ed.; ACS

1164

(19)

(20) (21) (22) (23)

(24) (25) (26) (27) (28)

(29) (30) (31) (32)

(33) (34)

(35)

(36)

(37) (38) (39) (40) (41)

Biomacromolecules, Vol. 5, No. 4, 2004 Symposium Series; American CHemical Society: Washington, DC, 1981; Vol. 150, pp 111-124. Morris, E. R.; Gothard, M. G. E.; Hember, M. W. N.; Manning, C. E.; Robinson, G. Conformational and rheological transitions of welan, rhamsan and acetylated gellan. Carbohydr. Polym. 1996, 30, 165175. Lopes, L.; Milas, M.; Rinaudo, M. Influence of the method of purification on some solution properties of welan gum. Int. J. Biol. Macromol. 1994, 16, 253-258. Lopes, L.; Andrade, C. T.; Milas, M.; Rinaudo, M. Influence of aggregates on the sedimentation properties of welan gum. Polym. Bull. 1995, 34, 655-662. Campana, S.; Andrade, C.; Milas, M.; Rinaudo, M. Polyelectrolyte and rheological studies on the polysaccharide welan. Int. J. Biol. Macromol. 1990, 12, 379-384. Falk, C.; Jansson, P. E.; Rinaudo, M.; Heyraud, A.; Widmalm, G.; Hebbar, P. Structural studies of the exocellular polysaccharide from Sphingomonas paucimobilis strain I-886. Carbohydr. Res. 1996, 285, 69-79. Villain-Simonnet, A.; Milas, M.; Rinaudo, M. Comparison between the physicochemical behavior of two microbial polysaccharides: RMDP17 and rhamsan. Int. J. Biol. Macromol. 1999, 26, 55-62. Lee, E. J.; Chandrasekaran, R. X-ray and computer modelling studies on gellan-related polymers: molecular structure of welan, S-657, and rhamsan. Carbohydr. Res. 1991, 214, 11-24. Kwei, T. K.; Nakazawa, M.; Matsuoka, S.; Cowman, M. K.; Okamoto, Y. The concentration dependence of solution viscosities of rigid rod polymers. Macromolecules 2000, 33, 235-236. Rinaudo, M. Relation between the molecular structure of some polysaccharides and original properties in sol and gel states. Food Hydrocoll. 2001, 15, 433-440. Rinaudo, M. Advances in characterization of polysaccharides in aqueous solution and gel state. In Polysaccharides: Structural DiVersity and Functional Versatility, 2nd ed.; Dumitriu, S., Ed.; Dekker: New York (in press). Ganter, J.; Milas, M.; Rinaudo, M. Study of solution properties of galactomannan from the seeds of Mimosa scabrella. Carbohydr. Polym. 1992, 17, 171-175. Kapoor, V. P.; Milas, M.; Taravel, F. R.; Rinaudo, M. Rheological properties of seed galactomannan from Cassia nodosa buch.-hem. Carbohydr. Polym. 1994, 25, 79-84. Kapoor, V. P.; Milas, M.; Taravel, F. R.; Rinaudo, M. Rheological properties of a seed galactomannan from Cassia siamea lamk. Food Hydrocolloids 1996, 10, 167-172. Kapoor, V. P.; Taravel, F. R.; Joseleau, J. P.; Milas, M.; Chanzy, H.; Rinaudo, M. Cassia spectabilis DC seed galactomannan: structural crystallographical and rheological studies. Carbohydr. Res. 1998, 306, 231-241. Petkowicz, C.; Reicher, F.; Mazeau, K. Conformational analysis of galactomannans: from oligomeric segments to polymeric chains. Carbohydr. Polym. 1998, 37, 25-39. Petkowicz, C. L. O.; Rinaudo, M.; Milas, M.; Mazeau, K.; Bresolin, T.; Reicher, F.; Ganter, J. L. M. S. Conformation of galactomanan, experimental and modeling approaches. Food Hydrocolloids 1999, 13, 263-266. Harada, T. Production, properties and applications of Curdlan. In Extracellular Microbial Polysaccharides; P. Sandford, P., Laskin, A., Eds.; ACS symposium series No 45; American Chemical Society: Washington, D.C, 1977; pp 265-283. Fulton, W. S.; Atkins, E. D. The gelling mechanism and relationship to molecular structure of microbial polysaccharide, T. In Fiber Diffraction Methods; French, D. A., Gardner, C. K., Eds.; ACS Symposium Series No. 141; American Chemical Society: Washington, DC, 1980; pp 385-409. Rinaudo, M.; Vincendon, M. 13C NMR structural investigation of the scleroglucan. Carbohydr. Polym. 1982, 2, 135-144. Bluhm, T. L.; Deslandes, Y.; R. Marchessault, H.; Perez, S.; Rinaudo, M. Solid-state and solution conformation of scleroglucan. Carbohydr. Res. 1982, 100, 117-130. Bo, S.; Milas, M.; Rinaudo, M. Behaviour of scleroglucan in aqueous solution containing sodium hydroxide. Int. J. Biol. Macromol. 1987, 9, 153-157. Aasprong, E.; Smidsrod, O. B.; Stokke, T. Scleroglucan gelation by in situ neutralization of the alkaline solution. Biomacromolecules 2003, 4, 914-921. Franz, G.; Hensel, A.; Kraus, J. Structure-actiVity relationship of immune modulating polysaccharides with an antitumor effect, in Biomedical and Biotechnological AdVances in Industrial Polysaccharides; Crescenzi, V., Dea, I. C. M., Paoletti, S., Stivala, S. S.,

Rinaudo

(42)

(43) (44) (45) (46)

(47)

(48) (49) (50) (51) (52)

(53) (54) (55) (56) (57)

(58)

(59)

(60)

(61) (62)

Sutherland, I. W., Eds.; Gordon and Breach Science Publishers: Langhorne, PA, 1989; pp 241-249. (a) Gravanis, G.; Milas, M.; Rinaudo, M.; Tinland, B. Comparative behaviour of the bacterial polysaccharides xanthan and succinoglycan. Carbohydr. Res. 1987, 160, 259-265. (b) Rinaudo, M.; Milas, M.; Tinland, B. Relation between molecular structure and physicochemical properties for some microbial polysaccharides. In NoVel Microbial Polymers; Dawes, E. A., Ed.; Kluwer Academic Publishers: Norwell, MA, 1990; pp 349-369. Milas, M.; Rinaudo, M. Conformational investigation on the bacterial polysaccharide xanthan. Carbohydr. Res. 1979, 76, 186-196. Rinaudo, M.; Milas, M. Xanthan properties in aqueous solution. Carbohydr. Polym. 1982, 2, 264-269. Milas, M.; Rinaudo, M. Properties of xanthan gum in aqueous solutions: role of the conformational transition. Carbohydr. Res. 1986, 158, 191-204. Milas, M.; Rinaudo, M. Investigation on conformational properties of Xanthan in aqueous solutions. In Solution properties of polysaccharides; Brant, D., Ed.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981; Vol. 150, pp 25-30. Moorhouse, R.; Walkinshaw, M. D.; Arnott, S. Xanthan gummolecular conformation and interactions. In Extracellular Microbial Polysaccharides; Sandford, P. A., Laskin, A., Eds.; ACS symposium series No 45; American Chemical Society: Washington, DC, 1977; pp 90-100. Tinland, B.; Rinaudo, M. Dependence of the stiffness of the xanthan chain on the external salt concentration. Macromolecules 1989, 22 (2), 1863-1865. Tinland, B.; Maret, G.; Rinaudo, M. Reptation in semidilute solutions of wormlike polymers. Macromolecules 1990, 23, 596-602. Rinaudo, M. Wormlike chain behaviour of some bacterial polysaccharides. In Macromolecules; Kahovec, J., Ed.; VSP: Zeist, The Netherlands, 1992; pp 207-219. Chazeau, L.; Milas, M.; Rinaudo, M. Conformations of xanthan in solution: analysis by steric exclusion chromatography. Int. J. Polym. Anal. Charact. 1995, 2, 21-29. (a) Goycoolea, F. M.; Richardson, R. K.; Morris, E. R.; Gidley, M. J. Stoichiometry and conformation of xanthan in synergistic gelation with locust bean gum or konjac glucomannan: Evidence of heterotypic binding, Macromolecules 1995, 28, 8308-8320. (b) Goycoolea, F. M.; Richardson, R. K.; Morris, E. R.; Gidley, M. J. Synergistic gelation of galactomannans or konjac glucomannan: binding or exclusion? In Gums and Stabilisers for the Food industry 7; Phillips, G. O., Williams, P. A., Wedlock, D. J., Eds.; IRL Press: Oxford, U.K., 1994; pp 333-344. Bresolin, T. M.; Sander, P. C.; Reicher, F.; Sierakowski, M. R.; Rinaudo, M.; Ganter, J. L. Viscometric studies on xanthan and galactomannan systems. Carbohydr. Polym. 1997, 33, 131-138. Bresolin, T.; Milas, M.; Rinaudo, M.; Ganter, J. Xanthan-galactomannan interactions as related to xanthan conformations. Int. J. Biol. Macromol. 1998, 23, 263-275. Rinaudo, M.; Milas, M.; Bresolin, T.; Ganter, J. Physical properties of xanthan galactromannan and their mixtures in aqueous solutions. Macromol. Chem. Phys., Macromol. Symp. 1999, 140, 115-124. Lopes, L.; Andrade, C. T.; Milas, M.; Rinaudo, M. Role of conformation and acetylation of xanthan on xanthan-guar interaction. Carbohydr. Polym. 1992, 17, 121-126. Goycoolea, F. M.; Milas, M.; Rinaudo, M. Heterotypic interactions of deacetylated xanthan with a galactomannan of high galactose substitution during synergistic gelation. In Gums and Stabilizers for the Food Industry 10; Phillips, G. O., Williams, P. A., Eds.; RSC: Cambridge, 2000; pp 229-240. Shatwell, K. P.; Sutherland, I. W.; Ross-Murphy, S. B.; Dea, I. C. M. Influence of the acetyl substituent on the interaction of xanthan with plant polysaccharides. II. Xanthan-guar gum systems. Carbohydr. Polym. 1990, 14, 115-130. Shatwell, K. P.; Sutherland, I. W.; Ross-Murphy, S. B.; Dea, I. C. M. Influence of the acetyl substituent on the interaction of xanthan with plant polysaccharides. I. Xanthan-locust bean gum systems. Carbohydr. Polym. 1991, 14, 29-51. Shatwell, K. P.; Sutherland, I. W.; Ross-Murphy, S. B.; Dea, I. C. M. Influence of the acetyl substituent on the interaction of xanthan with plant polysaccharides. III. Xanthan-konjac mannan systems. Carbohydr. Polym. 1990, 14, 131-147. Ojinnaka, C.; Jay, A. J.; Colquhoun, I. J.; Brownsey, G. J.; Morris, E. R.; Morris, V. J. Structure and conformation of acetan polysaccharide. Int. J. Biol. Macromol. 1996, 19, 149-156. Ojinnaka, C.; Morris, E. R.; Morris, V. J.; Brownsey, G. J. Effect of acetyl substituents on the conformational stability and functional



(63) (64)

(65)

(66) (67)

(68) (69)

(70) (71) (72) (73)

(74) (75)

(76) (77)

interactions of acetan, polysaccharide. In Gums and Stabilisers for the Food industry 7; Phillips, G. O., Williams, P. A., Wedlock, D. J., Eds.; IRL Press: Oxford, U.K., 1994; pp 15-26. Ojinnaka, C.; Brownsey, G. J.; Morris, E. R.; Morris, V. J. Effect of deacetylation on the synergistic interaction of acetan with locust bean gum or konjac mannan. Carbohydr. Res. 1998, 305, 101-108. Gravanis, G.; Milas, M.; Rinaudo, M.; Clarke-Sturman, A. J. Conformational transition and polyelectrolyte behaviour of a succinoglycan polysaccharide. Int. J. Biol. Macromol. 1990, 12, 195200. Gravanis, G.; Milas, M.; Rinaudo, M.; Clarke-Sturman, A. J. Rheological behaviour of a succinoglycan polysaccharide in dilute and semidilute solutions. Int. J. Biol. Macromol. 1990, 12, 201206. Gravanis, G.; Milas, M.; Rinaudo, M.; Tinland, B. Comparative behaviour of the bacterial polysaccharides xanthan and succinoglycan. Carbohydr. Res. 1987, 160, 259-265. (a) Boutebba, A.; Milas, M.; Rinaudo, M. Order-Disorder. Conformational transition in succinoglycan: calorimetric measurements. Biopolymers 1997, 42, 811-819. (b) Boutebba, A.; Milas, M.; Rinaudo, M. On the interchain associations in aqueous solution of a succinoglycan bacterial polysaccharide. Int. J. Biol. Macromol. 1999, 24, 319-327. Boutebba, A. Proprie´te´s du succinoglycane: Transition conformationnelle et ge´lification en milieu aqueux. Ph.D. Thesis, Grenoble, 1998. Ridout, M. J.; Brownsey, G. J.; York, G. M.; Walker, G. C.; Morris, V. J. Effect of O-acetyl substituents on the functional behaviour of Rhizobium meliloti succinoglycan. Int. J. Biol. Macromol. 1997, 20, 1-7. Balnois, E.; Stoll, S.; Wilkinson, K. J.; Buffle, J.; Rinaudo, M.; Milas, M. Conformations of succinoglycan as observed by atomic force microscopy. Macromolecules 2000, 33, 7440-7447. Milas, M.; Shi, X.; Rinaudo, M. On the physicochemical properties of gellan gum. Biopolymers 1990, 30, 451-464. Milas, M.; Rinaudo, M. The gellan sol-gel transition. Carbohydr. Polym. 1996, 30, 177-184. Sandford, P. A.; Conrad, H. E. The structure of the Aerobacter aerogenes A3 (Sl) polysaccharide. A reexamination using improved procedures for methylation analysis, I. Biochemistry 1966, 5, 15081517. Dutton, G. G. S.; Merrifield, E. H. The Capsular Polysaccharide from Klebsiella Serotype K54; Location of the O-acyl Groups, and a revised Structure. Carbohydr. Res. 1982, 105, 189-203. Franze´n, L. E.; Aman, P.; Darvill, A. G.; McNeil, M.; Alberstheim, P. The Structure of the acidic polysaccharide secreted by Klebsiella Aerogenes Type 54 Strain A3. Carbohydr. Res. 1982, 108, 129138. Dell, A.; Dutton, G. G. S.; Jansson, P. E.; Lindberg, B.; Lindquist, U.; Sutherland, I. W. Absence of O-formyl groups in Klebsiella Polysaccharide. Carbohydr. Res. 1983, 122, 340-343. Elloway, H. F.; Isaac, D. H.; Atkins, E. D. T. Review of the structures of Klebsiella Polysaccharides by X-ray diffraction, in Fiber Diffrac-

(78)

(79)

(80)

(81) (82)

(83) (84)

(85)

(86)

(87)

(88)

(89) (90)

(91) (92) (93)

tion Methods; French, A. D., Gardner, K. C., Eds.; ACS Symposium Series 141; American Chemical Society: Washington, DC, 1980; pp 429-458. Guetta, O.; Milas, M.; Rinaudo, M. Structure and properties of a bacterial polysaccharide from a Klebsiella strain (ATCC 12657). Biomacromolecules 2003, 4, 1372-1379. O′Neill, M. A.; Morris, V. J.; Selvendran, R. R.; Sutherland, I. W.; Taylor, I. T. Structure of theExtracellular Gelling Polysaccharide produced by Enterobacter (NCIB 11870) Species. Carbohydr. Res. 1986, 148, 63-69. Nisbet, B. A.; Sutherland, I. W.; Bradshaw, I. J.; Kerr, M.; Morris, E. R.; Shepperson, W. A. XM-6: A New Gel-forming Bacterial Polysaccharide. Carbohydr. Polym. 1984, 4, 377-394. Vigouret M.; Rinaudo, M.; Desbrieres, J. Thermogelation of methylcellulose in aqueous solutions. J. Chim. Phys. 1996, 93, 858-869. Hirrien, M.; Desbrieres, J.; Rinaudo, M. Physical properties of methylcelluloses in relation with the conditions for cellulose modification. Carbohydr. Polym. 1996, 31, 243-252. Desbrie`res, J.; Hirrien, M.; Rinaudo, M. A calorimetric study of methylcellulose gelation. Carbohydr. Polym. 1998, 37, 145-152. Desbrie`res, J.; Hirrien, M.; Rinaudo, M. Relation between the conditions of modification and the properties of cellulose derivatives: thermogelation of methyl-cellulose. In Cellulose deriVatiVes; ACS Symp. Series, 688; Heinze, T. J., Glasser, W. G., Eds.; American Chemical Society: Washington, DC, 1998; pp 332-348. Rinaudo, M.; Le Dung, P.; Gey, C.; Milas, M. Substituent distribution on O, N-carboxymethylchitosans by 1H and 13C NMR. Int. J. Biol. Macromol. 1992, 14, 122-128. Mazeau, K.; Perez, S.; Rinaudo, M. Predicted influence of N-acetyl group content on the conformational extension of chitin and chitosan chains. J. Carbohydr. Chem. 2000, 19, 1269-1284. Va¨rum, K. M.; Ottoy, M. H.; Smidsrod, O. Water solubility of partially N-acetylated chitosans as a function of pH: effect of chemical composition and depolymerisation. Carbohydr. Polym. 1994, 25, 65-70. Thibault, J. F.; Rinaudo, M. Interactions of mono- and divalent counterions with alcali- and enzyme-deesterified pectins in salt-free solutions. Biopolymers 1985, 24, 2131-2134. Thibault, J. F.; Rinaudo, M. Gelation of pectinic acids in the presence of calcium counterions. Br. Polym. J. 1985, 17, 181-184. Thibault, J. F.; Rinaudo, M. Interactions of mono- and divalent counterions with alcali- and enzyme-deesterified pectins in salt-free solutions. Biopolymers 1985, 24, 2131-2134. Thibault, J. F.; Rinaudo, M. Chain association of pectic molecules during calcium-induced gelation. Biopolymers 1986, 25, 455-468. Wirick, M. G. A study of the enzymic degradation of CMC and other cellulose ethers. J. Polym. Sci. A1 1968, 6, 1965-1974. Norton, I. T.; Goodall, D. M.; Frangou, S. A.; Morris, E. R.; Rees, D. A. Mechanisms and dynamics of conformational ordering in xanthan polysaccharide. J. Mol. Biol. 1984, 175, 371-394.

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