JPH0793878B2 - Polysaccharide A-1845, method for producing the same and microorganism producing the same - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel polysaccharide A-1845 which can be widely used in the fields of foods, pharmaceuticals, cosmetics, etc., a method for producing the same and a microorganism producing the same.

[0002]

BACKGROUND OF THE INVENTION Polysaccharides, especially acidic heteropolysaccharides, are widely used as thickeners, emulsion stabilizers, gelling agents and the like in the fields of foods, detergents, pharmaceuticals, cosmetics and the like. Among them, various products obtained by culturing microorganisms have been found (Carbohydrate Res
earch, 45 , p275 to 282 (1975)) However, since the physicochemical properties of acidic heteropolysaccharides differ depending on the polysaccharide, there are various fields in which they are used and various purposes of their use. It is desirable to find a wide variety of polysaccharides with properties.

[0003]

Means for Solving the Problems In view of the above situation, the present inventor has conducted diligent research to find out a novel acidic heteropolysaccharide, and as a result, actinomycetes isolated from the soil of Itsukaichi City, Tokyo have been found to act on amylase. The present invention has been completed by finding out that a novel acidic heteropolysaccharide that does not undergo the treatment is produced.

That is, the present invention provides a novel polysaccharide A-1845, a method for producing the same and a microorganism producing the same.

The polysaccharide A-1845 of the present invention is produced, for example, by culturing a polysaccharide A-1845-producing bacterium belonging to the genus Streptomyces and collecting it from the culture. A typical example of the polysaccharide A-1845-producing bacterium is the A-1845 strain. The mycological properties of this strain are as follows.

(1) Morphological properties Sporulating hyphae are formed on aerial hyphae and branched. 2 spores
Zero or more are linked in a spiral (2 to 3 rolls). The spore size is (0.7-0.9 μm × 0.9-1.3 μm) and the surface is smooth. No spores and flagellates are observed.

(2) Growth state in various media (1) Sucrose / nitrate agar medium A rich colony of powdery aerial hyphae of yellowish ash is formed and a raised colony is formed. The basal hyphae show pale yellow tea and do not produce soluble pigments. (2) Glucose / asparagine agar medium Aerial hyphae do not settle, but swelling colonies are formed. The basal hyphae show pale yellow tea and do not produce soluble pigments. (3) Glycerin / asparagine agar medium White powdery aerial mycelium is slightly settled to form a raised colony. The basal hyphae show pale yellow tea and do not produce soluble pigments. (4) Starch agar medium An abundant powdery aerial mycelium of light brown ash is settled to form a raised colony. The basal hyphae show pale yellow tea and do not produce soluble pigments. (5) Tyrosine agar medium An abundant powdery aerial mycelium of light brown ash is settled to form a raised colony. Basal hyphae show dark brown color and do not produce soluble pigment. (6) Nutrient agar medium White powdery aerial mycelia are slightly settled to form flat colonies. The basal hyphae show pale yellow tea and do not produce soluble pigments. (7) Yeast / malt agar medium A powdery aerial mycelium of light brown ash is thickly settled to form a raised colony. Basal hyphae show dark brown color and do not produce soluble pigment. (8) Oatmeal agar medium A rich colony of powdered aerial mycelium of light brown ash is formed and flat colonies are formed. The basal hyphae show pale yellow tea and do not produce soluble pigments. (9) Bennett's agar medium The powdery aerial mycelium of light brown ash is abundantly settled to form a raised colony. The basal hyphae show pale yellow tea and do not produce soluble pigments. (10) Calcium malate agar medium White powdery aerial hyphae are abundantly formed and swelling colonies are formed. The basal hyphae show pale yellow tea and do not produce soluble pigments.

(3) Physiological properties (1) Formation of melanin-like pigment: Negative (2) Starch hydrolysis: Positive (3) Nitrate reducibility: Negative (4) Gelatin liquefaction: Negative (5) Malic acid Use of calcium: Positive (6) Salt tolerance: Grows at 4%. (7) Growth pH: 5.0 to 9.0 (8) Growth temperature: 15 to 34 ° C. (9) Utilization of carbon source (28 ° C. cultivation on Pridham-Gotrieve agar medium)

[Table 1]

From the above mycological properties, the A-1845 strain belongs to the genus Streptomyces, and since the same strain does not exist even when compared with known strains, it was judged to be a new strain and industrial technology was adopted. Institute of Microbial Science and Technology, Institute of Microbiology
Deposited as No. 043 (FERM P-12043). A-18 of the present invention
The 45 strains easily mutate naturally or by ultraviolet rays, X-rays, chemical agents and the like. Therefore, these mutants can also be used for the production of the polysaccharide of the present invention.

Using the strain of the present invention, the polysaccharide A-18 of the present invention
To obtain 45, the strain is inoculated into a medium, cultured according to a conventional method, and the polysaccharide A-1845 is collected from this culture. It is preferable that an appropriate amount of assimilable carbon source, nitrogen source and other nutrient sources are contained in this medium. These are not particularly limited, but specifically, the carbon source includes glucose, sucrose, starch, glycerin and the like, and the nitrogen source includes yeast extract, meat extract, peptone, soybean powder and other organic nitrogen sources. Other examples include inorganic nitrogen sources such as ammonium sulfate and sodium nitrate, and other additives include, if necessary, sulfates, nitrates and chlorides of metals such as sodium, potassium, magnesium, calcium, zinc and iron. , Carbonates, phosphates and the like; organic acid salts such as sodium citrate and the like.

The culture is preferably carried out under aerobic conditions, and any of stationary culture, shaking, and aeration-agitation culture are possible, but shaking or aeration-agitation culture is advantageous. The culture temperature is preferably about 15 to 34 ° C, particularly about 20 to 30 ° C. The pH of the medium is preferably about 5.0 to 9.0,
Particularly, about 6.0 to 8.0 is preferable. The culture period varies depending on the culture conditions such as the composition of the medium and the temperature, but it is usually about 2-1.
It takes about 4 days, preferably about 4 to 6 days, and the culture is terminated at an appropriate time in consideration of the time when the above-mentioned production amount reaches the maximum.

In order to isolate and purify the polysaccharide A-1845 accumulated in the culture thus cultivated, a commonly used isolation / purification means may be applied. For example, the culture solution is centrifuged or filtered to remove the bacterial cells, and then an organic solvent such as ethanol, methanol, propanol, or acetone is added to precipitate the desired crude product of the polysaccharide of the present invention. Purification of this crude product is preferably carried out, for example, as follows. That is, first, the culture filtrate is ultrafiltered and then desalted and concentrated. Then trichloroacetic acid 10% (v / v)
To be added. After removing the precipitate, 4 times the amount of ethanol was added, the precipitated polysaccharides were collected, dissolved in pure water, added again with ethanol, dissolved in water, gel-filtered several times, and the obtained fraction was dialyzed. Subsequent freeze-drying yields a cotton-like purified polysaccharide A-1845.

The polysaccharide A-1845 of the present invention thus obtained is
It has the following properties. (1) Color: White (2) Constituents: Hexose 47.4% Anthuron-sulfuric acid method (Morris method). Uronic acid 9.9% By carbazole sulfate method (Bitter & Muir method). Pentose 4.6% Cysteine Sulfate method (Dische method). Methyl pentose 5.0% Cysteine sulfate method (Dische & Shettles method). Amino sugar 8.4% According to Elson-Morgan method (Blix method). Phosphorus 2.6% By a modification of the method of Burton et al. (3) Constituent sugar: Mannose, galactose, galacturonic acid, xylose, rhamnose, glucose, fucose, and ribose are composed in a ratio of 7.6: 4.0: 3.4: 3.1: 1.9: 1.7: 1.1: 1.0. (4) Elemental analysis: C 36.5%, H 5.5%, N 4.0% (5) Melting point: No clear melting point is shown. (6) Infrared absorption spectrum: Fig. 1 (7) Ultraviolet absorption spectrum: Fig. 2 (8) Solubility: insoluble in general organic solvents, soluble in water (9) Color reaction: anthrone reaction positive ninhydrin reaction negative (10 ) Basic, acidic, and neutral: It seems to be acidic because it adsorbs on DEAE Toyopearl 650W (Cl ^- type), which is an anion exchanger. (11) Action of amylase: Not affected. (12) Specific rotation: [α] ²⁴ _D ＋ 57.0 ° (c, 0.1, in water)

[0014]

The polysaccharide A-1845 of the present invention is used for foods, pharmaceuticals,
Although it can be applied to many fields such as cosmetics, it can be used as a low-calorie dietary fiber in foods because it is not affected by amylase.

[0015]

The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1 Glucose 1.0%, corn starch 3.0%, peptone 0.
80 ml of a medium containing 5%, soybean flour 1.0%, yeast extract 0.5% and calcium carbonate 0.2% was dispensed into a 500 ml Sakaguchi flask, sterilized by a conventional method, and then grown on a starch-casein medium A-1845. The cells of the strain were scraped and inoculated, and shake culture was carried out at 28 ° C. for 3 days on a reciprocal shaker. 60 ml of this culture solution was added to 3000 ml of the same sterilized medium, and the mixture was cultivated in a 5 l volume jar-famenter at 300 rpm and an aeration rate of 2 l per minute at 28 ° C. for 6 days. Approximately 3000 ml of the obtained culture broth was centrifuged and filtered through Toyo Filter Paper No. 2 to remove bacterial cells, and 2200 ml of the filtrate was subjected to ultrafiltration (molecular weight limit of 10,000) for desalting and concentration. 625 ml of this concentrate
90 ml of 80% trichloroacetic acid was added to and the resulting precipitate was removed by centrifugation. Ethanol 2600 in 650 ml of supernatant
ml was added, the resulting precipitate was dissolved in 200 ml of reverse osmosis water, and 800 ml of ethanol was added again to obtain a precipitate in the same manner. This operation was performed again, and the obtained precipitate was dialyzed in running water and reverse osmosis water. Ethanol (800 ml) was added to this internal solution, and the resulting precipitate was dried under reduced pressure, dissolved in ion-exchanged water (50 ml) and freeze-dried to obtain a crude polysaccharide product (1.57 g).

Next, 300 mg of the crude product was added to 20 mM sodium phosphate buffer (pH: 0.02% sodium azide).
7.0) Dissolved in 60 ml, the same buffer was used, and gel filtration was performed using a Toyopearl HW-75F column (2.6 × 97 cm). Gel filtration has a flow rate of 200 ml / hr and an aliquot of 5 ml / tube.
And detection was by absorbance at 280 nm. The sample amount was 10 ml (50 mg) once, and a total of 6 times. After concentrating the obtained fraction (330 ml) under reduced pressure,
It was made up to 60 ml with ion-exchanged water and again subjected to gel filtration under the same conditions. Further, the obtained fraction (210 ml) was concentrated in the same manner as above and then subjected to Toyopearl HW-65S column (2.6 × 97).
cm) gel filtration twice (conditions are Toyopearl HW
Same as with -75F column). The obtained fraction (50 ml) was dialyzed in ion-exchanged water, concentrated under reduced pressure to 5 ml, and freeze-dried to obtain polysaccharide A-1845 (76.5 mg). The infrared absorption spectrum and the ultraviolet absorption spectrum of the polysaccharide obtained in this example are shown in FIGS. 1 and 2.

[Brief description of drawings]

FIG. 1 is a diagram showing an infrared absorption spectrum of the polysaccharide of the present invention.

FIG. 2 is a diagram showing an ultraviolet absorption spectrum of the polysaccharide of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12R 1: 465) (C12P 19/26 C12R 1: 465)

Claims (3)

[Claims] 1. A polysaccharide A-18 having the following physicochemical properties:
45. (1) Color White (2) Constituents Hexose, uronic acid, pentose, methyl pentose, amino sugar and phosphorus (3) Constituent sugar Mannose, galactose, galacturonic acid, xylose, rhamnose, glucose, fucose and ribose (4) Melting point No clear melting point (5) Infrared absorption spectrum Figure 1 (6) Ultraviolet absorption spectrum Figure 2 (7) Solubility Water-soluble, insoluble in methanol, ethanol, propanol, and acetone (8) Color reaction Anthrone reaction Positive Ninhydrin reaction Negative (9) Distinction between basic, acidic and neutral Acid (10) Not affected by amylase (11) Specific rotation [α] ²⁴ _D + 57.0 ° (c, 0.1, H ₂ O) ) 2. A polysaccharide A-1 belonging to the genus Streptomyces
845-producing bacteria were cultured, and the polysaccharide A-184 was collected from the culture.
5. Polysaccharide A- according to claim 1, characterized in that 5 is collected.
1845 manufacturing method. 3. Streptomyces sp. A-1 having the ability to produce the polysaccharide A-1845 of claim 1.
845.