JPH0468918B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel method for producing a polysaccharide that forms a gel at room temperature. (Prior Art) Regarding polysaccharides produced by microorganisms, so far, there have been polysaccharides produced by bacteria belonging to the genus Alcaligenes, Xanthomonas, Arthrobacter, or Bacillus, yeasts of the genus Hansenula, and fungi such as the genus Burularia. Are known. However, among these polysaccharides, there are only a few that exhibit high viscosity and form gels at room temperature. (Problems to be Solved by the Invention) The present inventors collected a wide range of soil bacteria for the purpose of obtaining particularly highly viscous polysaccharides, and searched for their polysaccharide-producing ability. As a result, the present inventors found that a bacterium belonging to the genus Bacillus, tentatively designated as SP-1, accumulates a highly viscous polysaccharide-like substance in the culture. (Means for Solving the Problems) The present invention was completed based on the above findings, and the gist thereof is that the present invention belongs to the genus Bacillus and gels at room temperature, consisting of mannose, glucose, xylose, and glucuronic acid as its main constituents. This is a method for producing polysaccharides, which is characterized by culturing bacteria capable of producing polysaccharides that form polysaccharides in a medium to produce the polysaccharides, and collecting the polysaccharides. The present invention will be described in detail below. In the present invention, bacteria having the ability to produce a polysaccharide whose main components are mannose, glucose, xylose, and glucuronic acid and which form a gel at room temperature are cultured. An example of such a bacterium is SP-1 bacterium, which was collected from soil by the present inventors, and its mycological properties are as follows. <1> Morphological properties Microscopic observation Vegetative cell Form: Bacillus Animal: Yes Size: 0.8 to 1 x 2 to 5 μm Spores: Forms spores from the center to the edges. The sporangium swells up. Staining Gram staining: Positive Acid-fast staining: Negative Growth status in medium (1) Broth agar plate culture White, wrinkled, and lack luster. (2) Glucose agar plate culture It is white with a hint of yellow, glossy, and sticky. (3) Meat juice liquid culture A film forms on the top and becomes cloudy. (4) Glucose broth liquid culture Glucose grows better than meat juice and forms a film on the top. (5) Meat juice gelatin puncture culture Slow liquefaction. (6) Growth in litmus milk Coagulates and converts into peptone. Reducing litmus. <2> Physiological properties 1 Growth temperature: 15-45℃ 2 Optimum growth temperature: 30℃ 3 Growth PH: 5.5-9.5 4 Optimum growth PH: 7.0 5 Oxygen requirement: aerobic 6 Nitrate reducing ability: Yes 7 Desorption Nitrogen reaction: None 8 Methyl Red reaction: Positive 9 Indole generation: None 10 Hydrogen sulfide generation: None 11 Starch hydrolysis ability: Yes 12 Citric acid availability: Yes 13 Pigment formation: None 14 Urease generation: Yes 15 Oxidase production: Yes 16 Catalase production: Yes 17 O-F test: Oxidative fermentation <3> Fermentability of carbohydrates The results shown in Table 1 were obtained.

[Table] According to the above properties, Bergeys Manual of Determinative Bacteology
Bacteriology) 8th edition.
This bacterium was identified as Bacillus. Furthermore, considering that the sporangium swells sharply, the spores are oval, the spores are formed from the center to the edges, and the spores produce acid from glucose and do not produce gas, Bacillus circulans. circulans). However, when comparing its characteristics strictly, it differs from Bacillus circulans in that it has a positive Gram stain, produces acetoin, has a positive MR test, and produces urease. Therefore, this bacterium was recognized as a new bacterium belonging to the genus Bacillus, named Bacillus SP-1, and deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiological Research Institute No. 8017. In the present invention, the polysaccharide-producing ability belonging to the genus Bacillus is cultured in a medium. For this culture, a medium containing nutrients such as a carbon source, a nitrogen source, and various inorganic salts necessary for growth that can be assimilated by the microorganisms used is used. Specifically, carbon sources include starch, grapes, etc., and sucrose, and nitrogen sources include urea, meat extract, peptone, cornstarch liquor, yeast extract, ammonium sulfate, ammonium nitrate, and other organic or inorganic substances. Examples of inorganic substances include phosphates, sulfates, and carbonates of sodium chloride or magnesium, manganese, potassium, iron, calcium, and the like. Culture at pH 5.5-9.5 preferably 6.5-7.0, temperature 15
The culture is carried out at a temperature of 45°C to 45°C, preferably 25°C to 35°C, and usually only takes a few days. The culture thus obtained contains the polysaccharide targeted by the present invention. Therefore, it is necessary to extract and purify polysaccharides from the culture. When purifying the polysaccharide, it is preferable to collect the polysaccharide after removing bacterial cells and other solids from the culture in advance. Purification may be carried out by any means commonly used to recover polysaccharides from impurities, such as heating, centrifugation, washing, drying, fractional precipitation with solvents, and extraction, either singly or in appropriate combinations. . For example, a solvent such as acetone is added to the solution obtained by removing the solid content to precipitate the polysaccharide, the resulting polysaccharide is dissolved in water, and then acetone etc. The polysaccharide is precipitated using a solvent. After repeating this treatment, a purified polysaccharide can be obtained by dialysis and freeze-drying. The properties of the polysaccharide thus obtained are shown below. (1) Component Hydrolyzate of the polysaccharide of the present invention (hydrolysis conditions:
5% (V/V) sulfuric acid at 100°C for 12 hours) was concentrated and analyzed and quantified by thin layer chromatography, liquid chromatography, gas chromatography, and carbazole sulfuric acid colorimetric method. As a result, it was found that mannose, glucose, xylose, and glucuronic acid were the main components. The respective components are comprised in a molar ratio of 7:2:2:1. On the other hand, the hydrolyzate of this polysaccharide was
Colorimetric determination was performed using the Morgan method, but no hexosamine was detected. (2) General composition analysis (per dry matter) Crude protein (6.25×N): 2.6% Ash: 12.5%
Crude fat: 0.0% Carbohydrate: 84.9% (3) Molecular weight 750000 or more (measured by gel filtration method using Sephacryl C-300 (manufactured by Pharmacia Fine Chemical)) (4) Melting point No melting point is observed. (5) Infrared absorption spectrum As shown in Figure 1. (6) Solubility in solvents Soluble in water, insoluble in organic solvents such as methanol, ethanol, and acetone. (7) Color reaction Positive for Anthrone reaction and carbazole sulfate reaction, negative for Elson-Morgan reaction. (8) Distinction between basic, acidic, and neutral It is an acidic polysaccharide because it forms a precipitate with cetilitrimethylammonium bromide. (9) Color of substance White. (10) Viscosity It dissolves in water at room temperature, forming a highly viscous solution. The relationship between viscosity and concentration is shown in FIG. (11) Stability against acids and alkalis It exhibits maximum viscosity at pH 5 and relatively stable viscosity in the PH range of 6 to 10. (12) Stability against salts No decrease in viscosity is observed in the presence of divalent metal salts such as calcium, and the product exhibits high viscosity. In addition to the above characteristics, this polysaccharide also has the following characteristic properties. That is, it not only has the property of melting by heating and solidifying by cooling at a relatively low concentration (0.7% W/V), but also completely loses fluidity and becomes jelly-like just by contacting with water at room temperature. This polysaccharide having such characteristic properties can be used in a wide range of fields as a thickener, gelling agent, excipient, etc. It can also be used as a sizing agent, flocculant, etc., and can also be used in the agricultural field as a soil conditioner, fertilizer granulating agent, etc. Example: 100 ml of a medium with the following composition in a 500 ml Sakaguchi flask.
I put it in and sterilized it. (Medium composition) Glucose 5%, polypeptone 0.5%, yeast extract 0.5%, potassium phosphate 0.1%, magnesium sulfate (heptahydrate) 0.1%, calcium carbonate 0.75%,
PH7.0. Bacillus SP-1 (Feikoken Bacterial Serial No. 8017) was added to this medium.
No.) was inoculated and cultured with shaking at 30°C for 9 days. Add distilled water to 100 ml of culture solution, dilute 5 times, and incubate at 80℃.
After heating for 10 minutes, immediately centrifuge (14000G,
20 minutes) to remove other unnecessary components from the bacterial cells, and add twice the amount of acetone to the supernatant while stirring to precipitate the polysaccharide into fibers. After dissolving this crude polysaccharide in distilled water again and repeating the above acetone precipitation procedure twice, the polysaccharide solution was placed in a cellulose tube (molecular weight cut off: 10,000) and dialyzed against running water for 2 days and distilled water for 1 day. Afterwards, it was freeze-dried to obtain 4.4 g of cotton-like purified polysaccharide. This had the properties described above.

[Brief explanation of drawings]

FIG. 1 shows the infrared absorption spectrum pattern of the Bacillus SP-1 polysaccharide, and FIG. 2 is a graph showing the relationship between the concentration and viscosity of the polysaccharide.

Claims (1)

[Claims] 1. Cultivating in a medium a bacterium that belongs to the genus Bacillus and has the ability to produce a polysaccharide that forms a gel at room temperature and whose main components are mannose, glucose, xylose, and glucuronic acid to produce the polysaccharide,
A method for producing polysaccharides, which comprises collecting the polysaccharides.