JPS61239897A - Production of polysaccharide - Google Patents

Abstract

PURPOSE:To produce a polysaccharide which is dissolved under heating at low concentration, forms gel at normal temperature and usable in a wide range, obtained by cultivating a specific bacterium belonging to the genus Bacillus in a given medium. CONSTITUTION:A bacterium (e.g., Bacillus SP-1) belonging to the genus Bacillus separated from soil is inoculated into a medium consisting of a carbon source (e.g., starch), a nitrogen source (e.g., urea), an inorganic substance (e.g., NaCl), etc., and cultivated at 5.5-0.5pH at 15-45 deg.C for several days to form a polysaccharide having main constituent components consisting of mannose, glucose, xylose and glucuronic acid. Then, a mold and other solid contents in the culture mixture are removed and a polysaccharide is separated and purified.

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) Polysaccharides produced by microorganisms have so far been produced by alkaline earth metals, bacteria belonging to the genus Xanthomonas, Arthrobacterium, or Bacillus, yeasts such as Hansenula, and molds such as Burularia. something is 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 5P-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. Such bacteria include 5P-, which was collected from soil by the present inventors.
There is one bacterium, and its mycological properties are as follows.

1> Morphological properties, microscopic observation Vegetative cell morphology: Bacillus Motility: Yes Size: 0.8-I x 2-5 μB Spores: Spores are formed from the center to slightly toward the edges.

The spore ring is clearly swollen.

■、Dyeing Durham staining: positive Acid-fast staining: negative ■. Growth status in culture medium (1) Meat juice agar plate culture It is white, wrinkled, and lacks luster.

(2 glucose agar plate culture It is yellowish white, shiny, and sticky.

(3) Both juice liquid culture A film forms on the top and becomes cloudy.

4) Glucose broth liquid culture It grows better than meat juice and forms a film on the top.

[5] Meat juice gelatin puncture culture Slow liquefaction.

(6) Growth in lidmus milk: Coagulates and becomes peptonized. Return Ridmus.

2〉Physiological properties 1. Growth temperature = 15-45℃ 2゜Optimal temperature for growth = 30℃ 3. Growth PH: 5.5-9.5 4. Optimum growth pH near, 0 5. Oxygen requirement: aerobic 6. Nitrate reducing property: Yes 7. Denitrification reaction: None 8. Methyl red reaction double positive 9. Indole generation: None 10. Generation of hydrogen sulfide: None 11゜Starch hydrolysis ability: Yes 12. Usability of citric acid: Yes 13. Pigment formation: None 14. Urease generation: Yes 15. Oxidase production: Yes 16. Catalase production: Yes 17.0-F test Dioxide fermentation <3> Fermentability of carbohydrates The results shown in Table 1 were obtained.

(hereinafter referred to as the margin) Table 1 (hereinafter referred to as the margin) Based on the above characteristics, the Burgess Manual of
Determinative batateology
s M annual of D etermin
A search using tive8 acterioloOV > 8th edition revealed that this bacterium belongs to the genus Bacillus (3acill).
us). Furthermore, considering that the sporangia are clearly swollen, 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. ) close to. However, if we strictly compare their characteristics, Bacillus
It differs from Circulance in that it has a positive Durham stain, produces acetoin, has a positive MR test, and produces urease. Therefore, this bacterium was recognized as a new bacterial species belonging to the genus Bacillus, named Bacillus 5P-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, such polysaccharide-producing bacteria belonging to the genus Bacillus are cultured in a medium. In 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. in particular,
Carbon sources include starch, grapes, sucrose, etc.1
As nitrogen sources, urea, meat extract, peptone, cornstarch liquor, yeast extract, ammonium sulfate, ammonium nitrate, and other organic or inorganic substances are used, and inorganic substances include sodium chloride or magnesium, manganese, potassium, iron, calcium, etc. phosphate,
Examples include sulfates and carbonates.

Culture 1 &t PH 5, 5-9.5 Favorable L, <ha 5-7.0, temperature 15°C-45°C, preferably 25°C
Cultivation is carried out at ~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. In purifying the polysaccharide, it is preferable to collect the polysaccharide after removing bacterial cells and other solids in 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 a solvent such as acetone is added to the solution obtained by removing the solid content. 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) Constituent 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, when the hydrolyzate of this polysaccharide was colorimetrically determined by the Erlan-Morgan method, no hexosamine was detected.

(2J, - Membrane composition analysis (per dry matter) Crude protein (a25xN): 26% Ash content: 125%
Crude fat: 0.0% Carbohydrate: 84.9% (3) 0 Molecular weight 750.000 or more (5ephacryl G-3
(Measured by gel filtration method using 00 (7 Lumacia Fine Chemicals)) (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) Positive for color reaction Anthrone reaction and carbazole sulfate reaction, negative for Elson-Morgan reaction.

(8) It is an acidic polysaccharide because it forms a precipitate with cetilitrimethylammonium bromide.

(9), color of matter White.

ω), Viscosity It dissolves in water at room temperature, forming a highly viscous solution. The relationship between viscosity and concentration is shown in FIG.

al), stability against acids and alkalis It exhibits the highest viscosity at pH 5 and relatively stable viscosity in the pH range of 6 to 10.

(121, Stability against salts: No decrease in viscosity is observed in the presence of divalent metal salts such as calcium, indicating 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 50C) 100 ml of medium with the following composition in a Sakaro flask.
g+I and sterilized.

(Medium composition) Glucose 5%, polypeptone 0.5%, yeast extract 0
．． 5%, potassium phosphate 0.1%, magnesilla sulfate (
Heptahydrate) 081%, calcium carbonate 0875%, P H
7.0° This medium was inoculated with Bacillus 5P-1 (Feikoken Bacteria No. 8017) and cultured with shaking at 30°C for 9 days. Culture solution 10
01I was diluted 5 times with distilled water, heated at 80°C for 10 minutes, and then immediately centrifuged (14,0OOG, 20
) to remove bacterial cells and other unnecessary components, and add twice the amount of acetone to the supernatant while stirring to precipitate the polysaccharide in the form of fibers. After dissolving this crude polysaccharide in distilled water again and repeating the above acetone precipitation treatment twice, the polysaccharide solution was put into a cellulose tube (molecular weight cut off: 10,000) and soaked with running water for 20 minutes.
After dialysis against distilled water for one day, the product was freeze-dried to obtain 4.4 g of cotton-like purified polysaccharide. It had the properties described above.

[Brief explanation of drawings]

FIG. 1 shows the infrared absorption spectrum pattern of the Bacillus 5p-i fungal saccharide, and FIG. 2 is a graph showing the relationship between the concentration and viscosity of the present polysaccharide.

Claims (1)

[Claims] Cultivating bacteria that belong to the genus Bacillus and have the ability to produce polysaccharides that form a gel at room temperature and whose main components are mannose, glucose, xylose, and glucuronic acid in a medium to produce the polysaccharides, and then harvesting the polysaccharides. A method for producing polysaccharides characterized by: