Influence of the pyruvate content of xanthan on macromolecular association in solution

Influence of the pyruvate content of xanthan on macromolecular association in solution I. H. Smith, K. C. Symes and C. J. Lawson Tate and Lyle Ltd, Group Research and Development, P.O. Box 68, Reading, Berkshire, RG6 2BX, UK

and E. R. Morris Unilever Research, Colworth House, Sharnbrook, Bedford MK44 1LQ, UK

(Received 28 April 1980)

The unusual increase in viscosity and pseudoplasticity often observed when salts are added to moderately concentrated aqueous solutions of xanthan gum is shown to arisefrom an increase in the extent of macromolecular association. The fractional change in viscosity on addition of KCI to salt-free 1% (w/v) solutions of purified polysaccharide in the K + salt form isfound to be positive only when the degree of p yruvate substitution (fraction of side chains which carry pyruvate ketal substituents) exceeds ~0.31. Above this value, the fractional change in viscosity increases withfurther increase in the degree of p yruvate substitution. These differences cannot arisefrom different degrees of conformational ordering, since the magnitude of the thermally induced order-disorder transition (monitored by optical rotation at low ionic strength) is independent of pyruvate content. The temperature at the transition midpoint, however, falls with increasing degree of pyruvate substitution. 7-his is attributed to destabilization of the ordered structure by intramolecular electrostatic repulsion between pyruvate groups, and stabilization through apolar interactions of acetate methyl groups. 14scosit~concentration relationships show changes of slope which mark the onset of macromolecular association. Association commences at lower concentrations when ionic strength and degree of pyruvate substitution are high. It is suggested that once electrostatic repulsions have been diminished at high ionic strength, association is promoted by intermolecular apolar interactions of pyruvate methyl groups, which are suitably situated near the periphery of the helical conformation.

Introduction The commercial importance of xanthan, the extracellular polysaccharide produced by fermentation of Xanthomonas campestris, has largely stemmed from its unusual rheological properties in aqueous solution 1. The primary structure of the macromolecule has been shown z-4 (Figure 1) to consist of a cellulose-like main chain with fl-D-Manp (I~,)-fl-D-GIcA p (1-2)-~-D-Manp side chains linked glycosidically at the 0-3 position of alternate glucose units. The internal mannose of the side chain is substituted stoichiometrically at 0-6 with acetyl groups. However, deacetylation can be effected without further modification of the polymeric structure by mild alkaline treatment 5 and it is also possible for partial deesterification to occur during post-fermentation processing in which the dried product is recovered from culture broths. The terminal mannose of the side chain may be substituted with a pyruvate ketal, but the degree of substitution occurring during biosynthesis is apparently less than stoichiometric. Usually, about one in two or one in three side chains carry pyruvate substituents, the actual proportion being dependent mainly on culture conditions 6. The effect of added salt on solution viscosity is atypical of a polyelectrolyte. At low gum concentration, Jeanes et al. 5 observed an unusually small decrease of viscosity by addition of salt whilst a substantial increase in viscosity 0141 8130/81/0201294)6502.00 ©1981 IPC BusinessPress

occurred at higher gum concentrations (> 0.2-0.5~o, w/v). In each case, the viscosities reached plateau levels at salt concentrations < 1~o. The effect of added salt on viscosity at low xanthan concentrations was attributed to the reduction in molecular dimensions resulting from diminished intramolecular electrostatic repulsion. It was suggested s that at higher xanthan concentrations the apparent increase in macromolecular dimensions was indicative of mutual interactions or entanglement. More recently, deviations from normal polyelectrolyte behaviour have been interpreted 7 in terms of a rod-like ordered conformation in solution, which melts out only OH HO

CH.2OH 0

CH2OH

HO" HO"~-

oHO

OH

0

.o

OH. 0

~.

CHX2OH 0

\

CH2OH

0

2 ~.oO~.~

.o

0 OH

0

0

2 ~.oO~

/o.

CH3

Figure 1 Xanthan primary structure 2-4

Int. J. Biol. Macromol., 1981, Vol 3, April

129

Xanthan pyruvate: I. H. Smith et al. under conditions of elevated temperature and low ionic strength. Evidence of molecular order came initially8 from optical rotation studies of the thermally induced cooperative order~tisorder transition, and from reinterpretation of published rheological data, and was subsequently confirmed by nuclear magnetic resonance relaxation ~, circular dichroism9, and further studies of the temperature course of optical rotation 9'~° and solution viscosity10. The temperature at the transition midpoint (T,,) increases linearly with the logarithm of ionic strength, but is independent of polymer concentration l°'x 1. X-ray diffraction studies 12 on xanthan fibres have shown that in the solid state the macromolecule adopts a helical conformation having five-fold symmetry and a pitch 0f4.7 nm. It has not yet proved possible, however, to distinguish between two alternative models, which are almost equally consistent with observed diffraction intensities, and have both found some support from other evidence. These are, respectively, a coaxial double helix, suggested by electron microscopya 3 and hydrodynamic14 evidence, and a single-stranded structure, stabilized by alignment and packing of side chains along the polymer backbone, suggested 9 from the concentration independence 9,~0 and first-order kinetics ~5 of the disorder-order transition, and from sedimentation 16 and light scatteringl v studies. With increasing temperature, solution viscosity, measured at a relatively low rate of shear (7 s- ~), shows two anomalous increases 18. The larger of these coincides with the order~tisorder transition, but the second occurs at lower temperature, and has been attributed to thermal dissociation of intermolecular aggregates. The different rheological behaviour of solutions of xanthans varying in pyruvate content has been studied by Sandford et alJ9.They showed that solution viscosity increases with increasing pyruvate content at 0.1 and 0.5~o xanthan in 1% aqueous KC1 concluding that high and low pyruvate types interact differently. Our studies show that the extent of association of xanthan in solution under given conditions of concentration, ionic strength, temperature and shear rate is directly related to the degree of pyruvate substitution. A rapid increase in the extent of macromolecular association with increasing ionic strength for xanthans substituted above a critical degree of pyruvate substitution provides a rationale for the atypical increase in viscosity observed when salt is added to concentrated (above ~0.2~) aqueous solutions of the polysaccharide. The degree of pyruvate substitution and the ionic strength also influence the polysaccharide concentration above which macromolecular association becomes significant.

Experimental Materials Six polysaccharide samples (I-VI) were prepared from commercially produced xanthans available from Kelco Co. Inc., San Diego, California, USA. (Keltrol, Kelzan), Rhon~Poulenc Industries, Paris, France (Rhodopol, Rhodigel) and Lohmann Fermentations, GmbH, Cuxhaven, W. Germany (Viskotan). An aqueous solution (0.5%) of each industrial gum was centrifuged at 38 000g for 1 h at 10°C, and the polysaccharide remaining in the supernatant was converted to the potassium salt form and purified by dialysis against 0.1 M KC1 and then exhaus-

130

Int. J. Biol. Macromol., 1981, Vol 3, April

tively against distilled and deionized water. The final samples were recovered by freeze drying each dialysate. A seventh sample, VII, was derived from a shake flask culture of a pyruvate-free xanthan producing strain of Xanthomonas campestris (ATCC 31313) grown 2° on MYGP medium at 30°C. The fermentation broth was then treated in the same was as the solutions of the commercial products to yield the purified potassium salt of the polysaccharide.

Analyses The pyruvic acid content, P (~o,W/w,CH3COCO2H) of each polysaccharide was determined by hydrolysis and the lactate dehydrogenase NAD/NADH method 21. The ratio R of the degree of pyruvate substitution (x) to the degree of acetylation (y) was obtained from the corresponding ratio of peak areas at 6 1.5 (pyruvate methyl) and 6 2.1 (acetate methyl) in the n.m.r, spectra of the polysaccharides. The value of x, defined as the fraction of side chains bearing pyruvate ketal substituents, was then calculated (assuming the primary structure shown in Figure 1) using the relationship: x = 862 P R/(8800 R - 108 P R - 42P) The degree of acetylation y = x / R calculated.

(1)

could then be

Physical methods Flow curves were obtained at 25.0_+0.5°C with a Contraves Rheomat 30 viscometer and cone and plate geometry MS-KP (53.1 mm diameter, 0.5 ° cone angle, shear rate range 0.536-3950 s-1) on 1~o (w/v) solutions of the polysaccharide in distilled, deionized water and in 0.1 M KCI solution. Rheology-concentration relationships on polysaccharides II and V were also determined in the concentration range 0.002-1.0% (w/w) with the twin-gap double concentric cylinder geometry MS-O (shear rate range 0.237-1740 s -1) for solutions <0.8% (w/w). Cone and plate geometry was used for higher concentrations. Intrinsic viscosities, [q], were obtained by extrapolating plots of reduced viscosity versus concentration at 25.0 +_0.1°C determined with a Cannon-Fenske capillary viscometer on filtered (3 ktm Millipore) solutions in the concentration range 0.004-0.02% (w/v) in 0.1 M KC1. High resolution proton magnetic resonance spectra were recorded at 100 MHz at 0.5% (w/v) in D20 solution at 95°C on a Varian XL 100 Fourier transform spectrometer, as described previously 9. Optical rotation was measured at 436 nm on a Perkin-Elmer 241 polarimeter using a 10 cm thermostatted cell, a sample concentration of ~0.5% (w/v) and a constant ionic strength obtained by dialysis against 0.01 M KC1 solution.

Results and discussion Degrees of substitution Purified polysaccharide samples I-VI prepared from the six commercial xanthan samples had degrees of pyruvate substitution in the range 0.324).44 whilst sample VII was confirmed to be pyruvate-free (Table 1). Degrees of acetylation range from 0.90 (sample I) to 0.35 (sample VI).

Xanthan pyruvate." I. H. Smith et al. Table 1 Analytical and physical parameters of the purified polysaccharides

1% (w/v) in salt-free solution Purified xanthan sample I II III IV V VI VII

o]

1% (w/v) in 0.1 M KCI solution

range of (s-1)

Derived form

R

x

y

It/] (dl g-l)

K (Pa s")

n

Keltrol Kelzan Rhodopol Viskotan Rhodigel Keltrol Pyruvate-free xanthan

0.43 0.40 0.65 0.43 0.73 0.93 0.00

0.39 0.34 0.44 0.34 0.39 0.32 0.00

0.90 0.85 0.67 0.79 0.53 0.35 --

25.0 23.5 25.5 19.9 23.4 25.3 --

6.88 6.86 4.98 7.43 6.46 8.34 1.02

0.19 0.18 0.19 0.16 0.18 0.27 0.39

K (Pa s")

n

range of (s -1)

21.9 18.8 21.6 10.0 23.9 15.3 0.842

0.04 0.08 0.11 0.13 0.13 0.11 0.40

3.39-99.1 3.39-99.1 3.39 339 0.536-183 4.60460 2.49-183 0.40-3950

0.991-53.6 0.991-39.5 0.729-53.6 0.991-53.6 0.536 39.5 0.536-460 8.50-3950

80

75

-100

0

:o v

70

"5 E oJ

E u

65 -200

© "u v

s

60 0.0

I

I

0.2

0.4

I

I

0.6

0.8

1.0

R

Figure 3 Variation in the temperature (Tin)at the midpoint of the order-disorder transition ( s e e Figure 2) with pyruvate/acetate ratio (R)

-30C

-4001 30

I 40

I 50

I 60

I 70

I 80

I 90

10 0

T (*C)

Figure 2 Temperature dependence of optical rotation (436 nm) for samples II (o) and V1 (e), which have pyruvate/acetate ratios (R) of 0.40 and 0.93, respectively

Intrinsic viscosities The intrinsic viscosities of polysaccharides I-VI ranged from 19.9 to 25.5 dl g - ' (Table 1). Since the parameter is determined from measurements of reduced viscosities at low concentrations the extent of interchain association is assumed to be minimal. Therefore the intrinsic viscosity is dependent upon the size and shape characteristics of singly dispersed species at infinite dilution.

Chiroptical studies In view of the established ionic strength dependence of the stability of the ordered helical conformation 9J°, all chiroptical studies were at constant ionic strength (0.01 tool k g - x). As illustrated in Figure 2, the magnitude of the

optical rotation change which accompanies the orderdisorder transition is essentially independent of pyruvate and acetate levels, while the temperature course of the transition shows a marked systematic variation with the ratio R = x / y from n.m.r, spectra. Figure 3 shows the dependence of Tm on R, indicating that the ordered conformation becomes more stable as x decreases and y increases. The destabilizing effect of pyruvate is consistent with intramolecular electrostatic repulsions which oppose the formation of a compact helix. However, the apparent stabilizing influence of acetate implies that it takes part in an intramolecular attractive interaction.

Rheoloyy of 1% (w/v) solutions The flow curves of 1% (w/v) solutions in the absence and presence of 0.1 M KC1 were represented as log-log plots of apparent viscosity (r/) versus shear rate (i). For example, the curves for sample I in Figure 4 show that the solutions are highly pseudoplastic fluids tending towards more Newtonian behaviour at low and high shear rates and approximating power law behaviour 22 over a central shear rate range. Table i shows the K (consistency index) and n (flow behaviour index) values together with the

Int. J. Biol. Macromol., 1981, Vol 3, April

131

Xanthan pyruvate." I. H. Smith et al.

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~0

o.. °\o\

o\ 0'~'0\ O\

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. .~')~~ .

'~0-~o ..

::q"o. -2

0

! 1

I 2 Log ~7

I 3

Figure 4 Flow curves at 25°C for l ~o(w/v) solutions of sample I in distilled and deionized water (o) and in 0.1 M KC1 ( e ) Extrapolations of the regions approximating power law behaviour are indicated (.-.) shear rate range in which the power law given by r/= K);"-1 can be fitted to the data. The fractional change in viscosity (At/) resulting from the addition of 0.1 M KC1 to previously salt-free I ~ (w/v) solutions was calculated at several shear rates. Figure 5 shows that the value of At/is small and negative from zero degree of pyruvate substitution up to a critical value of x at ~0.31. When x exceeds this critical value, At/becomes positive and increases rapidly with further increases in x. Also, in this region A~/becomes more sensitive to shear rate because salt addition increases the pseudoplasticity of the solutions so that At/is greater at lower shear rates. Negative values of Aq in the range 0 < x < 0 . 3 1 imply that the reduction of electroviscous effects occurring at higher ionic strength has the predominant influence on At/. As the degree of pyruvate substitution rises above the critical value, this effect is at first offset and then completely surpassed by a positive contribution to the viscosity from macromolecular association. The enhanced pseudoplasticity of solutions containing associated macromolecules may derive from the progressive breakdown of structure under the influence of increasing shear rate. A fast rate of structure build-up and breakdown would then account for the very small amount of thixotropy observed in the xanthan solutions studied. The increase in pseudoplasticity due to salt addition can be attributed to an increase in the extent of association. Clearly, the degree of pyruvate substitution has a powerful effect on polymer-polymer interaction, which controls the extent of association in solution and hence the viscosity particularly at low shear rates. The effect of molecular weight on solution viscosity, not included in this discussion, has apparently been largely separated from the effect of pyruvate content by considering the viscosity change on addition of salt as a fraction of the viscosity in salt-free solution.

Rheology-polysaccharide concentration relationships Flow curves were determined at fifteen different concentrations on samples II and V, which have almost identical intrinsic viscosities (23.5 and 23.4 dl g - l ,

132

Int. J. Biol. Macromol., 1981, Vol 3, April

respectively) but differ significantly in their degrees of pyruvate substitution (0.34 and 0.39, respectively). Viscosities in water and 0.1 M KC1 interpolated from the flow curves at three selected shear rates (1, 10 and 100 s-1) are plotted on a log-log basis against concentration C in Figures 6 and 7. Each polysaccharide displays a different critical concentration in each solvent, at which a change in slope of the log r/versus log C relationship occurs (Table 2). Critical concentration, Co, and critical molecular weight, Me, are both well-known phenomena for polymer solutions and indicate the onset of network formation due to macromolecular entanglement 23-z7. At low concentrations, < C¢, the polysaccharide may be regarded as unassociated, as previously reported ~6. The similarity between the intrinsic viscosities of the two polysaccharides suggests a similarity of molecular weight for their unassociated macromolecules. Therefore, the effect of molecular weight on C~, can effectively be ignored. Comparison of Figures 6 and 7 indicates that the polysaccharide with the higher degree of pyruvate substitution (V, x = 0.39) has the lower critical concentration both in water and in 0.1 M KC1 (Table 2). This constitutes further evidence that association becomes increasingly favourable as the degree of pyruvate substitution rises. Above C~ in each solvent the slopes of the lines at a given

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x Figure 5 Fractional change in viscosity Aq at 25°C caused by addition of 0.1 M KC1 to sal.t-free 1~o (w/v) solutions of samples I-VII, as a function of the degree of pyruvate substitution of the polysaccharide. At/= 100{~/(in 0.l M KCI)- ~/(in water)}/r/(in water). Data calculated at three shear rates: (a) 4.61 s- 1 ( o); (b) 39.5 s -t (A); (c) 461 s -1 (~)

X a n t h a n pyruvate." I. H. Smith et al.

/ /o C

D

C0 O .d

d / O/ / / /o

9

/

-2 ¸

/

Onogi et al. 27 found that cto was smaller for polymers in good solvents than in poor solvents, the upper limit being ~ 6.8. Thus the higher values of ~t0 in each solvent for polysaccharide V (3.04 in 0.1 M KC1, 1.96 in water), compared with polysaccharide II (2.08 in 0.1 M KC1, 1.30 in water) indicate that polymer-polymer interactions become more significant when the degree of pyruvate substitution is high and the electrostatic repulsion between chains is minimized by high ionic strength. The two polysaccharides also display a crossover concentration C x independent of shear rate where the solution rheology in water is identical to that in 0.1 M KC1. At concentrations C~ are sufficient for the viscosity-enhancing effect of macromolecular association to predominate, causing a net increase in viscosity when KC1 is added to salt-free solutions. C x occurs at a lower concentration for polysaccharide II (0.15~o, w/w) than for polysaccharide V (0.25~o, w/w) due mainly to the low value of C c in water exhibited by polysaccharide V, which

//49

.g -3

I

I

-2

-1 Log C

I

0

Figure 6 Dependence of viscosity on concentration for sample II (x = 0.34) at 25°C in water ( o ) and 0.1 M KC1 ( • ). Data shown at three shear rates; A, 1 s-1; B, 10 s-1; C, 100 s-1 shear rate are greater for polysaccharide V, indicating a more rapid build up of association with concentration at the higher degree of pyruvate substitution. Polymer solution viscosity (r/) varies with concentration and molecular weight according to the following general relationship27:

£ ~1= k ( C p ) ~ M ~

(2)

'/o" o

._1

where C is the weight fraction of polymer, p is the solution density, k is a constant at a given temperature and ct and fl are the slopes of log r/versus log (Cp) and log r/ versus log M curves, respectively. When r/is measured at zero shear rate, the value of fl above Mc is generally 3.4 for all polymer solutions and melts. Values of ~ for aqueous solutions of disordered polysaccharides 2a are also normally around 3.4, although values of 5 or greater have been reported 27 for several synthetic polymers in various solvents. Since the solution densities within the range of polysaccharide concentrations investigated deviated very little from unity in both solvents, then Cp ~ C in equation (2). Figures 6 and 7 therefore show that ~ increases with decreasing shear rate. Values of ~ for C > C¢ were determined as the slopes of log r/ versus log C curves at the lower shear rates 0.1, 0.2, 0.5 and 1 s - 1 obtained from the relevant flow curves (extrapolated where necessary). Figure 8 shows these values of ~ as a function of ~1/2 for polysaccharides II and V in both solvents. Linear extrapolation of the points then gives the m a x i m u m value of ~ at zero shear rate, ~o. The polysaccharide with the higher degree of pyruvate substitution has the highest values of c~0 in each solvent and also shows a greater dependence of ct on shear rate.

C

/

P

/

o/ /o

/

9"

/ /

,09 9"

-2

/

/

c.-

J°Cp -3

.-I~ --'-°''~

-

-3

I

I

I

-2

-1 Log C

0

Figure 7 Dependence of viscosity on concentration for sample V (x = 0.39) at 25°C in water ( o ) and 0.1 M KC1 ( • ). Data shown at three shear rates; A, 1 s-l; B, 10 s-l; C, 100 s -1

Table 2 Critical and crossover concentrations for samples II and V Sample II V

x

Cc (water) (~o, w/w)

0.37 0.46

0.20 0.09

Cc (0.1 M KCI) Cx (To, w/w) (~o, w/w) 0.048 0.014

0.15 0.25

Int. J. Biol. Macromol., 1981, Vol 3, April

133

Xarlthan pyruvate:

I. H. Smith et al.

close enough in the helix to act cooperatively, forming apolar regions stretching not only around the helix for pyruvate groups on consecutive side chains, but also parallel to the axis of symmetry for pyruvate groups on every fifth side chain. The acetate substituents also contain methyl groups but these are located closer to the centre of the helix and are less available for intermolecular interaction. Apolar interactions of acetate groups can be invoked as an intramolecular binding force contributing to the stability of the ordered helical conformation. Finally, the ability of Xanthomonas campestris to change the degree of pyruvate substitution of its exopolysaccharide in response to a change in environment or fermentation conditions represents a possible controlling mechanism on the rheology of the polysaccharide which may be an important survival factor for the bacterium.

Acknowledgements We thank Dr D. A. Rees for discussions, Mr J. E. Rudland for rheological measurements, Mr J. M. Brown and Mrs R. Leeke for analyses and sample preparation, Mr E. J. Murray for chiroptical studies and Mr S. Bociek for n.m.r. measurements.

.. ... ... ... . . . ...p

References

rl ‘(3_

-

-

I)

_

_

_

a -

1

2 3

1.c .”

4 5 Figure 8 Determination of czO, the slope of log q versus log C curves at zero shear rate, by extrapolation (. . .) of tl uersus +“’ plots obtained in water ( o ) and 0.1 M KC1( l ) on samples I and

V enables the viscosity in water to surpass that in 0.1 M KC1 at a lower concentration.

Conclusions These results indicate that pyruvate substituents on the xanthan macromolecules promote association and structure formation by increasing the polymer-polymer affinity relative to the polymer-solvent affinity. The effect must be regarded as being additional to that of molecular weight on intermolecular interactions, and does not arise from differences in the extent of$onformational ordering with different degrees of pyruvate substitution. Intermolecular association may be enhanced by apolar interactions between pyruvate methyl groups, which are located on the periphery of the five-fold helical structure in either the double or single helix models suggested by Xray diffraction evidence I2 The association phenomenon would then be analogous to the micellization of a surfactant in solution above the critical micelle concentration, a process which is made thermodynamically favourable by a large positive entropy change in the system. When the degree of pyruvate substitution becomes sufficiently high ( >0.31) there is a greater possibility that pyruvate groups will occur on sufficient numbers of side chains so that they are spatially located

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Int. J. Biol. Macromol., 1981, Vol 3, April

6 I 8 9 10 11 12 13 14 15 16 17 18

‘Extracellular Microbial Polysaccharides’, (Eds P. A. Sandford and A. Laskin) ACS Symposium Series, vol. 45, Washington DC, 1977 Jansson, P. E., Kenne, L. and Lindberg, B. Carbohydr. Rex 1975, 45, 275 Melton, L. D., Mindt, L., Rees, D. A. and Sanderson, G. R. Carbohydr. Res. 1976, 46, 245 Lawson, C. J. and Symes, K. C. in Ref. 1, p. 183 Jeanes, A., Pittsley, J. E. and Senti, F. R. J. Appl. Polym. Sti. 1961, 5, 519 Cadmus, M. C., Knutson, C. A., Lagoda, A. A., Pittsley, J. E. and Burton, K. A. Biotechnol. Bioeng. 1978, 20, 1003 Morris, E. R. in Ref. 1, p. 81 Rees, D. A. Biochem. J. 1972, 126, 257 Morris, E. R., Rees, D. A., Young, G., Walkinshaw, M. D. and Darke, A. J. Mol. Biol. 1977, 110, 1 Holzwarth, G. Biochemistry 1976, 15, 4333 Milas, M. and Rinaudo, M. Carbohydr. Rex 1979, 76, 189 Moorhouse, R., Walkinshaw, M. D. and Arnott, S. in Ref. 1, p. 90 Holzwarth, G. and Prestridge, E. B. Science 1977, 197, 757 Holzwarth, G. Carbohydr. Res. 1978, 66, 173 Norton, I. T., Goodall, D. M., Morris, E. R. and Rees, D. A. J. Chem. Sot. Chem. Commun. 1980, 545 Rinaudo, M. and Milas, M. Biopolymers 1978, 17, 2663 Rees, D. A. Pure Appt. Chem. 1981, 5, 1 Dea, I. C. M., Morris, E. R., Rees, D. A. and Welsh, E. J. Carbohydr. Rex 1977, 57, 249

19 20 21

22 23 24 25 26 27 28

Sandford, P. A., Pitt&y, J. E., Knutson, C. A., Watson, P. R., Cadmus, M. C. and Jeanes, A. in Ref. 1, p. 192 Wernau, W. C. UK Pat. Appl. GB 2008 6OOA,(1979) Jeanes, A., Rogovin, P., Cadmus, M. C., Silman, R. W. and Knutson, C. A. ‘Polysaccharide (xanthan) of Xanthomonas campestris NRRL B-1459; Procedures for Culture Maintenance and Polysaccharide Production, Purification and Analysis, ARSNC 51, USDA, November 1976 Whitcomb, P. J., Ek, B. J. and Macosko, C. W. in Ref. 1, p. 160 Graessley, W. W. Adu, Pofym. Sci. 1974, 16, 1 Krumel, K. L. and Sarkar, N. Food Technoi. 1975, 29, 36 Chinai, S. N. and Schneider, W. C. J. Polym. Sci. (A) 1965,3,1359 Onogi, S., Kobayashi, T., Kojima, Y. and Taniguchi, Y. J. Appl. Polym. Sci. 1963, 7, 847 Onogi, S., Kimura, S., Kato, T., Masuda, T. and Miyanaga, N. J. Polym. Sci. (C) 1966, 15, 381 Morris, E. R. and Ross-Murphy, S. B. in ‘Carbohydrate Metabolism’, (Ed. D. Northcote), Techniques in Life Sciences, Elsevier, London, 1980