JPH0597903A - Polysaccharide and its production - Google Patents

Abstract

PURPOSE:To industrially advantageously produce a new polysaccharide having a high viscosity and useful as a thickening agent or a gelling agent by culturing a bacterium belonging to Pseudomonas genus and extracellularly producing ICC-001 polysaccharide and collecting the product from the cultured material. CONSTITUTION:A bacterium [e.g. Pseudomonas ICC-001 strain (Bikoken stipulation No. 12453)] belonging to Pseudomonas genus and extracellularly producing ICC-001 polysaccharide is shake cultured in a culture medium at 30 deg.C for 24hr and the cultured solution is subsequently filtered. To the filtrate 10wt.% cetylpyridinium chloride, etc., is added for generation of precipitates and the generated precipitates are filtrated and then purified by reprecipitation, thus obtaining the objective high viscous polysaccharide ICC-001 having about 700000 molecular weight (gel filtration method) and -0.16 deg. specific rotatory power (C0.4-1M-NaCl) and containing, as the main constituting components, D-glucose, D-galactose and pyruvic acid in an about 3.3:1 molar ratio of D- glucose to D-galactose.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel polysaccharide having a high viscosity and a method for producing the same.

[0002]

BACKGROUND OF THE INVENTION As useful polysaccharides, plant-derived cellulose, pectin, amylose, animal-derived heparin, chondroitin sulfate and the like are well known. It is also known that certain kinds of microorganisms produce polysaccharides, and among the polysaccharides produced by these microorganisms, hyaluronic acid, dextran, xanthan gum, pullulan and the like have already been or are being industrially utilized.

[0003]

Although various polysaccharides produced by microorganisms are industrially used, new materials having a stronger thickening property and gelling ability than these known compounds are available. The emergence of is always desired. The present inventors pay attention to these points, and try to solve this problem by collecting soil fungi and searching their polysaccharide-producing ability to provide polysaccharides having high viscosity.

[0004]

[Means for Solving the Problems] In order to meet the above-mentioned demand, the inventors of the present invention continued to search for a microorganism that produces a highly viscous substance on an agar medium, and found that a strain belonging to the genus Pseudomonas was found on the agar medium. The present invention has been completed by discovering that transparent colonies were produced in and that the transparent viscous substance was a polysaccharide having a novel structure. An object of the present invention is to provide a novel highly viscous polysaccharide and a method for producing the same.

The gist of the first aspect of the present invention is to provide a polysaccharide having high viscosity, which comprises glucose, galactose and pyruvic acid as constituent components. And second
The present invention provides a method for producing a polysaccharide, which comprises culturing a microorganism that belongs to the genus Pseudomonas and has the above-mentioned polysaccharide-producing ability, and collecting the above-mentioned polysaccharide from the culture solution. ..

1. Bacteriological Properties of Producing Bacterium ICC-001 Strain As an example of the microorganism used in the present invention, IC newly isolated from soil in Jonan City, Kyoto Prefecture by the present inventors
There is C-001 strain. The mycological properties of the ICC-001 strain are as follows. I. Microscopic morphology Morphology: Bacillus does not form spores. Motility: Movement with polar flagella Size: 0.6-0.8 x 1.2-2.0 μm II. Staining Gram stain: Negative III. Physiological properties Oxygen requirement: Absolute aerobic oxidase production: Positive β-galactosidase production: Positive Ornithine decarboxylase production: Negative urease production: Positive Tryptophan deaminase production: Negative acetylmethylcarbinol production: Negative fluorescence Dye formation: Negative Hydrogen sulfide formation: Negative Indole formation: Negative Starch hydrolysis: Negative Gelatin hydrolysis: Negative Nitrate reducibility: Positive Arginine degradation: Negative Lysine degradation: Negative Denitrification: Negative MR Test: Negative Casein degradation: Positive Use of citric acid: Positive Catalase reaction: Positive OF test: O type According to the above properties, the Vergiz Manual of
Systematic Bacteriology (Bergeys)
Manual of Systematic Bacte
As a result of searching by riology) Vol 1, this bacterium was a Gram-negative bacillus, motivated by polar flagella, absolutely aerobic, and oxidase-positive, so that this bacterium was identified as Pseudomonas. The ICC-001 strain was deposited at the Institute of Microbial Technology, Institute of Industrial Technology, August 2, 1991.
It was entrusted as Microbiology Research Institute No. 12453 on 8th.

2. Culturing Method of Polysaccharide-Producing Strain ICC-001 Strain In the present invention, the polysaccharide-producing bacterium belonging to the genus Pseudomonas is cultured in a medium. In the culturing, a medium containing a carbon source, a nitrogen source, and nutrient sources such as various inorganic salts necessary for growth that can be assimilated by the microorganism used is used. Specifically, glucose, sucrose, and the like are used as carbon sources, and meat extract, yeast extract, peptone, ammonium sulfate, and other organic or inorganic substances are used as nitrogen sources.
Culturing is performed at pH 5.5 to 9.5, preferably 6.5 to 7.5,
It is carried out at a temperature of 15 ° C. to 45 ° C., preferably 25 ° C. to 35 ° C., and usually a culture for several days may be performed.

3. Method for Purifying ICC-001 Polysaccharide The thus obtained culture contains the polysaccharide of interest of the present invention. Therefore, it is necessary to extract and generate polysaccharides from the culture. In purifying the polysaccharide, it is preferred to remove the bacterial cells and other solids in the culture in advance and then recover the polysaccharide.

The purification may be carried out by heating, centrifugation, precipitation, washing, drying or the like, which is usually used for recovering the polysaccharide from impurities, alone or in combination. As an example, a solvent such as ethanol is added to the solution obtained by removing the solid content to precipitate the polysaccharide. The polysaccharide thus obtained is dissolved in water containing salt, and the polysaccharide is precipitated again with a solvent such as ethanol. After repeating this treatment, a purified polysaccharide can be obtained by dialysis and freeze-drying.

4. Physicochemical Properties of ICC-001 Polysaccharide The properties of the ICC-001 polysaccharide thus obtained are shown below. (1) Molecular weight of about 700,000 (measured by gel filtration method with Toyopearl HW65S) (2) Specific rotation -0.16 ° (c 0.4, 1M NaCl) (3) Infrared absorption spectrum As shown in FIG. (4) Constituents ICC-001 Polysaccharide is hydrolyzed (hydrolysis condition: 5%
(V / V) sulfuric acid, 100 ° C., hour), the decomposed solution was concentrated, and analyzed and quantified by thin layer chromatography and gas chromatography carbazole sulfuric acid colorimetric method. As a result, it was found that D-glucose and D-galactose were the main constituents. And, each component is about 3.
It was 3: 1. Also, the polysaccharide solution is p-trifluoroacetic acid.
It was adjusted to H2 and heated at 100 ° C. for 2.5 hours for weak acid decomposition. After that, dialysis was performed, and the external solution was concentrated and separated by paper chromatography, and pyruvic acid was detected. Pyruvic acid was quantified using lactose dehydrogenase, and the amount of NADH consumed when pyruvic acid was reduced to lactic acid was measured by measuring the decrease in absorption at 340 nm. As a result, pyruvic acid contained 4% by weight. I found out that

(5) Binding mode In order to know the binding mode of the polysaccharide, methylation analysis by the Hakomori method was performed. Each methylated monosaccharide was alditol acetate, and the column of OV-1 capillary (0.25 mm
Gas chromatographic analysis (150 ℃ -230 ℃, 2 ℃)
/ Min). Each peak was identified by comparing the retention times of GC-MS and the standard sample. The results are shown in Table 1. Comparing the present polysaccharide with the depyruvated polysaccharide, the amount of sugar residues forming the straight chain portion was not significantly changed (Table 1 (1), (2), (3), (4),
(7)). On the other hand, the amount of dimethylgalactose (8) is decreasing. In addition, the terminal residue (6) of galactose is increased. From this, it is considered that the keto group at the 2-position of pyruvic acid is ketal-bonded to the 4- and 6-positions of galactose. On the other hand, since dimethyl glucose (5) is unchanged, it is considered that this forms the branch point of this polysaccharide, and the galactose residue to which pyruvic acid is bound is located at the end of the branch part correspondingly. Be done. In addition, depyruvic polysaccharide reacts with lectins that specifically bind to β-D-galactose (such as azuki bean lectin and castor bean lectin) to form a precipitation line. Therefore, galactose to which pyruvate is bound is β-linked. I understood.
In addition, by smith decomposition including mild hydrolyzation (0.1N acid, 25 ° C), glycerol, erythritol and other 2-O-
Since β-D-glycosylerythritol is produced, it is found that there is a sequence of → 3Glcl → 4Glcl → glucose residues in the main chain.

[0012]

[Table 1] Table 1 Mol ratio O-methylated sugar polysaccharide Depyruvic acid polysaccharide 2,3,4,6-Me ₄ -Glucose (1) 0.1 0.2 0.2 2,4,6-Me _3- Glucose (2) 2.1 2.0 2,4,6-Me ₃ -Glucose (3) 2.4 2.6 2,3,4-Me ₃ -Glucose (4) 0.9 0.9 2, 3-Me ₂ -glucose (5) 1.0 1.0 2,3,4,6-Me ₄ -galactose (6) 0.1 0.6 2,4,6-Me ₃ -galactose (7) 1 1 1.0 2,3-Me ₂ -galactose (8) 0.9 0.2

From the above results, the partial structure shown in the following equation was estimated. The present polysaccharide having such a partial structure can be obtained from Klebsiella and E. It has been found that this is a novel polysaccharide having a polysaccharide skeleton different from various pyruvic acid-containing polysaccharides produced by E. coli and the like.

Examples of the present invention will be shown below. Since the properties of the polysaccharide have been clarified by the present invention, various methods for producing the polysaccharide can be devised based on the properties. Therefore, the present invention is not limited to the examples, and the polysaccharides can be produced by applying known means based on the properties of the polysaccharides revealed by the present invention as well as the modifying means of the examples. It includes all methods of concentration, extraction, and purification.

[0015]

【Example】

Example 1 Beer infusion broth (Veal) as a seed medium
Infusion Broth, manufactured by Difco) 2.0
%, Glucose 0.1% was used as the medium.
Yeast extract 0.3%, meat extract 0.3 as production medium
%, Glucose 2.0%, agar 2.5% was used as the medium. The pH before sterilization was adjusted to pH 7.0 before use. The seed medium (40 ml) was dispensed 25
Sterilize a 0 ml Erlenmeyer flask at 120 ° C for 15 minutes.
Two to three platinum loops of a slope agar culture of CC-001 strain were inoculated and shake cultured at 30 ° C. for 24 hours for seed culture. Next, 500 ml of the above-mentioned production medium was dispensed and 10 ml of the above seed culture solution was inoculated into a large plate (35 cm × 35 cm) which was solidified to a thickness of about 0.6 cm, and static culture was carried out at 30 ° C. for 24 hours. After the culture was completed, the viscous material on the surface of the medium was scraped off and filtered to obtain a filtrate.

Example 2 Beer infusion broth (Vea) was used as a production medium.
l Infusion Broth, manufactured by Difco) 2.
A medium having a composition of 0% and glucose 0.1% was used. 700 ml of the production medium was dispensed into a 2 L Erlenmeyer flask, sterilized at 120 ° C. for 15 minutes, 35 ml of the seed culture solution prepared as in Example 1 was inoculated, and shake culture was performed at 30 ° C. for 24 hours to culture. After the completion, the culture solution was filtered to obtain a filtrate. Example 3 2 L of culture filtrate obtained by static culture or shaking culture
80 ml of 10% cetylpyridinium chloride was added to and a precipitate was obtained. The precipitate was separated by centrifugation, dissolved by adding 1 L of 2M saline solution, and then precipitated by adding 3 L of ethanol. The precipitate is collected by filtration and water 1L
It was then dissolved in the above, put into a cellulose tube, dialyzed in running water, and freeze-dried to obtain 6.6 g of ICC-001 polysaccharide.

[0017]

The polysaccharide of the present invention is a novel viscous polysaccharide and is expected to be used as a thickener / gelling agent.

[0018]

[Brief description of drawings]

FIG. 1 shows an infrared absorption spectrum of ICC-001 polysaccharide in KBr.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C12R 1:38) (72) Inventor Hidebe Takebe Meiji Confectionery Co., Ltd. 760 Shimooka-cho, Kohoku-ku, Yokohama Company Pharmaceutical Research Institute

Claims (2)

[Claims] 1. A highly viscous polysaccharide (ICC-001 polysaccharide) having the following physicochemical properties. (1) Molecular weight: about 700,000 (gel filtration method) (2) Specific rotation: -0.16 ° (c0.4 1M NaC
l) (3) Infrared absorption spectrum: Fig. 1 (4) Main constituent components are D-glucose, D-galactose and pyruvic acid, and the molar ratio of D-glucose and D-galactose is about 3.3: 1. 2. An IC that belongs to the genus Pseudomonas and is extracellular
The method for producing a polysaccharide according to claim 1, wherein a bacterium that produces C-001 polysaccharide is cultured, and ICC-001 polysaccharide is collected from the culture.