Purified Alteromonas macleodii polysaccharide and its uses

Obtaining a Preparation of Purified Polysaccharide

The exopolysaccharides secreted by the Alteromonas macleodii subsp. fijiensis strain C.N.C.M. I-1627 are obtained from cultures of this bacterium by precipitating with ethanol, as described by RAGUENES et al., [Applied and Environmental Microbiology, 62(1), pp. 67-73 (1996)].

After having been dissolved in Tris buffer (50 mM, pH 7.5), the exopolysaccharide mixture is loaded onto an anion exchange column (SEPHAROSE CL-6B gel). The column is eluted with a gradient of from 0.1 to 1M NaCl in the same buffer.

The elution profile is depicted in FIG. 1 . The fractions from the main peak, corresponding to an NaCl concentration of 450 mM, were pooled, dialyzed and lyophilized.

The analyses carried out on these fractions show that no proteins are present and enable the contents of uronic acids and hexoses to be calculated to be 38% and 37%, respectively.

Determining the Physicochemical Characteristics of the Polysaccharide

Composition

The polysaccharide obtained as described in Example 1 is subjected to an acid methanolysis and the methylglycosides are converted into corresponding trimethylsilylated derivatives in accordance with the method of MONTREUIL et al. [Carbohydrate analysis: a practical approach, Chaplin M. F and Kennedy J. K. (Eds), I.R.L. Press Oxford, Washington D.C., Glycoproteins pp. 143-204 (1986)], after which they are analyzed by gas chromatography coupled to a mass spectrometer.

The neutral monosaccharides are also analyzed in the form of alditol acetates, after hydrolysis of the polysaccharide followed by a reduction and an acetylation in accordance with the method of BLAKENEY et al., [Carbohydr. Res., 113, pp. 291-299 (1983)].

The content of pyruvic acid is calculated with the aid of a calorimetric assay method using 2,4-dinitrophenylhydrazone [SLONEKER et al., Nature, 194, pp. 478-479, (1962)].

The absolute configuration of each of the residues making up the polysaccharide was determined in accordance with the method of GERWIG et al. [Carbohydr. Res., 62, pp. 349-357, (1978)]. The glycosidic residues derived from hydrolyzing the polysaccharide react with (+)2-butanol in the presence of trifluoroacetic acid. The resulting chiral derivatives are trimethylsilylated and then analyzed by gas chromatography.

Analysis of the residues in the form of alditol acetate and trimethylsilylated methyl glycosides indicate the presence of glucose, galactose, mannose, glucuronic acid and galacturonic acid in the relative proportions of 1:1:1:2:1 (1:1.1:0.6:1.1:0.6 in the case of the crude polymer). It was established that each of the residues had the D configuration. The content of pyruvic acid is calculated by colorimetric assay to be 7% by weight, a value which indicates that all the mannose residues are substituted.

The results of the gas chromatographic analysis, coupled to the mass spectrometry of the trimethylsilylated methlyglycosides, are illustrated by FIG. 2 . This analysis shows that the pyruvate groups are linked at positions 4 and 6 of the mannose residue.

Nature of the Glycosidic Bonds

Analyzing the polysaccharide by methylation made it possible to determine the cyclic form (pyranose or furanose), and the nature of the glycosidic bonds, of each of the residues of which it is composed. Using the method of HAKOMORI [J. Biochem, 55(2), pp. 205-208, 1964)], modified by D'AMBRA et al. [Carbohydr. Res., 177, pp. 111-116, (1988)], the free hydroxyl functions of the polysaccharide are methylated by the action of methylsulfinyl lithium carbanion and methyl iodide in dimethyl sulfoxide. The hydroxyl functions involved in bonds are then liberated by hydrolysis. The alditol acetates derived from the reduction, and then the acetylation, of the partially methylated residues are analyzed by coupling gas chromatography and mass spectrometry. Their nature, characterized by the position of their methyl groups, defines the cyclic form and the positions of the glycosidic bonds of the corresponding residues.

This analysis shows the presence of glucose and galactose which are substituted in the 4 position, of mannose which is disubstituted in the 4 and 6 positions (confirming the substitution sites of the pyruvate group), of glucuronic acid which is substituted in the 3 position, of glucuronic acid which is substituted in the 4 position and of galacturonic acid which is disubstituted in the 3 and 4 positions. This latter therefore constitutes a branching point of a branched, repetitive hexasaccharide unit, the end of whose side chain is the pyruvate-substituted mannose residue. All the residues are in the pyranose cyclic form.

Location of the Uronic Acids

In order to locate the uronic acids which are present in the repetitive unit, the previously methylated polysaccharide is treated in alkaline medium in accordance with the method of ASPINALL et al. [Carbohydr. Res. 57, pp. C23-C26, (1977)].

This analysis demonstrates the presence of a single compound possessing a O-ethyl group, i.e. the alditol acetate derivative of the galactose residue. The repetitive unit therefore comprises a sequence of three uronic acid residues, which sequence is bonded in position 4 of the galactose residue.

Nuclear Magnetic Resonance Spectroscopy

After the polysaccharide had been depyruvated (by treating it in 2% acetic acid for 2 hours at 100° C.) and dissolved in deuterated water containing added NaCl, the NMR spectra were recorded at 320K on BRUKER AMX-500 and AMX-600 appliances. The “TOCSY” (Total Observed Correlation SpectroscopY) and “COSY” (Correlation Observed SpectroscopY) high field analyses in homonuclear mode, and the spectrum obtained at high field in “HSQC” (Heteronuclear Single Quantum Coherence) heteronuclear mode enabled the signals to be attributed to the protons and carbons of each residue.

The α/β configuration of the glycosidic bonds is defined by the values of the chemical shifts of the signals corresponding to the anomeric carbons and protons. The “NOESY (Nuclear Overhauser Enhancement SpectroscopY) analysis in homonuclear mode determines the arrangement of the residues in the repetitive unit.

Analysis of the HSQC spectrum made it possible to identify six separate residues constituting the repetitive unit, which was consistent with the results of the methylation. The configuration of the glycosidic bonds is determined from the values of the chemical shifts of their anomeric protons and carbons. Most of the signals are attributed to the protons of the different residues by analyzing the TOCSY and COSY spectra. Interpretation of the NOESY spectrum, as a supplement to the results from the β-elimination, made it possible to define the arrangement of the residues:

Determining the Rheologic Properties of the Polysaccharide

The flow properties of the polysaccharide in solution were studied in a semi-dilute system for polymer concentrations of 1 g/l ( ) and 2 g/l (♦) (that is 0.1% and 0.2%) in water to which NaCl had been added to a concentration of 0.1M. The results of the study are expressed by drawing up flow curves which depict the variation in the apparent viscosity as a function of the shear rate during the application of a continuous cycle of increasing and decreasing the rate. FIG. 3 demonstrates the thickening character of the polysaccharide in solution, with a viscosity which depends on the concentration of the polymer. Furthermore, the appearance of the flow curve demonstrates a rheofluidifying behavior, with the existence of a so-called “Newtonian” plateau in the area of the low shear rate values, followed by a decrease in the apparent viscosity as a function of the shear rate in the case of the high rate values.

Plotting such dilute system flow curves for polysaccharide concentrations of from 0.02 to 0.2 g/l (0.002% to 0.02%) enabled its intrinsic viscosity [η] (hydrodynamic volume occupied by one gram of polymer in a given solvent) in a 0.1 M solution of NaCl to be determined. The intrinsic viscosity was thus calculated to be 2 600 ml/g.

All the measurements were carried out at 20° C. using a LOW SHEAR 40 (CONTRAVES) viscosimeter having coaxial cylinders.

The flow curves for 2 g/l solutions of the purified polysaccharide in accordance with the invention in an aqueous solvent having an anionic strength of 0.1M were plotted for the presence of various ions. These curves, which are depicted in FIG. 4 (Mg 2+ : ♦; K + : ; Na + : □; Ca 2+ :□) show that, while the viscosity of the polysaccharide increases markedly in the presence of Ca 2+ ions, it is not, on the other hand, influenced by the presence of K + or Mg 2+ ions.

FIG. 5 depicts the flow curves obtained for 2 g/l concentration solutions of polysaccharide according to the invention in 0.1M NaCl and at different pH values (□=pH3; =pH4.5; □=pH6; □=ph7; □=pH9). These curves make it possible to conclude that the thickening properties of the polysaccharide according to the invention are stable over a range of pH values of between 4.5 and 9.

Use of a Polysaccharide According to the Invention for Preparing Foodstuffs

Table I below gathers together some examples of the incorporation of a polysaccharide according to the invention into foodstuffs, indicating the proportion by weight, based on the total weight of the ingredients, which is generally preferred for each of these foodstuffs.