WO1990011363A1

WO1990011363A1 - Polysaccharide compositions

Info

Publication number: WO1990011363A1
Application number: PCT/GB1990/000458
Authority: WO
Inventors: Victor John Morris; Barry James Herbert Stevens; Jane Elizabeth Harris; Geoffrey John Brownsey; Allan Patrick Gunning
Original assignee: Biotechnology and Biological Sciences Research Council
Current assignee: Biotechnology and Biological Sciences Research Council
Priority date: 1989-03-29
Filing date: 1990-03-27
Publication date: 1990-10-04
Anticipated expiration: 1991-09-29
Also published as: GB8907057D0

Abstract

Acetan is an extracellular polysaccharide secreted by Acetobacter xylinium. It has been found to form thixotropic aqueous dispersions and is thus useful in compositions such as foodstuffs, adhesives, cleaners, toothpastes and paints.

Description

POLYSACCHARIDE COMPOSITIONS

The present invention relates to polysaccharide compositions, particularly to polysaccharides from bacteria and polysaccharides for food use.

Many polysaccharide compounds are used in industry, particularly the food industry, for thickening or gelling compositions. Such compounds include starch, pectin, alginate, carrageenans, galactomannans and xanthan gum. A more recently disclosed compound is acetan, the isolation and partial characterisation of which are given in Tayama je aJL, Aqric. Biol. Chem. 49(4), 959-966 (1985) and Couso et al, J. Gen. Microbiol. 133, 2123-2135 (1987). These disclosures established that an aqueous solution of acetan is viscous and that it has a structure which is similar to that of xanthan. However, these properties do not in themselves indicate that acetan is suitable for industrial use, particularly in situations where the thickening agent must be thixotropic. Firstly, most polysaccharides produce viscous solutions. However, the viscosity is Newtonian. At low shear rates the viscosity- exhibits a plateau. At high shear rates the viscosity shows a small decrease in value. Carboxymethyl cellulose is an example of such a typical polysaccharide. Thixotropy requires a high viscosity at low shear rate and a continuous and reversible decrease in vicosity with increasing shear rate. Secondly, quite small changes in the chemical structure of polysaccharides can alter their properties in unpredictable ways. For example, 2

the capsular polysaccharide ("K54") secreted by lebsiella aerogenes serotype K54 has the same carbohydrate structure as the extracellular polysaccharide produced by an Enterobacter species (NCIB 11870) and called XM6. This structure is shown

_* below (A) .

^'3)βDGlc(l->4) ^■ DGlcA(l—>3) o^LFuc(l > ) (A)

Λ * ).

4

1 βDGlc

However, in 54 there is an O-acetyl substituent on alternate repeat units linked to the 2 position of the Fuc residue. Whereas XM6 will gel, the acetyl substituent in 54 prevents gelation; deacetylation of 54 results in gelation. Similarly, the polysaccharides produced by Alcaliqenes faecalis var.myxogenes (succinoglycan - structure B) and by Rhizobium meliloti IFO 13336 (structure C) have identical backbones but differ in the chemical composition of their sidechains:

4)βDGlc(1—>4)βDGlc(1—>4)βDGlc(1—>3)βDGal(1-

for structure B, R is

βDGlc(1—>3)βDGlc(1— 3)βDGlc(1— 6)βDGlc(1-

I I

4 6 V pyruvate

whereas, for structure C, R is

ø DRibA(1—>4 )SCOG1CA(1—>4)βDGlc(1— 6)βDGlc(1—>

Whereas the polysaccharide B has been reported to gel, the polysaccharide C has not been reported to gel. Clearly, changes in the side chain markedly affect rheology.

Hence, it was by no means obvious that acetan was suitable for food and other uses, as a thixotropic thickening and suspending agen .

Accordingly, one aspect of the present invention provides an industrially useful composition comprising an aqueous dispersion of acetan and other ingredients, wherein the composition is thixotropic at least partly due to the presence of the acetan. By "industrially useful composition", we mean a manufactured product, particularly produced in bulk, intended for and suitable for sale or for use in an industrial process.

Examples of such industrially useful compositions include: those with suspended abrasive particles; adhesives; herbicides, pesticides, fertilizers and fungicides; glazes for ceramics; cleaners, such as toilet cleaners and bleaches of the "Domestos" type ("Domestos" is a Registered Trade Mark); emulsions such as toothpastes; foodstuffs such as sauces, salad dressings, ice¬ creams, chilled desserts and instant soups; water-based inks; mineral ore suspensions; non-drip paints, rheology modifiers for paper-making; oil-drilling fluids; pigments, for example for textile printing or wall-paper printing; shoe, furniture and metal polishes; and welding rods.

Acetan is the trivial name given to the extracellular polysaccharide (EPS) secreted by Acetobacter xylinium. The two papers referred to above propose the structures I and II shown below:-

( 4 ) βDGlc ( 1 — ) βDGlc ( l -→ )

( ) n

3

R

wherein, in structure I, R is

LRha(l —>6)DGlc(l—>6)βDGlc(1— > 6 )DGlcA(l~τ>2)DMan(1 >

and, in structure II, R is

LRha(l—>6)βDGlc(1—>6) DGlc(l—>4)βDGlcA(l—s>2) DMan(l—>

The structures are the same with respect to sugar composition, sequence and linkages. Structure II is the latest and perhaps best structure to date. The ano eric configurations (alpha, beta etc) will require checking and this may explain the apparent difference between I and II. Structure I is claimed to contain one O-acetyl substituent per repeat unit and structure II has one-two O-acetyl groups per repeat unit.

The references above define the polysaccharide. The CTAB preparation described in Tayama et al_ is probably the best method of purification. Once purified, the polymer should contain the following sugars: glucose (DGlc), mannose (DMan), glucuronic acid (DGlcA) and rhamnose (LRha) in the ratio

DGlc:DGlcA:DMan:LRha: :4:1:1:1. Methylation analysis of the native polymer and the reduces polymer should reveal the following types of carbohydrate linkages:

Rha(1— > 1

→2)Man(l— > 1

->6)Glc(l—> 2

->4)Glc(l— 1

->4)GlcA(l—> 1

→4)Glc(l— > 1

A 3

T

These methods are sufficient to identify the polymer.

Acetan may be prepared from at least some strains of the organism Acetobacter xylinium (including the strains deposited as an open deposit with the U.S. Dept. of Agriculture's Agricultural Research Culture Collection, Peoria, Illinois, as NRRL B42; and with the National Collections of Industrial and Marine Bacteria on 20th March 1989 as deposit no NCIB 40123, the latter deposit having been made under the provisions of the Budapest Treaty for the purposes of this patent application) in the manner described hereinafter, by the methods of Tayama et a_l or by the methods of Couso et al_. The acetan can be precipitated with ethanol, washed and freeze-dried for storage. Other polymers may co-precipitate with the acetan. These do not usually affect the porperties of the acetan, but may of course dilute it.

Acetan may be used in industrially useful compositions in ways which are analogous to those involving xanthan. For example, the concentration of acetan may be from 0.1% to 5%. The pH of the composition is preferably between 2.5 and 10, for the thixotropic effect to be achieved optimally. It is surprising that acetan solutions remain viscous at pH's below 5. Such compositions form a preferred aspect of the invention, and include acidic vinegar based sauces .

It has been found, unexpectedly, that the viscosity of an acetan solution remains substantially constant or increases slightly when a salt concentration of over 0.1M is present. This is similar to what is found with xanthan solutions but is very different from what is found with most viscous charged polysaccharides. This, a preferred aspect of the invention provides acetan-containing industrially useful compositions having added excess salt concentrations.

Preferably, the salt concentration is between 0.01M and 2M. By "salt", we mean inorganic salts of strong or weak acids preferably containing monovalent or divalent cations and a soluble anion. As indicated above, acetan can be used in a freeze-dried form. However, suitably treated broths from A.xylinium fermentations can also be used. The untreated bifbtfes of most strains suffer two immediate disadvantages. Firstly the broths contain a cellulose-based pellicle structure which foπis at the top of the growth vessel and secondly the broths are coloured and odorous. The cellulose pellicles can be removed by filtration. The colour and odour present in the broths can be completely removed by treatment with charcoal. The broths can be filtered through a charcoal column, or granular charcoal can be added to the broth and then removed by filtration.

Thus, a further aspect of the invention provides a broth from the fermentation of Acetobacter xylinium which has been treated (a) to remove the pellicle without removing the acetan and/or (b) by substantial removal of colour and odour.

Clearly, the extent to which the pellicle, colour and odour must be removed will depend upon the use to which the acetan broth is to be put; generally, though, complete removal of the pellicle is desirable.

With currently-available strain of A.xylinium and growth conditions, the concentration of acetan in the broth usually needs to be concentrated, for example by rotary evaporation, before use. A concentration of at least 1.0 - 2.0% is generally useful and is, in fact, high enough to permit subsequently blending with vinegar, water or the like, whilst still yielding a useful concentration of acetan in the final product. Above 3%, the dispersion becomes very viscous and cannot be concentrated further by rotary evaporation.

Some strains of A. ylinium are naturally "pellicle-minus", at least when in a shaken culture which reduces the formation of pellicle even with the more common strains of A. ylinium. The yield of acetan can be increased with such "pellicle-minus" strains, at least when grown in shaken culture.

Preferred aspects of the invention will now be illustrated by way of example and with reference to the accompanying drawing:

Figure 1 is a viscosity/shear rate graph for varying strengths of aqueous acetan dispersions.

Example 1 : Preparation of Acetan

Acetan is prepared from cultures of Acetobacter xylinum NRRL B42 grown in AJ medium.

AJ medium contains (g/1): yeast extract (Difco) (10); glucose (50); 2HP04 (0.1); KH2P04 (0.1); MgS04.7H20 (0.25); FeC13 (0.005). The pH of the medium is adjusted to 6.0. Cultures are grown at 25oC for 7 days with shaking to ensure aeration. Cells are removed from the culture medium by centrifugation (23,000g, 30 ins). If necessary, the medium is diluted with water to facilitate sedimentation of the cells. Remaining cells may be removed by passage through a Millipore filter (pore size 0.65um).

The cell-free culture medium is then concentrated by rotary evaporation at 40oC to approximately half the original volume, and solid KC1 is added to a final concentration of 1% (w/v) . Acetan is precipitated from the medium with 2 volumes of industrial spirit. The precipitate is coarse and fibrous, and easily separated from the liquid. Acetan is washed twice with industrial spirit, and excess liquid squeezed out before re- dissolving in glass distilled water and freeze-drying.

Example 2: Alternative Preparation of Acetan

Acetan was prepared as in Example 1, except that the rotary evaporation was continued for longer, resulting in a highly visco-elastic liquid, from which the acetan was not precipitated. Differing strength of aqueous dispersions of acetan prepared according to Example 1 were tested in standard apparatus to yield the viscosity/shear rate data for Figure 1. Note the coincidence of the data, for each dispersion, whether the shear rate is being increased or decreased.

Example 3: Ink for Glass Marking

Barium sulphate 15 parts by weight Ammonium biphluoride 15 parts by weight Ammonium sulphate 8 parts by weight Acetan q.s. Water q.s.

Sufficient acetan is included to give the composition the desired physical properties. Glycerine may be employed as well. If too thick, add more water. If the action is too slow, up to 5% of sodium fluoride may be added. Use in a hood or well ventilated room.

Example 4: Aquarium Cement

Glazier's putty 4. 5 kg Litharge 0.45 kg Red lead 0.45 kg Asphaltum 112 cm³ Mix with boiled linseed oil and acetan solution to the proper consistency. Lamp black may be added to give a grey colour.

Example 5: Stabiliser for Particles or Emulsions in Food

Roughly 0.1% Acetan is used to suspend particles or droplets.

Food (e.g. Salad) Dressing 94.9% vinegar

5 % solids 0.1% Acetan

In a milk substitute 0.05% Acetan may be used to stabilise dispersions of dried whey or heat-processed soya protein in water.

Example 6: Ice Cream

Fat 10 %

Serum solids 11.7%

Sucrose 11.0%

Corn Syrup solids 4.8%

Acetan 0.5%

Water 62.0% Example 7 : All Purpose Cleaner

Water/perfume 91.3%

Alipal CD128 1.7%

Gafamide CDD518 0.5%

Butyl Cellosolve 3.5%

Sodium metasilicate 1.7%

Trisodium phosphate 1.0%

Acetan 0.3%

Example 8: Ceramic Glaze

Roughly 0.5% acetan is used to suspend particles in water.

Example 9 : Silver Polish

Water 78.6%

Snow Floss 15.0%

Triton X-102 5.0%

Anti-tarnish agent 1.0%

Acetan 0.4%

14

Example 10: Oven Cleaner

Acetan 0.1%

Trisodium phosphate 3.2%

Sodium metasilicate 1.1%

Sulfonate 0A5 2.1%

BRIJ 96 3.2%

Hexane 4.2%

Water 82.1%

Example 11: Buffing Compound or Stripper

0.2% Acetan

40.0% Abrasive

15.0% Grease*

3.0% Triethanolamine

2.0% Emulsifier

39.8% Water

(* The grease consists of 50% oleic or stearic or palmitic acid, 10% Triton XII and 40% Tallow mineral oil).

Example 12: Liquid Feed Supplement containing added suspended limestone

25% Molasses

25% Limestone Flour

0. 125% Acetan

19. 2% 50% Urea liquor

6. 75% Ammonium polyphosphate

5. 0% Salt (NaCL)

1. 5% Ammonium sulphate

0. 2% Trace minerals

Claims

1. An industrially useful composition comprising an aqueous dispersion of acetan and other ingredients, wherein the composition is thixotropic at least partly due to the presence of the acetan.

2. A composition according to Claim 1 wherein the composition comprises suspended abrasive particles, an adhesive, a herbicide, a pesticide, a fertilizer, a fungicide, a glaze for ceramics, a cleaner, toothpaste ingredients, a foodstuff, a mineral ore suspending agent, non-drip paint ingredients, paper-making ingredients, oil-drilling ingredients, a textile printing or wall-paper printing pigment, a shoe, furniture or metal polish, or welding rod ingredients.

3. A composition according to Claim 1 or 2 wherein the concentration of acetan is from 0.1% to 5%.

4. A composition according to any one of the preceding wherein the pH of the composition is between 2.5 and 10.

5. A composition according to any one of the preceding claims wherein the pH is below 5.0.

6. A composition according to any one of the preceding containing a salt concentration of 0.01 to 2.0M.

7. A broth from the fermentation of Acetobacter xylinium which has been treated (a) to remove the pellicle without removing the acetan and/or (b) by substantial removal of colour and odour.