PROCESS FOR THE RECOVERY OF A GELFORMING POLYSACCHARIDE, THE RECOVERED POLYSACCHARIDE AS WELL AS THE GEL FORMED THEREWITH

Abstract: Gel-forming polysaccarides, built up of galactose, glucose and mannose in a ratio of 4:1:1, obtainable by extracting cultures of strains of the genus Rhizobium and producing gels having a high gel strength, especially at low concentration.

Description: Process for the recovery of gelfoπαing polysaccharide, the recovered polysaccharide as well as the gel formed therewith. The invention relates to a process for obtaining a gel- orming polysaccharide by extraction of cultures of a microorganism. As is known, polysaccharide hydrocol loids which are obtained from plants and seaweeds are successfully employed in the foodstuff / petroleum and textile industries as well as in many other industries. The commercial use of these polysaccharides is inter alia based on the ability of these to modify the rheological properties of water as desired. Such polysaccharides are of particular impor¬ tance in the foodstuff industry and in oil production, especially with regard to methods for increasing the oil yield of dri I lings. Moreover, it is known that polysaccha ides are ext racel lular ly excreted by many microorganisms. In view of the fact that such microbial polysaccharides possess unique physically and chemically constant properties / and can be obtained on a regular basis, a number of these icrobially obtained polysaccharides have proved to be of commercial interest'. As is mentioned above / polysaccharides can be obtained from natural sources (vegetable or microbial) while at the same time synthetic sources are also of note. Examples of natural and synthetic sources are gum arabic, caraya and tragacanth (plant exudates), guar and locust bean gum (seed extracts), agar products*, alginates and carrageenan (seaweed extracts), starch (maize and potato) / pectin, gelatine / dextran and xanthan gum (natural pro¬ ducts), dextrins (starch)> carboxymethylcel lulose (cellu¬ lose), polyvinyl alcohol, polyacrylic acid and ethylene oxide polymers (synthetic products). In recent years, it is particularly the synthetic and microbially obtained gums which have been greatly in the ascendant at the expense of the vegetable gums. Characteristic uses of such gums, for example as stabili- sers and suspending agents, thickeners / film-forming agents, moisture-reta ning colloids, coagulants, lubricants and the like are to be found in the area of detergents / textiles / adhesivβS / paper, paints, feedstuffs, drugs and cosmetics. Moreover, some microbially prepared poly- sacchar des can, after hydrolysis, also be used as starting materials for the preparation of synthetic o ligosacchari des and polysaccharides. Examples of specific microbially prepared poly- sacchari des are: 1) Dextran (molecular weight about 100,000) prepared from sucrose under the action of culture filtrates of leuco- nostoc mesenteroides. 2) Xanthan gum, prepared from glucose with the aid of Xantho onas capestri s (Kelco Co.). 3) Alginates prepared ith the aid of Azotobacter vine- landi i (Tate and Lyle). 4) Curdlan, prepared with the aid of Alcaligenes faecalis (Takeda Co.) The first two products are characterised by a strong ability to increase the viscosity of aqueous solu¬ tions whilst the two last-mentioned products possess a considerable gel-forming capacity in aqueous solutions. An investigation has shown that varieties of Agro- bacteri urn predominantly produce two types of high molecular weight extracellular polysaccharides, namely (a) succi nog lycan, which is soluble in water and is res¬ ponsible for the high-viscosity behaviour of the cultures and (b) curdlan, which is insoluble in cold water but is soluble in 0.5M NaOH and hot water. The sue c i nog lycan is built up of glucose, galactose, pyruvate and succinate in a ratio of 7:1:1:1. Curdlan, which is built up of 8-(1 $ 3)-glucan and is reported to be insoluble in cold water but soluble in 0.5M NaOH and in hot water, gives with water, after neutralisa ion or cooling respecti ely, a fairly st i ff / resilient but ther o-irreversible gel. Moreover it has been found that the gelling properties of curdlan leave something to be desired, since, in order to obtain a gel, curdlan must be present in a concentration of at least 1% or more, sometimes even 2 to 3%. Moreover, it has been found that the gel strength of known agar products such as Gibco agar and Oxoid Ion agar No. 2 is undesirably low for a number of applications, especially if the concentration by weight is low. It has been found that the abovementioned disadvan¬ tages of known products can be overcome if a polysaccha¬ ride, built of galactose, glucose and mannose in the ratio of 4:1:1, is used, which polysaccharide is obtained by extraction of cultures of the genus Rhi zobium. In particular, using a polysaccharide according to the invention a stiff gel is obtained even at a weight concentration of only 0.2%, the gel strength of this gel being substantially greater than that of a gel obtained with a known agar such as Gibco agar. More especially, the varieties Rhi zobium- legumi nosarum, Rhizobiu -trifolii and Rhizobium-phaseoli of the genus Rhi zobium, are used. The extraction of the cultures of the genus Rhi zo¬ b um is carried out, for example, with the aid of an alkaline solution such as an M NaOH solution at room temperature, after which the extract obtained is neutralised with dilute acid. This gives a stiff gel having a high gel strength. The extraction of the cultures of the genus Rhi zo¬ um can also be carried out with almost boiling water, after which the suspension obtained is passed through a bacteriological fi lter. On cooling to room temperature, the clear filtrate solidifies to a stable and stiff gel. As mentioned, a stiff gel according to the inven¬ tion is obtained even at a concentra ion of only 0.2% of polysaccharide in water. The gel is thermorevers i b I e, with a transition temperature between the gel and sol o state of between 50 and 60 C (solidification point and melting point respectively ) . The process according to the invention is prefer¬ ably carried out with cells of the Rhizobium legumi nosa rum strains F-13 and RCR 1044 and also with Rhi zobium tri foli i strain TA-1 (deposited at the Laboratory for Microbiology in Delft under Nos. LMD 83.29, LMD 83.28 and LMD 83.30 ) . The polysaccharide according to the invention, which is bound to the cells as a capsule polysaccharide, is built up of repeat units having the following general formula: DtGal 4 DtGal 1 # 4)tDtGlct(l-*3)tDt __raτ(1→3) DτGalτ(1— > DtGal wherein D-Gal, D-Glc and D-Man stand for D-galactose, D-glucose and D-mannose and wherein the pol saccharide has a mean molecular weight of at least 150,000 (determined by gel c ro atography of ' the main chain over Ultragel A-4 ith dextrans of known molecular weight as reference substanc e .) Experimenta I The following strains were investigated: Rhizobium legumi nosarum F-13, TOM, RCR-1012 and RCR-1044. Rhizobium trifolii TA-1 and 0403 and Rhi zobium phaseoli 127 -82, all present in the public bacteria col¬ lection of the Laboratory for Microbiology in Wageningen. Carbohydrate analysis of the cultures 2 ml of a culture are pipetted into a centrifuge tube after which 2 ml of 2N NaOH are added. An extraction takes place by shaking for 2 hours at room temperature. Thereafter, the extracted cells, which contain non-extrac¬ ted glycogen, are centrifuged off and the supernatant liquor, which contains the extracted polysaccharide, is poured into a second centrifuge tube. The capsule poly¬ saccharide (CPS) is precipitated by adding 3 ml of 2N HCl, centrifuged off, decanted and redissolved in 4 ml of 2N NaOH. 0.1 ml thereof is taken for a determination of the carbohydrate content with the aid of the anthrone/ sulphuric acid reaction. The supernatant liquor, which contains separated-out acid, exopo lysac chari de (EPS) and β-1 ,2-g lucan, can be used further for the determination of these carbohydrate constituents. The invention is further explained with reference to the example which follows, but is in no way restricted thereto : Examp le Culturing of Rhizobium tri olii, strain TA-1, in glutamic acid/lactose/salts medium Composition of the medium: Glutamic acid 1 g C source: lactose 10 g K2HPO4 1 g in 100 ml of water MgS0 .7H 2 0 0.2 g Trace elements: FeC^.όH j O 2.5 mg ZnS0 .7H2° °- Q 9 MnCl 2 1 mg Vitamins: biotin 10 ug thia ine 100 μq demineraIised water 1000 ml After having dissolved the constituents in 1 litre of demineraIised water, the medium is brought to a pH of 7.0 by adding N NaOH. 50 ml amounts are metered into 200 ml Erlenmeyer flasks; larger quantities of 1 litre are metered into 5 litre Erlenmeyer flasks, then provided with cottonwool plugs and sterilised in an autoclave for 20 minutes at 120°C. The C source, lactose, is steri¬ lised separately and after it has cooled is added asepti- cally to the base medium. Culturing is carried out by inoculating a precul- ture of 50 ml of medium from a sloping tube containing a pure culture of Rhizobiurn trifolii strain TA-1 on Rhi zo¬ bium agar (yeast extract, 0.1%; mannitol, 0.5%; K H O4 0.05%; MgS0 .7H 2 0 / 0.025%; CaCl 2 .2H 2 0, 0.01%; agar, 1.0% in water). The culture is incubated on a rotary shaking machine at 25-30° for 5-7 days. This preculture is then used to inoculate 1 litre of the same medium in a 5 litre Erlenmeyer flask and the latter is incubated on a shaking machine at 30° for 7 days. Isolation of the gel-forming polysaccharide from the Rhizo¬ bium culture After the end of the culture, the bacteria are centrifuged off in a high speed (12,000 rpm ) centrifuge. The clear supernatant liquor thereby obtained can option- ally be used to isolate the acid exopo lysaccharide (EPS) excreted by the bacteria, by precipitation with alcohol or by forming a complex with cetylpyridi ni u chloride (L.P.T.M. Zevenhuizen. Methylation analysis of acidic exopo lysacchari des of Rhi zobium and Agrobacterium. Carbo- hyd. Res. 26, 409-419 (1973)). The neutral gel-forming capsule polysaccharide (CPS) is insoluble and is found in the cell pellet ob¬ tained. A ' determination of the quantities of EPS and CPS formed can be carried out in a simple manner by means of a hexose determination using the anthrone/sulphuri c acid method ' ; the consumption of the C source (lactose) can be determined by measuring the reducing capacity of the supernatant liquor, using the method of Somogy i-Ne Ison , The cell pellet obtained is suspended in 200 ml of water after which 200 ml of 2N NaOH are added. Extrac¬ tion takes place by stirring for 1-2 hours at room tempera¬ ture. The extracted cells are subsequently centrifuged off and the clear viscous supernatant liquor is acidified slightly with acetic acid or ith di lute hydrochloric acid. Hereupon, a voluminous gel separates out; this is centrifuged off, washed several times in the centrifuge tube, dialysed if appropriate, and f eeze-dri ed. Yield: about 2.3 g of dry polysaccharide material. After culturing in an 0.1% glutamic acid/0.5% manni to l/salts medium (see J. Microbiol. Serol. 47 (1981), 481-497 ) , the abovementi oned strains RCR 1044 and F-13 gave 200-300 mg of product containing more than 95% of hexose (calculated on D-ga lactos e) , determined by means of the anthrone/sulphuri c acid method. The abovement i oned strains RCR 1012, 0403, TOM, 127 K-82 and various other strains of the genus Rhi zobi urn yielded smaller quantities of the same product. Determination of the structure of the polysaccharide. Table I Sugar analysis of the original and modified capsule polysaccharide of Rhi zobium trifolii strain TA-1. Sugar component Molar ratio a ^ as a Idi to I acetates) A B C D E F Glycerol + Threitol 0.7 Erythri to I + D-Mannose 1.0 '1.00 1.00 1.00 1.00 0.17 D-Galactose 4.0 0.92 1.03 1.41 0.99 0.34 D-GLucose 1.0 0.97 0.96 0.96 0.14 0.11 a ' Po lysacchari de: A, original; B pe i odate-oxi i sed and borohydride- reduced; C periodate-oxidised, borohydride- reduced and Smith-cleavage (main chain); D, partially peri odate-oxi dised (0.5 hour), borohydri de-reduced and Smith-cleavage; E, peri odat e-oxi di sed and borohydride- reduced (main chain); and F, chromium tri oxide- oxidised, acetylated original polysaccharide. Table II Methylation analysis of the original and modified capsule polysaccharide of Rhizobium trifolii, strain TA-1 Methylated sugar a) T° Molar ratio c) A B C D E F 2,3,4,6-Man 1.00 1.00 2,3,4,6-Gal 1.07 1.80 0.34 2,4,6-Man 1.54 1.00 1.00 1.00 1.00 1.00 2,4,6-Gal 1.58 0.96 0.99 1.05 1.11 0.93 0.83 2,3,6-Gal 1.65 0.84 2,3,6-Glc 1.79 0.88 0.83 d ) 0.46 2,3-Glc 2.98 0.70 3-Glc 4.58 0.87 0.88 a) 2,3,4,6-Man = 2,3,4,6-tet ra-0-methy l-D-mannose, and so on, D*> Retention time of the corresponding alditol-acetates relative to that of 1 ,5-di-0-acety l-2,3,4,6-tet ra-0-meth l- D-glucitol on a "fused silica" WCOT-OV-225 column at 19Q°C. c) Polysaccharide: A, original; B, periodate-oxidised and borohydri de-reduced; C, peri odate-oxi di sed, boro¬ hydri de-reduced and Smith-cleavage; D, peri odate-oxi di sed, borohydri de-reduced, methylated, Smith-cleavage and tri- deuteri o-methy lated; E, partially per odate-oxi di sed (0.5 hour), borohydride-reduced and Smith-cleavage; F, perio- dat e-oxi di sed, borohydri de-reduced, Smith-cleavage main c ain. 2,6-Di-0-trideuteriomethyl-3-Q-methyl-D-glucose. The hydrolysis of the polysaccharide obtained (2M tri f luoroaceti c acid, 100°C, 6 hours) gave D-galac- tose, D-glucose and D-mannose in a molar ratio of 4:1:1 (Table I, column A), which ' suggests a hexasacchari de repeat unit. No uronic acids, or acyl-, (acetyl-, succinyl-) or acetal-bonded pyruvate substituents were detected. The methylation analysis of the polysaccharide (Table II, column A) shows two D-galactosyl end groups, one (1 *^ 3)-bonded D-mannosyl residue, one (1 ->3 ) -bonded D-galactosyl residue and one (1 ->4) D-galactosyl residue, and a doubly-branched D-glucosyl residue, via 0-1,2,4,6, to be present . The polysaccharide took up five moles of periodate per repeat hexasaccha ri de unit and gave two moles of formic acid, in the course of 5 hours at room temperature. The borohydride reduction of the polyaldehyde obtained, fol¬ lowed by hydrolysis in 2M t ri f luoroacet i c acid, gave D-glu- cose, D-galactose and D-mannose in a ratio of 1 :1:1 to¬ gether with glycerol and threitol (Table I, column B) . Methylation of the polyalcohol (Table II, column B) indi- cated that the peri odate-oxi dat i on had cleaved two D-galac¬ tosyl end groups and one (1 ->4 ) -bonded D-galactosyl resi¬ due. The Smith cleavage of the polyalcohol (90% formic acid, 40°C / 1 hour) gave a product which was sti ll macro- molecular and non-di a lysab le and contained equal quanti¬ ties of D-glucosβ / D-galactose and D-mannose (Table I , column C) . A methylation analysis of the Smith-cleaved polymer (Table II, column C) indicated that the latter was unbranched and consisted of (1 - 3)-bonded D-mannosyl residues, (1 ->3)-bonded D-galactosyl residues and (1-/4)- bonded D-glucosyl residues. The bonding points of the D-galactosyl side chains were fixed by methyla¬ tion of the polyalcohol, followed by Smith cleavage and t rideuteri omethy lati on of the cleaved polymer (Table II, column D) . The 2,3,6-tri-0-methyl-D-g lucose obtained was Me0-2,6-deuterated, indicating the positions of the D-galactosyl and the di-D-ga lactosyl side chains. The relative positions were established by methylation analy- sis of partially peri odat e-oxi di sed and Smith-cleaved polysaccharide (Table I, column D) . Since a D-galactosyl end group which possesses a vicinal triol group is con¬ sidered to be more sensitive to periodate attack than a (1 ->4)-bonded D-galactosyl residue which contains a vicinal diol group, each non-oxidised D-galactosyl residue which remains bonded to the main chain after such an oxida¬ tion will indicate the position of the di-D-ga lactosy I side chain. The formation of 2,3-d i-0-methy l-D-g lucose via methylation analysis of incompletely periodate-oxidised and Smith-cleaved polysacchar de (Table 11, column E) indicated that the di-D-galactosyl side chain occupied position 6 of the main chai n-D-g lucos I residue. The sequence of the hexose residues in the ma n chain was investigated by periodate oxidation of the poly- - -. - saccharide stripped of the side chains, since the now (1 ->4)-bonded D-glucosyl residues are sensitive to pe io¬ date oxidation (Table I, column E) . A successive reduc¬ tion and Smith cleavage of the polyalcohol gave a tri- saccharide product of D-mannose, D-galactose and erythri- tol, methylation analysis of which (Table II, column F) gave 2,3,4,6-tet ra-0-methy l-D-mannose and 2,4,6-tri-0- methyl-D-ga lactose, which indicated the following sequence: 3)-D-Man-(1 ->3) -D-Ga I- (1 ->4) -D-Gl c-(1 - > i n the main chain. A chromium trioxide oxidation of the acylated original polysaccharide followed by a sugar analysis ( Table I, column F) showed that all sugar residues were oxidised and consequently the residues were ai n ly -bonded . From the above data it was possible to deduce the following structure for the gel-forming polysaccharide according to the invention. D-Gal D-Gal 1 6 < <<? -» 4)-D-Glc-( 1 ->3)-D-Man-( l→3)-D-Gal-( 1 → 2 1 D-Gal The above structure i.ncludes some unusual aspects or a bacterial exopo lysaccha ri de which is built up of repeat units. It is a neutral heteropolysaccharide without carbonyl-contai ning substituents and therefore resembles the ext acellular polysaccharide gum from the slow-growing Rhi zobium strain CB 744 of the cowpea group (Arch. Biochem. Biophys. 124 (1968 ) 292-298). The structure also contains a doubly-branched sugar residue, which is an unusual fea¬ ture of bacterial polysaccharides. • Sim lar features are also present in the capsule polysaccharides of K lebsi e I la, types 33 and 38 (Carbohydrate Research 70 (1979) 134-144). The above structure of the gel-forming polysaccharide according to the invention is the first to be reported for an insoluble capsule polysaccharide of a fast-growing strain of Rhizobia. Methods of structure determination The sugar analysis and methylation analysis em¬ ployed in the structure determination are described in Carbohydrate Research 26 (1973) 409-419 and Chem. Commun. Univ. Stockholm, 8 (1976) 1-75. The periodate oxidation. Polysaccharide (100 mg) was dissolved in water (50 ml) at 60°C. 0.03M sodium metaperi odat e (50 ml) was added to the stirred solution after which the ixture was immediately cooled to room temperature. Samples (0.1 ml) were diluted at intervals to 25 ml and the periodate consumption was determined spect rophotomet ri ca I Ly at 223 nm. The liberation of formic acid was determined by ti- tration with 0.1M NaOH after reduction o: ' f unconverted periodate with ethylene glycol. The oxidation took place rapidly in the dark at 20oC; after 8 hours, an excess of ethylene glycol was added and the oxidised product was isolated by dialysis and subsequently reduced overnight with sodium borohydride (200 mg) . The excess reducing agent was cleaved with acetic acid and the mixture was dialysed and freeze-dri ed. The Smith cleavage. The Smith cleavage of the product (84.8 mg) was carried out for 1 hour at 40°c in 90% formic acid (40 ml). The formic acid was evaporated off in vacuo and a solution of the residue in water was dialysed and freeze- dried. A portion (33.1 mg) of the product (43.1 mg) was dissolved in 15 mM NaIθ4 (40 ml ) . After 4 days in the dark, the mixture was worked up in the manner described above. A part (10 mg) of the product (30 mg) was subjected to a sugar analysis and another part (10 mg) was subjected for 1 hour at 40°c to a Smith cleavage in 90% formic acid ( 5 ml ) . The solution was evaporated in vacuo and freeze-dried, after which the residue was subjected to a methylation analysis. The chromium trioxide oxidation. The chromium trioxide oxidation was carried out in the manner described by Lindberg and Lonngren (Methods Enzymol. 50 (1978), 20-24). Comparison of the gel strengths of the capsule polysaccha- ride of Rhizobium trifolii TA-1 and of two known agar products The res.ults of the gel strength determinations are shown in the attached figure. The gel strengths are deter ined by measuring the breaking strengths of the gels by means of an Overload Dynamics type S-100 gel tester; the plunger diameter of the load cell was 12.7 mm. The gels to be investigated were prepared by heating, followed by cooling in water at various weight concentrations (0.1-2.0% weight/volume). It can be deduced from the attached figure that the bacterial gel according to the invention has a higher gel strength, at the same weight concentrations, than two known agars used as comparison material, namely Gibco agar and Oxoid Ion agar No. 2. In particular, at low weight concentrations (0.1-0.4%) fairly stiff gels were still obtained with the bacterial polysaccharide according to the invention, while this was hardly any longer possible with the two agars in the said concentration range. Fur- ther, the bacterial product proves to be more resilient than the known agars, in that the bacterial gel permitted a greater deformation (8 mm penetration depth of the plun¬ ger) than the more brittle agars which only permitted 4 mm deformation before the break point o-f the gels was reached. Moreover, the bacterial gel was found to have shorter "setting times" (less than 1/2 hour) than the "setting times" of the two agars (more than 1 hour), for reaching maximum gel strength after melting and cooling to room temperature. The bacterial gel was stable over a pH range of 2-11 and a steri lisation (20 minutes at 120°C) had no adverse effect on the gel strength. The Gibco agar is marketed by Gibco Europe B.V. in Hoofddorp, Netherlands, and the Oxoid Ion agar No. 2 b-y Pharmachemie B.V. in Haarlem, Netherlands.

Claims: C l a i ms 1. Process for obtaining a gel-forming polysaccharide by extraction of cultures of a microorganism, characterised in that a polysaccharide built up of galactose, glucose and mannose in a ratio of 4:1:1 is obtained by extracting cultures of a strain of the genus Rhi zobium. 2. Process according to claim 1, characterised in that cultures of the varieties Rhizobium leguminosarum, Rhizobium trifolii or Rhi zobium phaseo li are used. 3. Process according to claim 1 or 2 , characterised in that cultures of the Rhizobium leguminosarum strain R 13 or RCR 1044 or of the Rhizobium trifolii strain TA-1 are used. 4. Process according to one or more of claims 1-3, characterised in that the cultures of the genus Rh i zobi urn are subjected to. an extraction with the aid of an alkaline solution and the extract obtained is subsequently neutralised with a di lute acid. 5. Process according to claim 4 / characterised in that M NaOH is used as the alkaline solution. 6. Process according to claim 4 or 5 , c aracterised in that the extraction is carried out at room temperature. 7. Process according to one or more of claims 1-3 / characterised in that the cultures of the genus Rh i zobi um are subjected to an extraction with almost boi ling water and the hot suspension obtained is passed through a bac- teri o logi cal filter. 8. Gel-forming polysaccharide built up of repeat units having the formula DtGal 1 I 4 DtGal 1 A i 6 5 4 4 4) DtGlct(l-*3) tDtMant( 1→3) tDtGalt( 1 —3 2 4 t 1 DtGal wherein D-Gal / D-Glc and D-Man stand for D-galactosβ / D-glucose and D-mannose / the polysaccharide having a mean molecular weight of at least 150,000. 9. Gel of a polysaccharide according to claim 8 / or obtained using a polysaccharide obtained by means of the process according to one or more of claims 1-7 respec¬ tive ly . 10. Process for the preparation of synthetic oligo- saccharides and polysaccharides / characterised in that the starting material used therein is a product which is obtained by hydrolysis of a polysaccharide obtained by means of the process according to one or more of claims 1-7 or of a polysaccharide according to claim 8.

Inventors: VAN NEERVEN ALEXANDER RAYMUNDU, ZEVENHUIZEN LUDOVICUS PETRUS T, VAN NEERVEN, ALEXANDER, RAYMUNDUS, WILHELMUS, ZEVENHUIZEN, LUDOVICUS, PETRUS, THEODORUS, MARIA
Applicants: SUIKER UNIE, COOPERATIEVE VERENIGING SUIKER UNIE U.A.
Application year: 1984
Main classification: C12P19
IPC codes: C08B1, C12P19, C12R1