GB2223503A - Gel-forming polysaccharides - Google Patents

Abstract

A number of polysaccharides of Formula (I): <IMAGE> can be gelled by exposing an aqueous solution thereof to sufficient electrolyte or cooling the aqueous solution. Such gels are useful in many contexts, for example food preparations. The polysaccharides can be extracted from Rhizobium leguminosarum species.

Description

GEL-FORMING MATERIALS This invention relates to gel-forming bacterial polysaccharides.

Microbial polysaccharides provide a source of new pol.rsaccharides and are increasingly being examined to discover potentially useful polymers with desirable physical and rheological properties. Up to the present time very few geJ-forming bacterial polysaccharides have been discovered. An exception is curdlan. a neutral 1-+3 linked beta-D-glucan synthesised by certain Agrobacterium spp. and by Rhizobium meliloti (T. Harada in "Polysaccharides in Foods'; eds. J.M.V. Blanshard and J.M. Mitchell. , Butterworths, London, p 283 (1979); T.

Harada and A. Amenura, Mem. lnst. Sci. Ind. Res. Osaka Unit. 38,37 (1981)). A second exception is the gel forming capsular polssaccharide produced by Rhizobium leguminosarum and Rhizobium trifolii spp (L.P.T.M.

Zevenhuizen and A.R.W. van Neerven, Carb. Res. 124, 166 (1983)). This is a neutral branched heteropolysaccharide containing gaiactose, glucose and mannose. A third exception is the bacterial polysaccharide called gellan gum, an acidic heteropolysaccharide containing glucuronic acid, glucose and rhamnose (M.A. O'Neill, R.R. Selvendran and V.J. Morris, Carb. Res., 124, 123 - (1983)). This polymer is esterified and in its native state forms soft elastic thermoreversible gels. Progressive deesterification of the polymer results in firmer, more brittle gels (R. Moorhouse et al, American Chemical Society Symposium Series 150, 111 (1981); V.J. Morris, M.J. Miles and M.A. O'Neill, in "Gums and Stabilisers for the Food Industry.2.Applications of Hydrocolloids", ed.

G.O. Phillips, D.J. Wedlock and P.A. Williamsons, Pergamon Press, Oxford, p.485 (1984)). A fourth exception is the bacterial polysaccharide XM6 elaborated by an Enterobacter sp. (NCIB 11870) (B.A. Nisbet et al, Carb.

Polvtn. A, 377 1984). This polymer is a branched acid heteropolysaccharide containing glucuronic acid, glucose and fucose (M.A. O'Neill et al, Carb. Res. 148, 63 (1985)) and is unusual because aqueous solutions of the polysaccharide do not gel unless sufficient electrolyte is added to the samples. Upon addition of electrolyte the samples form thermoreversible gels with sharp melting and setting temperatures. The melting temperature is sensitive to the type and concentration of electrolyte.

The fifth and last exception is the mixture of exocellular polysaccharides secreted by Bacillus polymyxa (J.K. Madden, I.C.M. Dea and D.C. Steer, Carb. Poly. 6, 51 (1986)). This mixture is stated to consist of three fractions: a minor neutral polysaccharide. a minor acidic polysaccharaide and a major acid polysaccharaide. The major acidic branched heteropolysaccharide containing glucuronic acid, glucose, mannose and galactose is said to be responsible for establishing a gel network.

It has now been discovered that the exocellular acidic heteropolysaccharides secreted by certain Rhizobium spp. will under appropriate conditions form thermoreversible gels. The purified polsaccharides can be dissolved to form aqueous solutions which do not gel.

However, in the presence of sufficient electrolyte the aqueous samples form thermoreversible gels. Gelatin ie observed for members of a family of polysaccharides possessing the same backbone structure but containing one of several types of sidechain.

Accordingly, one aspect of the invention provides a gel comprising a polvsaccharide of Formula (I):

where n is an integer between 10 and 101o but typically expected to be 105-106, and R is a straight or branched oligosaccharide side chain comprising from 0 to 20 sugar residues.

Preferably, R consists of one or more of the following sugar residues: D-Galp, D-GlcAp and D-Glcp.

Preferably, R is a straight chain of 5 to 8 residues. One or more of the residues, preferably the terminal residue remote from the main chain, may be modified by pyruvate substitution or b other noncarbohydrate substitutions such as other ester substituents (for example, succinyl or acetyl).

Most preferably, R is selected from:

The structure of the polysaccharide can be determined to various degrees of sophistication. The neutral sugars present in the polsaccharide can be determined as follows: The polysaccharide is degraded b a Saeman hydrolysis (using concentrated sulphuric acid) and the liberated sugars determined as their alditol acetates by has chromatography (Selvendran RR and Dufont.

N.S. in Development in Food Analysis Techniques - 3 (Ed.

R.D. King) Elsevier Applied Science, London (1984), 1- 68). The amount of uronic acid present can be determined colorimetrically by a modification (Selvendran RR and Dupont MS in Developments in Food Analysis Techniques - 3 (Ed RD King) Elsevier Applied Science, London (1984), 1- 68) of the method of N. Blumenbrantz and G. Asboe-Harsen AnalBiochem. 1973, 54, 484 - 489). Substituents such as acetate and pyruvate may be determined b an HPLC method (NWH Cheetham and A. Punruckrong. Carb. Polym. (1985),5, 399-406). The types of linkages between the sugars can be determined by methylation analysis.The polysaccharide is meths-lated by a modification of the Hakomori method (Rino: SG and Selvendran RR, Phyto- chemistry (1978) 17 745-752) and converted to partially methylated alditol acetates which are analysed by gas chromatography and gas chromatography-mass spectrometry.

Individual partially methylated alditol acetates are identified by their retention times relative to a standard 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol and from their mass-spectrometric fragmentation patterns (Jansson P-E. et al. Chem. Commun. University of Stockholm (1976) number 8.), Quantitative analysis of the data can be achieved using the molar response facters given by Sweet DP et al -(Carb. Res.(1975)40 217-225) The mosr sophisticated method of analysis of these pol-saccharides is described by McNeil et al (Carb. Res 146 (1986) 307-326) and by Hollingsworth et al (Carb.

Res. (1988) 172, 92-122). The polysaccharide is degraded using ar. enzyme obtained from a bacteriophage. This (1988) 172. 92-122). The polysaccharide is degraded using an enzyme obtained from a bacteriophage. This enzyme breaks the polysaceharide down into individual repeat units which can be separated and characterised by the methods described above. The sequence of the sugars in the repeat unit and the position of non-carbohydrate substituents (for example, acetate or pyruvate) can be defined by one dimensional and two-dimensional proton nuclear magnetic resonance studies. This method provides a unique method of defining each or any of the polysaccharides.

The polysaccharide may be obtained (a) bs isolation from any suitable source (b) by chemical synthesis, (c) by chemical degradion or (d) by the action of enzymes extracted from suitable orgamisms (such as Rhizobium bacteria) or prepared by recombinant DNA techniques. but suitably it is obtained (e) directly from a bacterium of the genus Rhizobium, especially from R. legominosarium phaseoli, R. leguminosarum bv. trifolii, or R.

leguminosarum bv. viciae, or by cultivation of an organism carrying the relevant genelsi from such Rhizobia.

The polysaccharides of Formula (I) may be obtained from a wide variety of readily obtainable leguminous Rhizobia and, in particular, from the following sources: Side chain (i) Rhizobium leguminosarum bv. trifolii 4S Side chain (ii) Rhizobium leguminosarum bv. phaseoli 127K36 Rhizobium leguminosarum bv. phaseoli LPR49 Rhizobium leguminosarum bv. trifolii NA30; 0403 Rhizobium leguminosarum bv. trifolii LPRS Rhizobium leguminosarum bv. trifolii TA-l Rhizobium leguminosarum bv. viciae 128c53; 128c63 Rhizobium leguminosarum bv. viciae LPR1 Rhizobium leguminosarum bv. viciae 8401 p RLlJl Rhizobium leguminosarum bv. phaseoli 8002 Rhizobium leguminosarum bv. phaseoli RW4 p1J142 Side chain (iii) Rhizobium leguminosar:lm bv. phaseoli 127K44 Side chain (iv) Rhizobium leguminosarum bv. phaseoli 127K38 Side chain (v) Rhizobium leguminosarum bv. phaseoli 12/K8, second aspect of the invention provides a process for preparing a gel from a polysaccharide of Formula (I) above, comprising admixing sufficient electrolyte and an aqueous solution o, the polysaccharide to form a gel- forming compound.

The amount of electrolyte which is sufficient will depend upon the nature of the polysaccharide, the nature of the electrolyte, the concentration of the polysaccharide, the presence of other components, the temperature and so on, but may readily be determined by the skilled man by routine and non-inventive experimentation.

For cold-setting gels the cations can be added (b- dialysIs or use of partially soluble salts etc.) to a solution of the polysaccharide at a temperature below the eventual melting point of the prepared gel. For thermosetting gels the cations can be added before dissolving the polysaccharide (as a mixed powder). to coid solutions which are subsequently heated and then recooled to cause gelation or to hot solutions which are cooled to cause gelation. Alternatively, the polysaccharide can be dissolved and the cations addedqseparately to either the hot or cold solution eithe-r as a solid powder or as a solution. The limitation on adding the cations cold is the melting point of the gel.If this is high (greater then about 60oc) then it is difficult to disperse the mixture properly and obtain a homogeneous gel unless the cations are added to a hot solution of the polysaccharide.

Generally speaking, the polysaccharide is exposed to an ionic strength of less than 4 for monovalent cations and less than 2?1 for divalent cations. Conveniently, in the situation wherein the polysaccharide is prepared from a bacterial culture, the electrolyte is caused to be present in the culture medium: the polysaccharide which is produced in that medium has been found then to have gel-formina properties without the need for any further electrolyte.

Cations nomally employed to promote gelation in the process of the invention include Group Ia (lithium to caesium) and Group IIa (beryllium to barium), although other cations may be used. An example of another cation is ammonium (NH4+) Any anion may be employed e.C.

chloride or nitrate. Preferably, the cation is monovalent or bivalent, most preferably sodium or calcium.

The unpurified or at least partially purified bacterial broth forms a third aspect of the invention.

Thermoreversible gels are formed b these polysaccharides. The gels have been found to be transparent and to melt and set sharply on addition of salts to aqueous solutions of the polysaccharides.

Divalent cations have been found to be more effective at gelling the polysaccharides than monovalent cations.

Trivalent cations, such as ferric cations, generally cause precipitation of the polysaccharides. The variation of the storage modulus of the gels with cation radius for monovalent cations of Group Ia and divalent cations of Group IIa of the Periodic Table is shown in Figure Both the storage modulus and the melting temperature increase with increasing concentration of added salt. At higher electrolyte concentrations than those investigated ( > 0.4N for divalent cations and > 3.2 for monovalent cations) it is thought that the storage modulus of the gels may begin to decrease due to the formation of heterogeneous gel structures.Thus, at a given polysaccharide concentration. and for a given tspe of salt, there may be an optimum-composition: for gelation.

Such an optimum concentration mas easily be identified for a given polysaccharide concentration and type of electrolyte by comparing a set of gels prepared at a fised polymer concentration but with a range of salt concentrations.

It has been found possible to obtain gels with properties comparable with gels used for normal industrial or food applications by using a composition consisting of about 1% - 10%, preferably about 3%, w/w of polysaccharide prepared in about 1.0 - 2.OM, preferably about 1.6M, sodium chloride.

The storage modulus of the polysaccharide gels has been found to increase with increasing polymer concentration (Figure 2). Generally speaking, it is desirable that the gel is formed from as low a concentration of polymer as possible.

Aqueous solutions of these polysaccharides can be prepared by dissolving the purified polysaccharides in hot water and cooling the samples to room temperature.

Such samples are non-gelling. However, it has been discovered that if these samples are stored at OoC to 6 C. preferably +4 C. in a refrigerator or other suitable cooling device. then the samples will gel. These samples melt in the vicinity of room temperature (aDout 20 - 25 C). The melting temperature of these gels increases with increasina concentration of added salt.

Thus, by addition of a suitable quantity of an appropriate salt. gels can be produced which will melt ar body temperature (about 30 - 35 C). Such gels have applications in the biomedical. pharmaceutical and food industries. The gels may be used as a basis for preparing commercial gels, creams or lotions.

Conventional gelling polysaccharides normally used for industrial and food applications are usually extracted from plant and/or animal tissue. Eamples include pectin. alginate, carrageenans and agar. The chemical structures of these polysaccharides are not regular. Although the polysaccharides are chiefly composed of long sequences based upon a regular repeating monosaccharide or disaconaride repeat units the contain irregular sugar substituents. the number of which and type of which depend on the nature of the plant tissue and the method of extraction.

The present polysaccharides differ f-rom the plant and algal polysaccharides described above in possessing a regularly repeating chemical structure. This has tne advantage of producing more reproducible physical and rheological properties. Certain facets of the gelation of the polysaccharides resemble the gelation of pectin and and alginate. However,the present polysaccharides differ from pectin and alienate in the following ways. Firstly, whereas both pectin and alienate form cold-setting thermoirreversible gels upon introduction of salts containing certain divalent cations, cold setting gels can be prepared from the present polysaccharides using salts containing both monovalent and divalent cations.

Secondly, the gels can be made thermallv reversible or thermally irreversible by varying the type of salt ane the salt concentration. Finall, hereas pectin and alginate can be gelled over onir a narrow range of divalent cation concentrations determined b the detailec structure of the polysaccharide. it has been found that the present polysaccharides can be gel led over a wide range of monovalent and divalent cation concentrations.

The presently described polysaccharides resemble agar and carrageenans in producing thermoreversible heat setting gels. They differ from agar and carrageenans in that the polysaccharides of the invention do not show the large hvsteresis effects observed upon melting ana setting or agar and. unlike agar and the carrageenans, the poly- saccharides of the invention can be used to prepare cold- setting thermoreversible and cold-setting thermoirreversible gels.

Cold setting involves introduction cations into a sol at a fised temperature below that of the eventual melting-point of the final gel. Heat setting involves mixing salt and polymer solution at room temperature and heating and then recooling to allow the gel to set.

Alternatively salt may be added to a hot solution of the polysaccharide and a gel produced upon cooling.

The invention will now be illustrated by reference to the following non-limitino Examples Example i. Preparation of crude extracollular polysaccharide from R.leguminosarum 8002 R.leguminosarum by phaseoli 8002 (obtained from John Innes Institute, Norwich, U.K.) as grown in Y medium at 290C. 750 ml of medium at a pH of 6.8 was used in 2 litre flasks shaken at 150 rpm. The samples were harvested at late exponential phase - usually 72 hrs incubation. The cultures were centrifuded at 23,000g for 30 mins. to remove bacterial cells.The -supernatant was poured off and filtered through two layers of Whatman GFIB glass fibre filter paper. The supernatant was then sequentially filtered through Millipore filters of decreasing pore size (1.2um. 0.8um and 0.65um). ("Millipore" and "Whatman" are Registered Trade Marks). The filtered supernatant was concentrated by rotary evaporation to approximately one third the original volume. The extracellular polysacchaiide (EPS) was precipitated using 4-5 volumes of industrial spirit. The precipitate was isolated by filtration through a glass sinter funnel and then several times with industrial spirit. The precipitate was redissolved in glass distilled water and freeze dried.

Example 2. Prenaration of crude extracellular poly- saccharide from R.1euminosarum 8401 R.leguminosarum bv viciae 8401 (also obtained from John Innes Institute) is near isoenic with R.leguminosarum 8002. The former is derived from the latter in two stages: first the strain 8002 was obtained in which the sym plasmid pRP2JI had been lost (J.W. Lamb, G. Hombrecher and A.W.B. Johnson, cool. Gen. Genet. 186.

449 (1982)) and then the viciae sym plasmid pRLlJI was introduced by conjugation (J.A. Downie, G. Hombrecher, Q-S Ma, C.D. Knight, B. Wells and A.h.B. Johnston, Mcl.

Gen. Genet. 190, 359 (1983)). The effect of changing the svm plasmid is to change the host plant infected by the bacterium from phaseoli (beans) to viciae (peas).

The bacteria were grown in Y medium at 290C as described in Example 1. The bacteria were harvested in late exponential phase and the crude EPS isolated as described in Example 1.

Example 3. Prenaration of crude extracellular poly- saccharide from R.leguminosarum RW4 p1J1427 R.leguminosarum by phaseoli RW4 pIJ1427 (also obtained from John Innes Institute) is a genetic mutant prepared from R.leguminosarum 8002. The strain Rl;4 was prepared from R.leguminosarum 8002 using transposon mutagenesis and selecting for non-mucoid colonies. RW4 is an EPS derivative of strain 8002. Polrsaccharide production has been restored br the introduction of the multi-copy plasmid pIJ1427 which also confers resistance to tetracycline (D. Borthakur et al, Nol. Gen. Genet.

203, 320; 1986).

The bacteria were grown in Y medium supplemented with DUG ml-l tetracycline. Growth conditions, separation of bacteria and preparation of crude EPS are as described in Example 1.

Example 4. Preparation of crude EPS from R.leduminosarum 8002 in a low calcium medium R.leguminosarum bv phaseoli 8002 was grown in Zevenhuizen medium (L.P.T.M. Zevenhuizen, Appl.

Microbiol. Biotechnol., 20, 393 (1984)). This medium differs from Y medium in containing a lower calcium concentration and in minor variations in the presence of trace elements. 750 ml of medium at a final pH of l.O was used in 2 litre flasks. The incubation temperature was 290C. The bacteria were grown, and the crude EPS separated and prepared as described in Example 1.

Example 5. Preparation of de-ionised crude EPS from R.leguminosarum 8401 Crude EPS from R.leaum1nosarum 8401 was prepared as described in Examples 1 and 2. A 0.2% polymer solution was prepared as follows: 0.5g of polysaccharide were dispersed in 250 ml of distilled water, heated to 92 C, stirred to aid dissolution, and then cooled to room temperature (220 C) . The mixture was ion-exchanged into the acid form by adding DOWER 50W-X8 and stirring the mixture for 15 minutes. The resin was removed by filtration through a glass sinter funnel. The crude EPS was converted into the sodium ion form by neutralisation with 0.1M NaOH.The de-ionised sodium salt form was recovered b freeze drying the sample.

Example 6. Preparation of the purified EPS from R.leguminosarum 8401 Crude EPS from R.leguminosarum 8401 was prepared as described in Examples 1 and 2. A 0.2% polymer solution was prepared as follows: 1.0g of crude EPS was dispersed in 500ml of distilled water. The mixture was heated to 920C and stirred to disperse and dissolve the poly- saccharide. The solution was cooled to room temperature (220C). The acidic polysaccharide was purified by precipitation with the quaternary ammonium salt CTAB (cetyltrimethyl ammonium bromide) as described by J .

Scott Methods in Carbohydrate Chemistry Vol. 5 Academic Press, ew York (Ed. R.L. Whistler) p. 38 (1965). Sodium sulphate was added to the polysaccharide solution to produce a final concentration of 10m.N1. 50ml of 3% CTAB solution was added and the mixture stirred for 2 hrs at 370C. The precipitated polysaccharide complex was recovered by filtration through a glass sinter funnel and washed three times with 650 ml distilled water. The precipitate was then dissolved in 400 ml of 10% NaCl solution, reprecipitated by addition of 2 volumes of acetone, redissolved in 400 ml 10% NaC1, dialysed extensively against 1% NaC1 solution and finally dialysed against distilled water.The purified EPS was collected b freeze drying.

Example 7. Gelation of the crude EPS from R.leguminosarum 8401 Crude EPS from R.leguminosarum 8401 was prepared as described in Examples 1 and 2. A 3% polysaccharide solution was prepared by dispersing 0.3g of poly- saccharide in 10 ml of distilled water. The mixture was contained in a sealed tube and heated to 9DoC until the pol-saccharide dissolved. The mixture was poured into a plastic mould, covered and allowed to cool to 4 C in a refrigerator overnight. The gelled sample was then removed from the refrigerator, warmed to 250C and the rheological properties examined. The rheological data obtained are shown in Table I. The flat frequency dependence of G', G' > G" and the low phase angle are characteristic of a gelled sample.

Example 8. Gelation of the crude EPS from R.leguminosarum 8002 grown in low calcium medium Crude EPS from R.leguminosarum 8002, grown in low calcium medium, was prepared as described in Examples 1 and 4. A 3% polysaccharide solution was prepared ana gelled as described in Example 7. The rheological data obtained are shown in Table II and are characteristic of a true gel.

Example 9. Gelation of deionised crude EPS from R.leguminosarum 8401 Crude EPS from R.leguminosarum 8401 was prepared as described in Examples 1 and 2. The deionised crude EPS was prepared as described in Example 5. A 3% poly- saccharide solution was prepared by dispersing ().3g of polysaccharide in lOml of distilled water. The mixture was contained in a sealed tube and heated -to 9500 until the polysaccharide dissolved. The mixture was poured into a plastic mould, covered and allowed to cool to 40C overnight.The rheological properties of the sample were measured after allowing the sample to warm to 250 C. The rheological data obtained on the sample are shown in Table III. The frequency dependence of G', G' > G" and the high phase angle are characteristic of an entangled liquid.

A second lOml sample of a 3% polysaccharide solution was prepared. The sample was heated to 950C and sufficient .NaCl added to produce a molarity of 1.6. This sample was poured into a plastic mould and stored overnight at 40C. The sample was warmed to 250C and the resultant rheological properties are shown in Table IV.

The data are characteristic of a gelled sample.

A third 10ml sample of a 3% polysaccharide solution was prepared. The sample was heated to 950C and sufficient CaC12.6H2V added to produce a polarity of 0.4M. This sample was poured into a plastic mould and stored at 40C overnight. The sample was warmed to 250C and the resultant rheological properties are shown in Table V. The data are characteristic of a gelled sample. Example 10. Gelation of the crude EPS from R.leguminosarum RW4 pIJ1427 The bacteria were grown and the crude EPS extracted as described in Example 3. A 10ml sample of a 4% polysaccharide solution was prepared by heating a dispersion of O.4g of crude EPS in 10 ml of distilled water contained in a sealed tube until the polysaccharide dissolved.The solution was poured into a plastic mould, covered, stored at 4 C overnight, warmed to 250C and the rheological properties measured. The rheological data are presented in Table VI. The data are characteristic of a gelled sample.

Example 11. Gelation of the crude EPS from R.

leguminosarum bv. trifolii TA 1.

The bacteria (obtained from L.P.T.M. Zevenhuizen, Laboratory of Microbiology, Agricultural University, Hesselink van Suchtelenweg 4, 6703-CT Wageningen, Holland) , were grown in Y medium using the conditions described for R.leguminosarum 8002 in Example 1. The crude EPS was extracted as described in Example 1. A 3% solution of the polysaccharide was prepared by dispersing a 0. 3g of crude EPS in lOmi of distilled water contained in a sealed tube and heating the mixture to 95C to dissolve the polysaccharide. The hot sample was poured into plastic moulds, covered and stored at 40C overnight.

The sample was warmed to 250C and the rheclogical properties measured. The rheological data shown in Table VII are characteristic of a gelled sample.

Example 12. Gelation of the crude EPS from R.leguminosarum b. phaseoli LPR49 The bacteria (from Peter Albersheim, Dept. of Chemistry, Campus Box 215, University of Colorado, Boulder, CO 8080309, USA) were grown and the crude EPS isolated as described for R.leguminosarum bv. trifolii TA-1 in Example 11. Gels were prepared as described for R.leguminosarum bv.trifolii TA-1 crude EPS in Example 11.

The rheological properties of a 3% gel are shown in Table VIII.

Example 13. Gelation or the crude EPS from R.leguminosarum by. phaseoli 127K87 (The bacteria were obtained from Peter Albersneim.

as above) . The crude EPS was extracted and gelled as described for R.leguminosarum by. phaseoli LPR49 in Example 12. The rheological properties of a 3% gel are shown in Table IX.

MATERIALS Growth Media Y medium contains mannitol 10gl-1; K2HPO4 0.22gl-1; CaCl2.6H2O 0.22gl-1; glutamic acid 1.1gl-1; MgSO4.7H2O 0.1gl-1; FeCl3.6H2O 0.02gl-1; biotin 750ugl-1; thiamine 750ugl-1; pantothenic acid 750ugl-1. Final pH=6.8 Zevenhuizen (low calcium) medium contains: mannito 10gl-1; K2HPO4 1.0gl-1; CaCl2 .6H2O 0.04gl-1; glutamic acid 1.0gl-1; MgSO4.7H2O 0.2gl-1; FeCl3.6H2O 2.5mgl-1; H3PO4 10ugl-1; ZnSO4.7H2O 10ugl-1; CaCl2.6H2O 10ugl-1; CuSO4.5H2O 10ugl-1; Na2NoO4.2H2O 10ugl-1; biotin 10ugl-1; MnCl2 1ugl-1; thiamine 100ugl-1. Final pH = 7.0.

Rheological Measurements Rheological measurements were made using an Instron 3250. G' is the storage modulus and G" the loss modulus.

A phase angle of 900 is the ideal value for a liquid whereas a true gel would ideallv have a phase angle of 00. Measurements were made using flat plates.

TABLE I Rheological Data for a 3% gel or R.leguminosarum 8401 crude EPS Frequency (Hz) G'(Nm-2) G"(Nm-2) Phase Angle (deg.) 0.099 232.8 1.625 0.4 0.137 233.4 1.630 0.4 0.192 234.1 2.043 0.5 0.267 234.4 2.455 0.6 0.371 235.1 2.872 0.7 0.516 235.7 2.880 0.7 0.719 236.3 3.712 0.9 0.999 237.2 4.140 1.0 1.388 238.2 4.575 1.1 1.929 239.8 5.860 1.4 2.681 242.8 7.205 1.7 3.726 245.6 7.290 1.7 5.178 250.4 9.620 2.2 7.195 256.5 9.405 2.1 10.000 269.7 9.890 2.1 TABLE II Rheological Data for a 3% gel of R.leguminosarum 8002 crude EPS Frequency (Hz) G'(Nm-2) G"(Nm-2) Phase Angle (deg.) 0.099 200.1 6.287 1.8 0.137 201.6 6.687 1.9 0.192 203.0 8.155 2.3 0.267 204.9 8.947 2.5 0.371 206.7 9.746 2.7 0.516 208.7 10.94 3.0 0.719 211.5 12.56 3.4 0.999 214.1 13.85 3.7 1.388 217.6 15.98 4.2 1.929 221.9 17.46 4.5 2.681 228.0 19.95 5.0 3.726 236.7 21.95 5.3 5.178 250.7 24.14 5.5 7.195 276.2 26.11 5.4 10.000 331.4 22.59 3.9 TABLE III Rheological Data for a 3% solution of R.leguminosarum 8401 deionised crude EPS Frequency (Hz) G'(Nm-2) G"(Nm-2) Phase Angle (deg.) 0.099 6.78x10-3 0.169 87.7 0.137 4.13x10-3 0.237 89.9 0.192 3.42x10-3 0.327 89.4 0.267 2.36x10-3 0.451 89.7 0.371 1.76x10-2 0.631 88.4 0.516 4.45x10-2 0.878 87.1 0.719 9.69x10-2 1.18 85.3 0.999 1.69x10-1 1.64 84.1 1.388 3.65x10-1 2.20 80.6 1.929 6.06x10-1 3.01 78.6 2.681 1.06 3.99 75.1 3.726 1.76 5.39 71.9 5.178 3.17 6.95 65.5 7.195 5.28 8.82 59.2 10.000 8.78 10.5 50.1 TABLE IV Rheological data on 3% R.leguminosarum 8401 deionised crude EPS gel plus 1.6 MNaCL Frequncy (Hz) G'(Nm-2) G"(Nm-2) Phase Angle (deg.

0.099 308.8 7.546 174 0.137 309.2 6.477 1.2 0.192 310.6 5.421 1.0 0.267 311.3 4.891 0.9 0.371 312.1 4.358 0.8 0.516 312.6 3.273 0.6 0.719 313.3 2.734 0.5 0.999 314.1 1.644 0.3 1.388 314.8 1.099 0.2 1.929 315.2 2.681 316.0 0.551 0.1 3.726 315.9 1.103 0.2 5.178 316.6 1.658 0.3 7.1695 317.2 3.322 0.6 10.000 319.4 3.903 0.7 TABLE V Rheological data on a 3% R.leguminosarum 8401 deionisewd crude EPS gel plus D.4MCaCl3 Frequency (Hz) G'(Nm-2) G"(Nm-2) Phase Angle (deg.) 0.099 320.3 0.951 1.7 0.137 320.1 0.950 1.7 0.192 319.1 0.170 2.1 0.267 319.8 1.004 1.8 0.371 320.0 0.950 1.7 0.516 320.8 0.784 1.4 0.719 321.7 0.674 1.2 0.999 322.1 0.562 1.0 1.388 322.5 0.450 0.8 1.929 323.4 0.395 0.7 2.681 324.1 0.283 0.5 3.726 325.8 0.284 0.5 5.178 329.1 0.230 0.4 7.195 332.6 0.232 0.4 10.00 342.3 0.179 0.3 TABLE VI Rheological data on a 4% R.leguminosarum RW4 pIJ1427 crude EPS gel Frequncy G'(Nm-2) G"(Nm-2) Phase Angle (deg.) 0.099 631.6 3.307 0.3 0.137 630.7 5.504 0.5 0.192 630.9 5.505 0.5 0.267 631.4 5.510 0.5 0.371 632.1 4.413 0.4 0.516 632.7 3.313 0.3 0.719 633.0 3.314 0.3 0.999 633.8 2.212 0.2 1.388 634.7 2.215 0.2 1.929 635.6 2.219 0.2 2.681 637.5 2.225 0.2 3.726 641.6 3.360 0.3 5.178 648.3 3.394 0.3 7.195 661.4 5.772 0.5 10.000 684.7 7.170 0.6 TABLE VII Rheological data on a 3% R.leguminosarum by trifolii TA-1 crude EPS gel Frequency (Hz) G'(Nm-2) G"(Nm-2) Phase Angle (deg.) 0.099 79.9 1.26 0.9 0.137 80.4 1.40 1.0 0.192 80.7 1.55 1.1 0.267 80.9 1.84 1.3 0.371 81.3 2.13 1.5 0.516 81.6 2.56 1.8 0.719 82.2 2.73 1.9 0.999 82.9 3.33 2.3 1.388 83.3 4.22 2.9 1.929 84.5 3.99 2.7 2.681 85.4 4.77 3.2 3.726 86.4 5.74 3.8 5.178 87.2 6.25 4.1 7.195 88.6 7.29 4.7 10.000 91.1 7.81 4.9 TABLE VII Rheological data on a 3% R.leguminosarum by.

phaseoli LPR49 crude EPS gel Frequency (Hz) G'(Nm-2) G"(Nm-2) Phase Angle (deg.) 0.099 69.5 1.09 0.9 0.137 69.9 1.10 0.9 0.192 70.1 1.35 1.1 0.267 70.5 1.35 1.1 0.371 70.5 1.72 1.4 0.516 70.9 2.23 1.8 0.719 71.3 2.74 2.2 0.999 71.7 3.26 2.6 1.388 72.1 3.78 3.0 1.929 73.1 4.86 3.8 2.681 74.2 5.97 4.6 3.726 75.4 7.39 5.6 5.178 77.0 8.23 6.1 7.195 79.6 10.61 7.6 10.000 82.6 12.52 8.6 TABLE IX Rheological data for a 3% R.leguminosarum by.

phaseoli 127K87 crude EPS gel Frequency (Hz) G'(Nm-2) G"(Nm-2) Phase Angle (deg.) 0.099 92.8 3.57 2.2 0.137 92.5 3.59 2.2 0.192 94.1 3.94 2.4 0.267 94.9 4.31 2.6 0.371 95.8 4.52 2.7 0.516 96.6 4.89 2.9 0.719 97.5 5.28 3.1 0.999 98.6 5.68 3.3 1.388 99.7 6.45 3.7 1.929 101.1 7.07 4.0 2.681 102.2 6.97 3.9 3.726 104.5 8.04 4.4 5.178 106.3 8.74 4.7 7.195 108.3 10.24 5.4 10.000 112.8 9.87 5.0 References cited 1. T. Harada in "Polysachharides in Foods" (eds J.M.V.

Blanshard and J.M. Mitchell), Butterworths, London, p 283 (1979).

2. T. Harada and A. Amenura, Mem. Inst. Sci. Ind. Res.

Osaka Univ. 38, 3, (1981).

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Res. 124, 166 (1983).

4. M.A.O'Neill, R.R. Selvendan and V.J. Morris, Carb.

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5. R. Moorhouse, G.T. Colegrave, P.A. Sandford, J.K.

Baird and K.S. Rang, ACS Symp. Ser. 150, 111 (1981).

6. V.J. Morris, M.J. Miles and M.A. O'Neill in "Gums and Stabilisers for the Food Industry.2.Application of Hydrocolloids" (Eds. G.O. Phillips, D.J. Wedlock and P.A. Williams), Pergamon Press, Oxford, p 485 (1984).

7. B.A. Nisbet, I.W. Sutherland, I.J. Bradshaw. M.

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Sutherland and I.T. Tailor, Carb. Res. , 148, 63 (1986) 9. J.K. Madden, I.C.M. Dee and D.C. Steer, Carb. Polym.

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Claims (11)

1. A gel comprising a polysaccharide of Formula (I):

where n is an integer between 10 and 101o and R is a straight or branched oligosaccharide side chain comprising from 0 to 20 sugar residues.

2. A gel accordIng to Claim 1 wherein R consists of one or more of the following sugar residue0' D-Galp, D-GlcAp and D-Glcp.

3. A gel according to Claim 1 or 2 wherein R is a straight chain of 3 to 8 residues.

4. A gel according to any one of the preceding claims wherein one or more of the residues is modified by noncarbohydrate substitutions.

5. A gel according to any one of the preceding claims wherein R is selected from:

6. A process for preparing a gel according to any one of the preceding claims comprising (a) exposing an aqueous solution of the said polysaccharide to sufficient electrolyte to form a del-forming compound or (b) cooling an aqueous solution of the said polysaccharide.

7. A process according to Claim 6 wherein the poly- saccharide is exposed to an ionic strength of less than 4M for monovalent ions and less then 2M for divalent cations.

8. A process according to Claim 6 or 7 wherein the polysaccharide is prepared from a bacterial culture and the electrolyte is caused to be present in the culture medium during growth of the bacteria.

9. X process according to any one of Claims 6 to 8 wherein the electrolyte comprises monovalent or bivalent cations.

10. An unpurified or at least partially purified bacterial broth comprising a polasaccharide of Formula (I) and sufficient electrolyte to cause the poly- saccharide to gel.

11. A foodstuff comprising a gel according to any one of Claims 1 to 5.