GB1570487A

GB1570487A - Mucilaginous polysaccharide ax

Info

Publication number: GB1570487A
Application number: GB42662/75A
Authority: GB
Original assignee: Takeda Chemical Industries Ltd
Current assignee: Takeda Pharmaceutical Co Ltd
Priority date: 1975-10-17
Filing date: 1975-10-17
Publication date: 1980-07-02

Description

(54) NEW MUCILAGINOUS POLYSACCHARIDE AX" (71) We TAKEDA YAKUHIN KOGYO KABUSHIKI KAISHA also known as Takeda Chemical Industries Ltd., a body corporate organised according to the laws of Japan, of 27, Doshomachi 2-chome, Higashi-ku, Osaka, Japan, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- This invention relates to a new mucilaginous polysaccharide, namely, Polysaccharide AX, and is particularly concerned both with the new Polysaccharide AX and with the microbial production thereof.

We have unexpectedly found that a microorganism belonging to the genus Acetobacter produces a polysaccharide which is soluble in water, and the viscosity of this polysaccharide does not decrease at pH 2 to 7 or in the presence of a salt. Research based on this fmd- ing culminated in the present invention.

The present invention, therefore, provides a new mucilaginous polysaccharide, herein referred to as Polysaccharide AX, the production of Polysaccharide AX, and food additives, carriers for pharmaceutical preparations, or cosmetic materials containing Polysaccharide AX.

The microorganisms which are employed in the process of this invention are those which belong to the genus Acetobacter and which are capable of producing Polysaccharide AX.

These microorganisms are isolated from natural sources or are obtained from certain type culture collections. Mutants which are spontaneously or artifically induced by mutagenic treatment can be used, provided they are able to accumulate the polysaccharides.

Acetobacter aceti, sub-species xylinum B-i IFO-13659 (ATCC-31174,FERM-P.

No. 3707) is a typical example employable in the process of this invention. The numbers preceded by the symbols IFO-, ATCC- and FERM- are the accession numbers at the Institute for Fermentation, Osaka, Japan (IFO), the American Type Culture Collection, Maryland, U.S.A. (ATCC) and the Fermentation Research Institute of the Agency of Industrial Science and Technology, Chiba, Japan (FERM), respectively.

The microbiological characteristics of this strain are as follows: 1. Morphological characteristics Microscopic observations (growth on agar slant, 48 hours): Shape: rods occurring singly or in chains; non-sporulating Motility: non-motile Size: 0.3 to 0.5 micron by 2.0 to 4.0 microns Gram staining: negative 2. Cultural characteristics 1) Nutrient broth: surface growth: the production of thick, leathery, cellulosic membranes.

2) Nutrient agar slant: no growth 3) Plain gelatin stab: no growth 4) 1% glucose bouillon agar plate: punctiform 1 mm. or less in diameter; pale brown in colour; convex, regular edge, and tough consistency; the whole colony lifted off the agar when touched with a needle.

3. Physiological characteristics 1) Temperature for growth 15 C to 420C; optimum, 280C to 300C.

2) pH for growth; pH 3.0 to 7.4; optimum, pH 4.5 to 6.0 3) Relationship with oxygen: aerobic 4) Gelatin: not liquefied 5) Litmus milk: no growth 6) Indole: not produced 7) Hydrogen sulphide: not produced 8) Nitrates: not reduced to nitrites 9) Ammonia: not produced 10) Methyl red test: positive 11) Voges-Proskauer test: negative 12) Starch: not hydrolysed 13) Catalase: produced 14) Citric acid: not utilised 15) Ammonium salts are utilised as the sole nitrogen source 16) Ketogenesis in glycerol: positive 17) Formation of gluconate: positive 18) Oxidation of acetate and lactate to carbon dioxide: positive The cultivation of the microorganisms may be carried out either on a solid medium or in a liquid medium, and it is recommended to employ a liquid medium for industrial purposes.

When the liquid medium is employed, the cultivation may be conducted under aerobic conditions with shaking or agitation.

The culture medium to be employed in this invention comprises assimilable carbon sources, digestible nitrogen sources, inorganic salts and so on.

Suitable carbon sources for the cultivation of these microorganisms are exemplified by glucose, galactose, fructose, sorbitol, mannitol, sucrose, starch hydrolysates and glycerol as well as organic acids such as fumaric acid or its salts. The concentration of such carbon sources in the culture medium may be varied provided an adequate growth of the particular microorganism used and a high production of the desired polysaccharide are ensured.

As the nitrogen source, inorganic ammonium salts (e.g. ammonium sulphate, ammonium phosphates, ammonium chloride or the like), and organic nitrogen sources (e.g. aminoacids, yeast extract, meat extract, fish extract, corn steep liquor, corn gluten, soybean meal or the like) may be effectively employed.

The useful inorganic salts as nutrients are those which are conventionally employed for the cultivation of microorganisms, including, for example, potassium phosphates and sodium phosphates as well as the sulphates, chlorides, or carbonates, for example, of manganese, iron, magnesium, calcium zinc, cobalt and so on.

The main fermentation is carried out at a temperature ranging from 20"C to 40"C, preferably from 24 C to 300C, for 48 to 388 hours, preferably 96 to 240 hours, at a pH value of from 3 to 8, preferably from 6 to 7.

Other conditions of cultivation such as aeration or agitation are as employed conventionally in the cultivation of microorganisms belonging to the genus Acetobacter.

Polysaccharide AX is elaborated and accumulated in the culture medium.

In harvesting the Polysaccharide AX, it is possible to apply one or more per se conventional separation and purification procedures, in any desired combination.

For example, there may be employed such techniques as filtration, centrifuging, drying (e.g. spray drying or lyophilising), precipitation (e.g. precipitation by using organic solvents such as alcohol, acetone and the like), or powdering.

A typical purification procedure is mentioned below. Thus, not less than 1% of filter aid (e.g. diatomaceous earth such as Celite Standard Super Cel (trade mark), (manufactured by Johns Manville, Ltd.) may be added to the culture broth, and the broth is filtered under suction or compression to remove insolubles.

Activated charcoal is added to the filtrate, and, after decolouring, the mixture is filtered to remove the particles of charcoal. An organic solvent such as ethanol, methanol, acetone, dioxane or dimethylsulphoxide is added to the filtrate, whereupon the desired Polysaccharide AX separates out as a gel. This gel fraction is collected and dried by freeze-drying.

The viscosity of the Polysaccharide AX which is obtained does not decrease in the acid state (pH 2 to 7) or in the presence of a salt (e.g. ammonium sulphate, ammonium chloride, ammonium nitrate, sodium chloride, potassium chloride, magnesium chloride, zinc chloride, copper chloride or aluminium chloride).

Therefore, the Polysaccharide AX is used as a food additive, as a carrier for pharmaceutical preparations or cosmetic materials, or as an industrial material such as a drilling agent or a tubulous friction reducer.

When Polysaccharide AX is used as a food additive, the resulting food is improved in terms of viscosity, palatability, binding quality, waterholding capacity, body, gloss, thickness and stability, and the drip loss is reduced.

The foods to which the Polysaccharide AX can be applied as a food additive may include various types of foods which need some increase in viscosity or emulsification property.

Among those foods are salad dressings, soups and soup mixes, sauces and sauce mixes, gravies, pastes for Japanese pickles (tsukemono toko), ketchups, tomato juice, nectar drinks, fruit drinks, jams, marmalade, peanut butter, flour paste, bean jam, instant curry mixes, instant stews and the like.

When Polysaccharide AX is used as a food additive, 0.01 to 20.0 (weight/weight)%, more preferably 0.08 to 6.0 (weightiweight)%, of Polysaccharide AX relative to the total amount of the finished foodstuff is added.

Polysaccharide AX is also employed for industrial purposes, e.g. in mobility control, tubulous friction reduction, suspension control, viscosity control and waterloss control.

The invention is illustrated by the following examples. In the examples, "part(s)"are based on weight unless otherwise specified, the relationship between "part(s)" and "part(s) by volume" corresponds to that between "gram(s)" and "milliliter(s)", and viscosity measurements are measured by rotating viscometer: Rotovisco (manufactured by Gebruder Hiake, West Germany).

EXAMPLE 1 (a) A fermentation tank of 100 parts by volume in capacity is filled with 50 parts by volume of a liquid seed culture medium (pH 7.0) composed of mannitol (5%), K2HP04 (0.01%), KH2P04 (0.09%), MgS04.7H20 (0.025 yto), FeC13 (0.00056Xo) and asparagine (0.1%). After sterilisation, the medium is inoculated with a slant culture ofAcetobacter aced sub-species xylinum B-i (IFO- 13659), and is incubated at 30"C for 48 hours under agitation at 280 r.p.m. and 100% aeration to obtain a seed culture broth.

(b) A fermentation tank of 50,000 parts by volume in capacity is filled with 30,000 parts by volume of a liquid main culture medium (pH 7.0) composed of mannitol (5%), K2 HP04 (0.01 /to), KH2P04 (0.09%) MgS04.7H20 (0.025%), Fecal3 (0.0005%) and asparagine (0.1%). After sterilising, 1000 parts by volume of the seed culture obtained as above are transferred to the main culture medium, and the main culture medium is incubated at 300C for 96 hours under agitation at 280 r.p.m. and 100% aeration.

The resulting culture broth is filtered with a filter aid (Celite Standard Super Cel (trade mark)). To the filtrate are added 300 parts by volume of activated charcoal and, after decolouring, the mixture is filtered to remove the particles of charcoal. The colourless and transparent filtrate is treated with three times as much pure ethanol, whereupon the desired polysaccharide separates out as a gel. The gel fraction is collected by filtration and washed twice with 7,000 parts by volume of ethanol, followed by drying with ether to give 150 parts of the Polysaccharide AX.

EXAMPLE 2 (a) A fermentation tank of 100 parts by volume in capacity is filled with 50 parts by volume of a liquid seed culture medium (pH7 .0) composed of sorbitol (5%), K2HP04 (0.01%) KH2I'04 (0.09%), MgS04 .7H20 (0.025%), FeCQ3 (0.0005aSo) and (NH4)2S04 (0.1%). After sterilisation, the medium is inoculated with a slant culture of Acetobacter aced subsp.

xylinum - B-i (IFO-13659), and is incubated at 30"C for 48 hours under agitation at 280 r.p.m. and 100% aeration to give a seed culture.

(b) One thousand parts by volume of the seed culture are transferred aseptically into 50,000 parts by volume in capacity containing 30,000 parts by volume of a sterilised main culture medium (pH 7.0) composed of sorbitol (5%), K2 HPO4 (0.01%), KH2 P04 (0.09%), MgS04 .7H2 0 (0.025%) FeCQ3 (0.0005%) and (NH4)2S04 (0.1%). Cultivation is carried out at 30"C for 96 hours under agitation at 280 r.p.m. and with 100% aeration.

The resulting culture broth is filtered with filter aid (Celite Standard Super Cel (trade mark .

To the filtrate are added 300 parts by volume of activated charcoal and, after decolouring, the mixture is filtered to remove the charcoal. The filtrate is treated with three times as much pure ethanol, whereupon the desired polysaccharide separates out as a gel. The gel fraction is collected by filtration and washed twice with 7,000 parts by volume of ethanol, followed by drying with ether to give 140 parts of the polysaccharide AX.

EXAMPLE 3 (a) A fermentation tank of 100 parts by volume in capacity is filled with 50 parts by volume of a liquid seed culture medium (pH 7.0) composed of mannitol (5%), K2HP04 (0.01%), KH2P04 (0.09%), MgS04 .7H20 (0.025%), FeCQ3 (0.0005 ,fo) and asparagine (0.1%). After sterilization, the medium is inoculated with a slant culture ofAcetobacteracetisubsp. xylinum B-i (1F0-13659) and incubated at 300C for 48 hours under agitation at 280 r.p.m. to give a seed culture.

(b) One thousand parts by volume of the seed culture are transferred aseptically into 50,000 parts by volume in capacity containing 30,000 parts by volume of a sterilized main culture medium (pH 7.0) composed of fructose (5%), K2HP04 (0.0l%),KH2P04 (0.09%) MgS04 .71120(0.025%), FEAR3 (0.0005%) and asparagine (0.1%). Cultivation is carried out at 30 C for 96 hours under agitation at 280 r.p.m. and with 100% aeration.

The resulting cultured broth is filtered with filter aid (Celite Standard Super Cel (trade mark)).

300 parts by volume of activated charcoal are added to the filtrate and, after decolouring, the mixture is filtered to remove the charcoal.

The filtrate is treated with three times as much pure ethanol, whereupon the desired polysaccharide separates out as a gel. The gel fraction is collected by filtration and washed twice with 7,000 parts by volume of ethanol, followed by drying with ether to give 200 parts of Polysaccharide AX.

The physico-chemical properties of the resulting Polysaccharide AX are given below: 1. Specific rotation: [a] 2D2 = +210 + 80 (c=0.5%, in water) 2. Average molecular weight: From 100,000 to 100,000,000, when measured by light scattering spectrometry in pure water. The molecular weight of the polysaccharide AX varies in accordance with the incubation period.

3. Elemental analysis: C=39.17+1%; H=6.28+0.5%; N=0.00%; 4. Colour reaction: Polysaccharide AX gives a positive reaction to the Molisch test, the phenol-sulphuric acid test and the anthrone test.

5. Ultraviolet absorption Polysaccharide AX has a weak absorption at 280 m (see Figure 5).

6. Infrared absorption: Significant absorption bands at the wave numbers (cm1) ) shown below, indicate the presence of the s-glucosidic bond at 890 cml and the presence of the acetyl group at 1240 cm and 1740 cm (Of the symbols given in parenthesis, S denotes a strong intensity, M a medium intensity and SH a shoulder). 3600-3200(5), 3000-2850(M), 1740(M), 1620(M), 1375(M), 1240(M), 890(SH), 1050(S) This spectrum is closely similar to that of acetylcellulose.

The infrared absorption spectrum is shown in Figure 1 of the accompanying drawings (KBr disk method).

7. Solubility: Polysaccharide AX is soluble in water, IN-hydrochloric acid, IN-sulphuric acid and IN-aqueous ammonia, and is insoluble in ethanol, ether, acetone, chloroform, dimethyl sulphoxide and dioxane.

8. Viscosity: A 0.25% (W/V) aqueous solution of Polysaccharide AX shows a viscosity within the range of from 4 ops. to 13 cps, inclusive, the temperature being 300C and the rate of shear being 1046.7 sec 9. Viscosity against pH: The viscosity of Polysaccharide AX does not decrease in the pH range of from 2 to 11.

The viscosity of Polysaccharide AX does not decrease after long storage in the pH range of from 2 to 11.

10. Viscosity against salts: The viscosity of Polysaccharide AX does not decrease in the presence of a solution of 5% (weight/volume) of potassium chloride, 5% (W/V) of magnesium chloride,5% (W/V) of zinc chloride, 5% (W/V) of copper chloride, 5% (W/V) of ammonium sulphate or a sodium chloride solution up to saturation point.

11. Decomposition reaction: Polysaccharide AX is hydrolysed with 2N-H2S04 at 110 C in a sealed tube for 17 hours. The reaction mixture is neutralised with barium hydroxide and the mixture is run onto a column of ionexchange resin (Dowex50W-X4 (trade mark) manufactured by Dow Chemical Co., Ltd.) to neutrality. The eluate is concentrated under reduced pressure.

The concentrate is subjected to paper chromatography with a developer (phenol: water = 5 1) to detect a spot for glucose (Rf value: 0.39+0.03).

12. Acetyl-value: 50% sulphuric acid is added to the Polysaccharide AX in a stoppered flask, and the mixture is kept standing at 200C for 24 hours.

The sulphuric acid is removed by distillation.

The product is subjected to analysis by gasliquid chromatography in comparison with an authentic sample of acetic acid.

The acetyl value is determined. as 0.5 to 5% (weight/weight).

13. Enzymatic reaction: Cellulase (manufactured by Merck & Co., Reaction temperature: 370C; Reaction time: 60 minutes) decreases the viscosity of Polysaccharide AX and releases reducing sugars.

14. Colour and shape Polysaccharide AX is white and amorphous. An aqueous solution of Polysaccharide AX is colourless and transparent.

15. Taste and odour An aqueous solution of Polysaccharide AX is of no taste and of no odour.

16. Comparison of the viscosity of Poly saccharide AXwith carboxymethylcellulose and guar gum (see Figure 2 of the accom panying drawings) Experimental conditions: 0.25% aqueous solution; temperature: 300C; rate of shear: 1046.7 second~'; rotating viscometer; Rotovisco (manufactured by Gebruder Haake, West Germany).

The experiment teaches that the polysaccharide AX has a higher viscosity than other mucilages, does not show an extreme decrease in viscosity, and shows a good stability in the acidic stage.

17. Thixotropic property: The relationship between viscosity and rate of shear is shown in Figure 3 of the accompanying drawings. Experimental conditions: 0.5 and 1.0% (W/V) aqueous solutions of the polysaccharide AX employed: temperature: 300 C; rotating viscometer: see item 16 above.

Figure 3 teaches that the polysaccharide AX has thixotropic properties.

18. Spinnability: Polysaccharide AX shows little spinnability.

The relationship between log (viscosity) and log (rate of shear) is shown in Figure 4 of the accompanying drawings.

Experimental conditions: a 0.5% aqueous solution of Polysaccharide AX is employed; tem perature: 300C; rotating viscometer: see item 16 above.

The slope of Figure 4 is substantially -0.67, and this means that Polysaccharide AX has little spinnability.

(See: Tsurutaro Nakagawa, Bulletin of the Chemical Society of Japan 25, 88 (1952)).

19. Structure: Based on the analytical data in items 6, 11 and 13, Polysaccharide Axis considered to be mainly composed of glucose connected through p-1,4-glucosidic linkages, and is partially acetylated.

EXAMPLE 4 Seventy-five parts by volume of vinegar are put in a bowl and are thoroughly stirred with the addition of 5 parts of salt, 0.5 part of white pepper, 0.5 part of sodium glutamate, 4 parts of paprika and 0.2 part of Polysaccharide AX.

160 parts by volume of salad oil in limited amounts are then added to the mixture. The procedure gives about 240 parts of salad dressing.

EXAMPLE 5 2,500 parts of unpasteurized milk are added to seven hundred and fifty parts of fresh cream and the resulting mixture is heated at 550C for 30 minutes. This is followed by the addition of 10 parts of Polysaccharide AX, 1,500 parts of defatted condensed milk, 220 parts of cane sugar, and 10 parts of sucrose palmitate. The mixture thus prepared is pasteurised, homogenised and frozen to yield approximately 5,000 parts of ice-cream.

EXAMPLE 6 Fifteen % of dextrin is blended with 80% of glucose and 5% of polysaccharide AX powder.

The mixture is a superior ice glazing agent which has a high viscosity and a high filmforming ability.

EXAMPLE 7 Three-hundred parts of butter are put in a bowl and externally heated in a water bath to a creamy state. The butter is thoroughly stirred with the addition of 240 parts of granulated sugar and 3 egg yolks, followed by the addition of a small amount of lemon essence. The mixture is blended with a homogeneous mixture of 450 parts of wheat flour and 9 parts of Polysaccharide AX. The resulting dough is spread evenly with a rolling pin and is stamped to size.

The stampings are baked in an oven for about 8 minutes. The procedure gives about 980 parts of biscuits.

EXAMPLE 8 21.5 parts cf wheat bran are blended with 2.5 parts of sodium chloride, 14 parts of a seasoning (mixture of 5 parts of sugar, 1.5 part of sodium glutamate, 0.7 part of hydrolysed plant protein, 6.3 parts of soy sauce and 0.5 part of lactic acid) and 6 parts of Polysaccharide AX powder.

56 parts of water are added with mixing.

The resulting mixture is usable as a paste for Japanese pickles {Tsukemono toko), keeps its quality stable with a proper viscosity during the curing process.

WHAT WE CLAIM IS: 1. The mucilaginous polysaccharide herein called Polysaccharide AX, which has the following properties and structure: (a) Specific rotation: [a] 2f=+21 +8 (C = 0.5% in water); (b) Molecular weight: 105 to 108 (determined as herein defined); (c) Elemental analysis: C:39.17% + 1%; H:6.28% + 0.5%; N:0.00%; (d) Colour reaction: positive in Molisch test, phenol-sulphuric acid test and anthrone test; (e) Infrared absorption: significant absorptions (cm)are ) are as follows: 3600-3200(S), 3000-2850(M), 1740(M), 1620(M), 1375(M), 1240(M), 890(SH) and 1050(S); (f) Solubility: soluble in water, IN-hydrochloric acid, IN-sulphuric acid and IN-aqueous ammonia, and insoluble in ethanol, ether, acetone, chloroform, dimethylsulphoxide and diox ane; (g) Viscosity: 4 to 13 cps. in a 0.25 weight/volume percent aqueous solution at 30 C and at a rate of shear of 1046.7 sec; (h) Viscosity against pH: viscosity does not decrease even after long storage in the pH range of from 2 to 11; (i) Vicsocity against salts: does not decrease in the presence of a solu tion of 5% (weight/volume) of potassium chloride, 5% (W/V) of magnesium chloride, 5% (W/V) of zinc chloride, 5% (W/V) of copper chloride, 5% (W/V) of aluminium chloride, 5% (W/V) of ammonium sulphate, or a sodium chloride solution (up to satura tion); (j) Decomposition reaction: the hydrolysis (as herein defined) of the polysaccharide AX gives glucose; (k) Acetyl-value: 0.5 to 5% (weight/weight) determined as herein defined; (1) Enzymatic reaction: the viscosity is lowered by the enzymatic re action of cellulase, and a reducing sugar is released thereby; (m) Thixotropic property; Polysaccharide AX has a high viscosity at low stress, but a low viscosity when an increased stress is applied; (n) Colour and shape: Polysaccharide AX is white and amorphous, and an aqueous solution thereof is colour less and transparent; (o) Taste and odour: an aqueous solution of polysaccharide AX is tasteless and odourless; (p) Spinnability: The Polysaccharide AX shows little spin nability; and (q) Structure: Polysaccharide AX is mainly composed of -1, 4-glucosidic linkages, and is partially acetylated, wherein the viscosity measure ments are determined as herein defined.

2. A method of producing Polysaccharide AX as claimed in Claim 1, which comprises cultivating a Polysaccharide AX-producing microorganism of the genus Acetobacter in a culture medium; allowing the Polysaccharide AX to be accumulated in the culture medium; and isolating the accumulated polysaccharide AX from the culture medium.

3. A method according to Claim 2, wherein the microorganism belongs to the sub-species Acetobacter aceti sub-species xylinum.

4. A method according to Claim 3, wherein the microorganism is Acetobacter aceti sub species xylinum IFO-13659.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims

**WARNING** start of CLMS field may overlap end of DESC **.

procedure gives about 240 parts of salad dressing.

EXAMPLE 5 2,500 parts of unpasteurized milk are added to seven hundred and fifty parts of fresh cream and the resulting mixture is heated at 550C for 30 minutes. This is followed by the addition of

10 parts of Polysaccharide AX, 1,500 parts of defatted condensed milk, 220 parts of cane sugar, and 10 parts of sucrose palmitate. The mixture thus prepared is pasteurised, homogenised and frozen to yield approximately 5,000 parts of ice-cream.

EXAMPLE 6 Fifteen % of dextrin is blended with 80% of glucose and 5% of polysaccharide AX powder.

The mixture is a superior ice glazing agent which has a high viscosity and a high filmforming ability.

EXAMPLE 7 Three-hundred parts of butter are put in a bowl and externally heated in a water bath to a creamy state. The butter is thoroughly stirred with the addition of 240 parts of granulated sugar and 3 egg yolks, followed by the addition of a small amount of lemon essence. The mixture is blended with a homogeneous mixture of 450 parts of wheat flour and 9 parts of Polysaccharide AX. The resulting dough is spread evenly with a rolling pin and is stamped to size.

The stampings are baked in an oven for about 8 minutes. The procedure gives about 980 parts of biscuits.

EXAMPLE 8 21.5 parts cf wheat bran are blended with 2.5 parts of sodium chloride, 14 parts of a seasoning (mixture of 5 parts of sugar, 1.5 part of sodium glutamate, 0.7 part of hydrolysed plant protein, 6.3 parts of soy sauce and 0.5 part of lactic acid) and 6 parts of Polysaccharide AX powder.

56 parts of water are added with mixing.

The resulting mixture is usable as a paste for Japanese pickles {Tsukemono toko), keeps its quality stable with a proper viscosity during the curing process.

WHAT WE CLAIM IS: 1. The mucilaginous polysaccharide herein called Polysaccharide AX, which has the following properties and structure: (a) Specific rotation: [a] 2f=+21 +8 (C = 0.5% in water); (b) Molecular weight:

105 to 108 (determined as herein defined); (c) Elemental analysis: C:39.17% + 1%; H:6.28% + 0.5%; N:0.00%; (d) Colour reaction: positive in Molisch test, phenol-sulphuric acid test and anthrone test; (e) Infrared absorption: significant absorptions (cm)are ) are as follows: 3600-3200(S), 3000-2850(M), 1740(M), 1620(M), 1375(M), 1240(M), 890(SH) and 1050(S); (f) Solubility: soluble in water, IN-hydrochloric acid, IN-sulphuric acid and IN-aqueous ammonia, and insoluble in ethanol, ether, acetone, chloroform, dimethylsulphoxide and diox ane; (g) Viscosity:

4 to 13 cps. in a 0.25 weight/volume percent aqueous solution at 30 C and at a rate of shear of 1046.7 sec; (h) Viscosity against pH: viscosity does not decrease even after long storage in the pH range of from 2 to 11; (i) Vicsocity against salts: does not decrease in the presence of a solu tion of 5% (weight/volume) of potassium chloride, 5% (W/V) of magnesium chloride, 5% (W/V) of zinc chloride, 5% (W/V) of copper chloride, 5% (W/V) of aluminium chloride, 5% (W/V) of ammonium sulphate, or a sodium chloride solution (up to satura tion); (j) Decomposition reaction: the hydrolysis (as herein defined) of the polysaccharide AX gives glucose; (k) Acetyl-value: 0.5 to 5% (weight/weight) determined as herein defined; (1) Enzymatic reaction: the viscosity is lowered by the enzymatic re action of cellulase, and a reducing sugar is released thereby; (m) Thixotropic property; Polysaccharide AX has a high viscosity at low stress, but a low viscosity when an increased stress is applied; (n) Colour and shape: Polysaccharide AX is white and amorphous, and an aqueous solution thereof is colour less and transparent; (o) Taste and odour: an aqueous solution of polysaccharide AX is tasteless and odourless; (p) Spinnability: The Polysaccharide AX shows little spin nability; and (q) Structure: Polysaccharide AX is mainly composed of -1, 4-glucosidic linkages, and is partially acetylated, wherein the viscosity measure ments are determined as herein defined.
2. A method of producing Polysaccharide AX as claimed in Claim 1, which comprises cultivating a Polysaccharide AX-producing microorganism of the genus Acetobacter in a culture medium; allowing the Polysaccharide AX to be accumulated in the culture medium; and isolating the accumulated polysaccharide AX from the culture medium.
3. A method according to Claim 2, wherein the microorganism belongs to the sub-species Acetobacter aceti sub-species xylinum.
4. A method according to Claim 3, wherein the microorganism is Acetobacter aceti sub species xylinum IFO-13659.
5. A food additive, which comprises Poly

saccharide AX as claimed in Claim 1.
6. A foodstuff composition, which comprises 0.01 to 20.0 (W/W)% of Polysacchadde AX as claimed in Claim 1, relative to the total amount of the complete foodstuff.
7. A foodstuff composition according to Claim 6, wherein the composition includes from 0.08 to 6.0 (W/W)% of Polysaccharide AX.
8. A method according to Claim 2, substantially as herein described with reference to any of the specific examples.
9. Polysaccharide AX when produced by a method as claimed in any of Claims 2 to 4, or in Claim 8.
10. A foodstuff composition comprising Polysaccharide AX as claimed in Claim 9.
11. A foodstuff composition substantially as herein described with reference to any of the specific examples.
12. A carrier for pharmaceutical formulations which comprises Polysaccharide AX as claimed in Claim 1.
13. A cosmetic material which comprises Polysaccharide AX as claimed in Claim 1.
14. Artificially produced mutants of the microorganism sub-species Acetobacter aceti, sub-species xylinum which are capable of producing and accumulating polysaccharide AX as claimed in claim 1.
15. Artificially produced mutants of the microorganism Acetobacter aceti sub-species xylinum IFO-13659 which are capable of producing and accumulating Polysaccharide AX as claimed in Claim 1.