IE46144B1 - A microbiological process for the preparation of a polysaccharide - Google Patents

Abstract

Process for preparing an exocellular polysaccharide comprises cultivating Pseudomonas sp. NCIB 11264 (ATCC 31260). Polysaccharides are formed from a repeating unit contg. 7 gluco-D units comprising 1 glucose unit substd. in the 6-posn., 2 glucose units substd. in posn. 4, 2 glucose units substd. in posn. 3, and 2 glucose units substd. in positions 4 and 6; 1 galacto-D unit comprising galactose substd. in position 3; 1 acetate and pyruvate unit; the units contain a branch point on the 4,6-disubstd. glucose and a lateral chain terminating in a 4,6-O-(1-carboxyethylidene)-glucose-D unit. The polysaccharide has an optical rotation a 22 of -15 degrees (C 0.68H2O). The polysaccharides have remarkable pseudoplastic flow properties and shearing fluidisation properties compared with xanthanes and alginates.

Description

This invention relates to a polysaccharide possessing useful flow and gel-forming properties, and to a process for its preparation.

Polysaccharides from microbiological sources 5 are becoming increasingly important in many different industrial applications where materials with particular flow properties are required. Microbial exopolysaccharidec can possess unique properties and furthermore can he more easily produced to a uniform specification than plant or algal polysaccharides.

Polysaccharides, such as locust bean gum and alginates, are widely used in industry, as emulsifiers, stabilizers and thickeners. In the food industry they are used as emulsion stabilizers for ice-cream, as gelling agents for milk puddings, as thickeners for sauces and as foam stabilizers for beer. They are also used in the manufacture of paper and textiles and for thickening drilling muds in oil drilling. Xanthan gums are also increasingly widely used in a range of applications.

Ve have discovered a polysaccharide possessing in aqueous systems pseudoplastic flow and shear thinning properties which are remarkably similar to those of xanthans and alginates, suggesting similar commercial applications. This - 2 46144 polysaccharide can be prepared by cultivating a polysaccharideproducing strain of Pseudomonas c_q deposited at the National Collection of· Industrial Bacteria, Torry Research Station, 135 •Abbey Road, Aberdeen under the Number NCIB 11264, in a nutrient medium therefor. This polysaccharide-producing strain has also been deposited at the American Type Culture Collection in Washington DC under the number ATCC 31260.

Pseudomonas sp NCIB 11264 (ATCC 31260) was isolated from a carbohydrate-rich industrial effluent. Its morphology and physiology may be summarised as follows (all temperatures in degrees centigrade): Morphology Oxoid CM3 Nutrient Agar 25° Gram-negative, small-medium parallel-sided rods, becoming short . rods and sometimes coccobacilli.

Motile. Plagella position: single, polar (electron micrographs). Colonies (6 days): 2mm, whitish, opaque (translucent confluent growth), circular, entire, low convex, smooth, soft, easily dispersed, no variation.

Physiology 30° Catalase Kovacs1 oxidase Growth at 37° + (colony growth rate at 37° approximately equal to· rate at 25°) Growth at temperatures in excess of 40° very poor - 3 4 614 4 Anaerobic growth, glucose agar Hugh & Leifson Glucose Peptone water sugars, Andrades' indicator ) Glucose, lactose, Pructoso ) ) Sucrose, Maltose, Mannitol ) Glycerol, Starch , ) Kosers' citrate Starch hydrolysis King et al A & B Arginine, Millers Gelatin hydrolysis Casein hydrolysis NIIj from tryptone NO' to NO' or H2 Christensen's urease DNAse Egg yolk plate reaction Toges - Proskauer test Methyl red Indole Polypectate degradation - (slight) oxidative no acid alkaline 1-J days The micro-organism can. be cultivated, under aerobic conditions, in any convenient medium in which it will grow and produce exocellular polysaccharide. Typical media include complex broths, e.g. a 1% nutrient broth, or a chemically 614 4 defined, medium such as that described by Gray et al. (Biochimica et Biophysics' Acta, 117, 22-32, 1966), with a supplementary carbon source of, for example, glucose or sucrose. A supplement of about 2% w/v in the medium is desirable.

A particularly preferred defined medium (the glucose- supplemented Gray et al medium) for use in the cultivation of Pseudomonas sp 1TC1B 11264 has the following composition: Glucose 20 g/litre nh4ci 2.66 g/litre kh2po4 5.44 g/litre NaOH to pH 7 approx. 1.5 g/litre solution of trace elements* 6 ml/litre * a solution containing MgSO^.THpO 10 g MnCl2.4H20 1 S PeS04.7H2O 0.4 g - CaCl2.2H20 0.1 g Distilled water to 1 litre The culture may be effected batch-wise or in a continuous manner, according to conventional practice, Continuous cultures are preferably conducted under nitrogenlimiting conditions, e.g. about 0.3 g NH^Cl/litre. With a glucose· supplemented medium, the glucose conversion is about 30% - 5 144 in batch cultures, but up to 75% in continuous cultures.

A temperature of from 25°C to 35°θ is satisfactory, a temperature of about 30°C being optimal.

In general, polysaccharide production is found to be enhanced when an excess of carbon source is present, under nitrogen-limited conditions. Preferably the pH of the medium should not fall below 6 and may conveniently be from 6.5 to 8.0.

The exocellular polysaccharide may be isolated from the culture supernatant (free from cells) by precipitation with an organic water-miscible solvent such as isopropoanol and deionized e.g by a conventional desalting using dialysis Conveniently, unwanted cellular matter can he removed by trypsin digestion, e.g by digesting an aqueous solution of the polysaccharide buffered to pH 7-8, e.g using 0.2M HEPES buffer, at about 30°C in the presence of a hacteriostat such as mercuric chloride. For example, 3.6 litres of solution, buffered to pH 7-8 with 0.2M HEPES, are treated with 20 mg of the enzyme and mercuric chloride (1.5 ml of a saturated alcoholic solution) for 5 days at 30°C.

After dialysis, the isolated material can be freezedried to give the purified dry exopolysaccharide. - 6 46144 Analysis has shown the: purified polysaccharide bo he a polysaccharide having a repeating unit containing the following components: D-gluco units comprising Ί unit of 6-substitutcd glucose, 2 units of 4-substituted glucose, 2 units of J-substituted glucose, and 2 units of 4,6-disubstituted glucose; 1 D-galacto unit comprising 3-substituted galactos; 1 acetate unit; and 1 pyruvate unit; the above components including 1 4,6-disubstituted glucose branch point and 1 side chain terminated by a 4,6-0~(1-carboxyethylidene)-D-glucose unit.

The purified polysaccharide has an optical rotation ^-^22 = (0 °*68Hg0^ which indicates that all the sugars are linked in the ^-D configuration.

The viscosity and flow properties of the polysaccharide of the present invention may be described in terms of the consistency index k and the flow behaviour index n, as suggested by Krumel and Sarkar, Flow Properties of Gums useful in the Pood Industry, in Food Technology, April 1975 pp 36-44, Vol. 29(4).

The apparent viscosity (-y) in centipoises was measured using a cone and plate viscometer at various rates of shear (D) in see~\ A plot of log y against log D for a 1% by weight solution of the polysaccharide according to the present invention at - 7 46144 °C gave a straight line graph which gave a k value —Ί ('Q extrapolated to a shear rate of 1 sec ) of 4600 cps and an n value (the slope of the graph plus 1) of 0.22. A commercial sample of food grade xanthan gum sold under the Trade Mark Keltrol by Kelco of San Diego, California gave under the same conditions values of k and n of 5,000 and 0.25 respectively. t The following Examples illustrate the invention: Example 1 litre Batch Fermentation Exopolysaccharide production by Pseudomonas sp NGIB 11264· was followed in a 10 litre batch fermentation, without pH control, for 50 hours at 50° with volume/volume aeration, and an impeller speed of 550 rpm. The doubling time of the organism under these conditions was 140 minutes.

Logarithmic growth continued for some 18 hours at which point (E^q O.O) all nutrients were apparently in excess, although the oxygen tensions were not determined. It is known, however, that exopolysaccharide synthesis is at a -846144 maximum when the oxygen tendon is non-limiting. At this stage,, polysaccharide could he detected hy isopropanol precipitation,. although exopolymer had beep detectable in increasing amounts in culture supernatants from 12-13 hours after inoculation utilising a more sensitive viscometric assay. Thus, although exopolysaccharide production apparently commenced during the .late, exponential phase of growth, the formation was maintained maximally for another 20 hours during the stationary growth phase, before the rate of production eventually began to decrease. This fermentation pattern is typical of a secondary metabolite.

Of the-glucose utilised, only 30% was converted into exopolysaccharide the other 70% being metabolised to establish and maintain the culture.

Example 2 Steady State Exopolysaccharide Production Exopolysaccharide-producing cultures of Pseudomonas sp NCIB 11264 were maintained in a steady state for up to 500 hourp,... The defined medium based on that described by Gray et al .(1966) supplemented with glucose (10 mg/ml),.was used in .all the previously described continuous culture studies. - 9 46144 As a result of those investigations, the initial concentrations of some of the components were reduced, and continuous polymer production followed under conditions of imposed nitrogen limitation. Conditions were optimised at pH 7.0-0.1 with a growth temperature of 30^1° and an aeration rate of 500 ml/min.

After inoculation, the culture was grown as a hatch and allowed to establish for 24 hours before the flow rate was adjusted to 44 ml/hour. The course of the fermentation, _ η run at a dilution rate of 0.08 hr , was followed for 500 hours. An impeller speed of 900Ϊ10 rpm was maintained throughout. Steady state values for total cell density, polysaccharide level and glucose conversion remained constant after 100 hrs these values were respectively 0.26 (Ε^θ x ), 1.6 mg/ml and 40%, while after 500 hours they were 0.26 (E^2q x 10 ), 1.6 mg/ml and 45%. There was no evidence to indicate cultural deterioration or development of mutant strains Polysaccharide samples analaysed were of a constant composition and solutions of the polymer (0.1 mg/ml) had a similar, relative viscosity (1.7^0.05) when measured at 25° with a modified Zimm-Crothers rotating cylinder type viscometer (55mA), indicating that there had been no change in the molecular weight of the exopolymer produced over the period of the fermentation.

Claims (13)

1. A process for the preparation of a polysaccharide which comprises cultivating Pseudomonas sjx NCIB 11264 (ATCC 31260) to yield an exocellular polysaccharide.

2. A process according to Claim 1 wherein cultivation is effected in a continuous manner.

3. A process according to Claim 2 wherein the continuous culture is conducted under nitrogen-limited conditions.

4. A process according to Claim 2 or 3 wherein the cultivation is effected in a medium according to Gray et al supplemented with a supplementary carbon source.

5. A process according to Claim 4 wherein the medium is a medium according to Gray et al supplemented with glucose or sucrose.

6. A process according to any one preceding claim wherein cultivation is effected at 25°θ to 35°θ·

7. A process according to any one preceding claim wherein the pH of the cultivation medium is maintained at above 6.

8. A process according to Claim 7 wherein the pH is maintained at 6.5 to 8. - u·

9. A process according to any one preceding Glajin, wherein the exocellular polysaccharide is isolated by precipitation with an organic water-miscible solvent and is then deionised.

10. A process according to Claim 9, wherein the isolated polysaccharide is freeze-dried.

11. A process for the preparation of a polysaccharide, which process is substantially as herein described in Example 1 or 2.

12. Polysaccharide when prepared by a method according to any One preceding claim.

13. A polysaccharide produced by Pseudomonas sp NCIB 11264 (ATCO 3Ί260) which has a repeating unit containing 7 D-gluco units comprising 1 unit of 6-substituted glucose, 2 units of 4-substituted glucose, 2 units of 3-substituted glucose, and 2 units of 4,6-disubstituted glucose; 1-D-galacto unit comprising 3-substituted galactose; 1 acetate unit; and 1 pyruvate unit; the units including 1 4,6-disubstituted glucose branch point and 1 side chain terminated by a 4,6-0-(1-carboxyethylidene)-D-glucose unit; the polysaccharide having an optical rotation of -15° (c 0.6¾ θ).