JPH06136003A - Substance bs-2 and its production - Google Patents

Abstract

PURPOSE:To obtain a substance BS-2, readily soluble in water, capable of providing a non-Newtonian transparent solution and manifesting excellent dispersibility at a low concentration and useful as a food additive, etc., by culturing a microorganism belonging to the genus Klebsiella. CONSTITUTION:Polysaccharides are obtained by culturing a microorganism, belonging to the genus Klebsiella and capable of producing a substance BS-2 (e.g. Klebsiella.oxytoca) in a carbon source, a nitrogen source, etc., as a culture medium at 20-37 deg.C preferably at about a neutral pH, separating and collecting the substance BS-2 from the resultant culture. The polysaccharides have the following properties: (A) white to light brown powder; (B) readily soluble in water, sparingly soluble in methanol, ethyl acetate, chloroform and benzene and hydrolyzed with a mineral acid; (C) 1-100cP viscosity; (D) principal constituent saccharide components: 17.6-27.9% mannose, 37-56.9% galactose, 0-6.8% glucose and 22.6-35.8% glucuronic acid; (E) positive to the phenol sulfuric acid, carbazole sulfuric acid and the Molisch and ninhydrin reactions; (F) 1000-10000000 molecular weight, etc.

Description

Detailed Description of the Invention

[0001]

The present invention relates to the genus Klebsiella (Kleb Sierra).
related to a polysaccharide produced by a microorganism belonging to siella) and useful as a dispersant or a thickener, and a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, various polysaccharides produced by microorganisms are known. For example, xanthan gum produced by Xanthomonas campestris, dextran produced by Leuconostoc mesenteroides,
Well-known are pullulan produced by Aureobasidium pullulans and curdlan produced by Alcaligenes faecalis. As a polysaccharide produced by a microorganism belonging to the genus Klebsiella, the BS-1 substance produced by Klebsiella pneumoniae is known (Japanese Patent Publication No. 2-19121). These polysaccharides are widely used, for example, as food additives such as thickeners, dispersants, and quality improvers in the food field, as concrete fluidizers in the industrial field, and as substitute plasma agents in the pharmaceutical field.

[0003]

However, these polysaccharides have drawbacks such that it is difficult to achieve the desired performance unless they are used in a relatively high concentration and the long-term stability is poor. Therefore, in various fields including food, industrial materials, pharmaceutical fields, etc., it shows good dispersibility at low concentrations,
Moreover, the development of a substance having excellent stability is desired.

[0004]

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned current situation, and it is produced by culturing a specific microorganism, which is satisfactorily improved with a relatively low concentration of an added amount, BS, which is a polysaccharide substance showing long-term stable dispersibility
-2 It was made based on the discovery of substance. That is, the present invention relates to a BS-2 substance obtained by culturing a BS-2 substance-producing bacterium belonging to the genus Klebsiella and collecting it from the culture and a method for producing the same.

The present invention will be described in detail below. The BS-2 substance according to the present invention is BS-2 belonging to the genus Klebsiella.
A polysaccharide produced by a substance-producing bacterium, the BS-
Separated and collected from the culture of the two substance-producing bacteria.

The BS-2 substance is characterized by having the following properties. a. Appearance: White to light brown powder. b. Solubility: Soluble in water, sparingly soluble in methanol, ethyl acetate, chloroform and benzene, hydrolyzed by mineral acid. c. Viscosity: 1-100 centipoise (0.4% aqueous solution, temperature 30
℃, shear rate 79.6sec ^-1 ). d. Main constituent sugar components: mannose 17.6 to 27.9%, galactose 37.0 to 56.9%, glucose 0.0 to 6.8%, glucuronic acid 22.6 to 35.8%. e. Color reaction: phenol-sulfuric acid reaction: positive, carbazole-sulfuric acid reaction: positive, Maurish reaction: positive, ninhydrin reaction: positive. f. Molecular weight: 10 to 10 million. g. Elemental analysis value: C 30.0 to 40.0%, H 4.0 to 6.0%,
N 0.1-1.0% h. Infrared absorption spectrum (KBr tablet method, Hitachi 260-50
(Measured with mold) (cm ^-1 ): 3400,2930,2120,1610,1410,131
0,1210,1130,1070,890,820,790,730

The BS-2 substance according to the present invention can be produced by culturing a BS-2 substance-producing bacterium belonging to the genus Klebsiella and collecting the BS-2 substance from the culture. As a BS-2 substance-producing bacterium belonging to the genus Klebsiella, Klebsiella oxytoca (Klebsiella o
xytoca) can be illustrated. The Klebsiella oxytoca strain is stored as JCM1665 strain in the microbial strain preservation facility of RIKEN.

[0008] As is well known, Klebsiella oxytoca is easily irradiated by irradiation with ultraviolet rays, X-rays, radiation or the like or by mutation means such as a mutation source (eg, nitrosoguanidine, nitrogen mustard, acridine orange). Therefore, it should be understood that all the mutants thus obtained having the ability to produce the BS-2 substance are included in the Klebsiella oxytoca strain in the present invention. is there.

Next, a method for producing a BS-2 substance using a Klebsiella oxytoca strain will be described. The BS-2 substance is obtained by subjecting the above strain to aerobic culture such as shaking culture or aeration stirring culture in a solid medium or a liquid medium containing a carbon source, a nitrogen source and other nutrients necessary for growth. Produced. Carbon sources used in the medium include lactose, sucrose, maltose,
Examples thereof include sugars such as galactose, glucose and fructose, and glycerol. Of these, lactose is preferable. The concentration of these carbon sources in the medium is not particularly limited as long as the strain used can grow, but is usually 1 to 10% (w / v), preferably 3 to 5% (w / v).
Examples of the nitrogen source include organic substances such as polypeptides, yeast extracts, trypticase peptone, and inorganic substances such as nitrates and ammonium salts.
From the viewpoint of productivity of the S-2 substance, an organic nitrogen source is preferable. Further, in the medium, if necessary, phosphates such as monopotassium phosphate and dipotassium phosphate, trace metals such as iron, copper, magnesium, manganese, molybdenum, zinc and boron, and biotin, thiamine and vitamin B12. You may add vitamins and nucleic acids.

The culture temperature in the above medium is 20 to 37 ° C., preferably 25 to 32 ° C., which is the optimum temperature range for the growth of the microorganism used. The pH of the medium for culturing is not particularly limited,
3 to 11, preferably around neutral. When cultured under the above-mentioned culture conditions, the viscosity of the medium increases with time, and B
It can be seen that the S-2 substance is produced and accumulated in the medium. The culture may be performed until the viscosity of the medium reaches the maximum, but in practice, it is usually 3 to 7 days from the viewpoint of production efficiency.

After completion of the culture, the BS-2 substance in the medium is separated and collected according to a conventional method. For example, when a liquid medium is used, the separation / collection is carried out by diluting the culture medium with water after the culture, removing the bacterial cells by centrifugation or the like, and then removing the cells with methanol, ethanol or isopropanol. The BS-2 substance precipitates when an organic solvent such as acetone, a quaternary ammonium salt such as cetyltrimethylammonium salt, or an acid amide such as butyl amide is added. The precipitate is washed and then dried to obtain the BS-2 substance.

The BS-2 substance thus obtained has the following properties. a. Appearance: White to light brown powder. b. Solubility: Soluble in water, sparingly soluble in methanol, ethyl acetate, chloroform and benzene, hydrolyzed by mineral acid. c. Viscosity: 1-100 centipoise (0.4% aqueous solution, temperature 30
℃, shear rate 79.6sec ^-1 ). d. Main constituent sugar components: mannose 17.6 to 27.9%, galactose 37.0 to 56.9%, glucose 0.0 to 6.8%, glucuronic acid 22.6 to 35.8%. e. Color reaction: phenol-sulfuric acid reaction: positive, carbazole-sulfuric acid reaction: positive, Maurish reaction: positive, ninhydrin reaction: positive. f. Molecular weight: 10 to 10 million. g. Elemental analysis value: C 30.0 to 40.0%, H 4.0 to 6.0%,
N 0.1-1.0% h. Infrared absorption spectrum (KBr tablet method, Hitachi 260-50
(Measured with mold) (cm ^-1 ): 3400,2930,2120,1610,1410,131
0,1210,1130,1070,890,820,790,730

The BS-2 substance obtained as described above can be further purified if necessary to obtain a purified BS-2 substance. In the purification treatment, the above-mentioned BS-2 substance is redissolved in water, and an organic solvent such as methanol, ethanol, isopropanol or acetone, a quaternary ammonium salt such as cetyltrimethylammonium salt or an acid such as butyl amide is added thereto. By repeating the operation of adding amide and reprecipitating, further dialysis treatment or chromatography treatment is carried out for purification. By performing the purification treatment in this manner, a purified BS-2 substance in which the low molecular weight portion is separated can be obtained. Purified BS-
The two substances have the following properties.

A. Appearance: odorless white powder b. Solubility: Easily soluble in water, methanol, ethyl acetate, black
Poorly soluble in loform and benzene, hydrolyzed by mineral acid. c. Viscosity: 10-200 centipoise (0.4% aqueous solution, temperature 30
℃, shear rate 79.6sec^{ー 1} ). d. Main constituent sugar components: Mannose 18.0-27.0%, Garaku
Tose 40.0-50.0%, Glucose 0.0-6.0%, Glucuro
Acid 23.0-35.0%. e. Color reaction: Phenol-sulfuric acid reaction: Positive, carbazo
Sulfuric acid reaction: positive, Mauritian reaction: positive, ninhi
Drin reaction: Negative. f. Molecular weight: 10,000-10 million. g. Elemental analysis value: C 31.0 to 40.0%, H 4.0 to 6.0%,
N 0.0-0.2% h. Infrared absorption spectrum (KBr tablet method, Hitachi 260-50
Measured with mold) (cm^{ー 1}): 3400,2930,2120,1610,1410,131
0,1210,1130,1070,890,820,790,730

The present invention will be specifically described below with reference to examples.

Example 1 (1) Production of BS-2 substance Klebsiella oxytoca JCM
1665 lactose 3 wt%, tryptic case peptone 0.35
wt%, monopotassium phosphate 0.6 wt%, magnesium sulfate 0.0
Those grown on a slant medium consisting of 7 wt% and agar 1.4 wt% were scraped with platinum loops, inoculated into 10 ml of liquid medium excluding the agar in the above medium, and precultured by shaking culture at 28 ° C for 1 day. . Then, a liquid medium of the same composition as used in the above pre-culture was heat-sterilized at 120 ° C for 15 minutes
100 ml of the above-mentioned precultured whole was aseptically inoculated and shake culture (180 rpm) was carried out at 28 ° C. for 1 day. As the culture progressed, the culture became viscous, and production and accumulation of the BS-2 substance was observed.

The resulting culture was centrifuged at 18000 rpm for 15 minutes to remove the cells, and then twice the amount of ethanol was added to precipitate a light brown solid (BS-2 substance). This light brown solid (BS-2 substance) was collected by centrifugation, washed repeatedly with ethanol, redissolved in distilled water and freeze-dried to obtain 1.45 g of a dried product. As a result of examining the physicochemical properties of the obtained dried product, it was confirmed to be BS-2 substance as shown below.

A. Appearance: White to light brown powder. b. Solubility: Soluble in water, sparingly soluble in methanol, ethyl acetate, chloroform and benzene, hydrolyzed by mineral acid. c. Viscosity: 6.4-80.0 centipoise (0.4% aqueous solution, temperature
30 ℃, shear rate 79.6sec ^-1 ). d. Main constituent sugar components: mannose 24.2%, galactose
42.9%, glucose 0.6%, glucuronic acid 32.3%. e. Color reaction: phenol-sulfuric acid reaction: positive, carbazole-sulfuric acid reaction: positive, Maurish reaction: positive, ninhydrin reaction: negative or positive. f. Molecular weight: 1-10 million. g. Elemental analysis values: C 36.7%, H 5.2%, N 0.4% h. Infrared absorption spectrum (KBr tablet method, Hitachi 260-50
(Measured with mold) (cm ^-1 ): 3400,2930,2120,1610,1410,131
0,1210,1130,1070,890,820,790,730

(2) Purification of BS-2 substance 1.45 g of the BS-2 substance obtained as described above was dissolved in distilled water and centrifuged. The operation of adding ethanol to this solution to reprecipitate a white solid (BS-2 substance) was repeated twice, and then the precipitate was dissolved in distilled water. This aqueous solution is put into a dialysis membrane, dialyzed against pure water for 24 hours, and then freeze-dried to give a white lump-like purified product.
0.9 g was obtained. The physicochemical properties of the obtained purified product were as shown below and were confirmed to be purified BS-2 substances.

A. Appearance: odorless white powder b. Solubility: Easily soluble in water, methanol, ethyl acetate, black
Poorly soluble in loform and benzene, hydrolyzed by mineral acid. c. Viscosity: 10-200 centipoise (0.4% aqueous solution, temperature 30
℃, shear rate 79.6sec^{ー 1} ). d. Main constituent sugar components: mannose 19.5%, galactose
54.1% glucose, 0.7% glucose, 25.7% glucuronic acid. e. Color reaction: Phenol-sulfuric acid reaction: Positive, carbazo
Sulfuric acid reaction: positive, Mauritian reaction: positive, ninhi
Drin reaction: Negative. f. Molecular weight: 2-10 million. g. Elemental analysis values: C 38.1%, H 5.2%, N 0.1% h. Infrared absorption spectrum (KBr tablet method, Hitachi 260-50
Measured with mold) (cm^{ー 1}): 3400,2930,2120,1610,1410,131
0,1210,1130,1070,890,820,790,730

[0020]

Example 2 The following experiment was conducted in order to examine the properties of the BS-2 substance according to the present invention as a dispersant. 20 parts of a 0.2% aqueous solution of the BS-2 substance of the present invention in 100 parts by weight of slaked lime powder
0 parts by weight was added, and the mixture was thoroughly stirred and homogenized with a homogenizer, and then the viscosity at 30 ° C. was measured by changing the shear rate. The results are shown in Table 1.

For comparison, the change in viscosity was also measured when a 0.2% aqueous solution of a known dispersant was used in place of the BS-2 substance. As is clear from Table 1, when the BS-2 substance was used, the viscosity was the lowest as compared with other dispersants at all shear rates. This indicates that the BS-2 substance is an excellent dispersant.

[0022]

[Table 1]

[0023]

INDUSTRIAL APPLICABILITY The BS-2 substance according to the present invention and the purified BS-2 substance obtained by further purifying it are easily dissolved in water to form a non-Newtonian transparent solution, which is excellent in various concentrations at low concentrations. It has the property of imparting excellent dispersibility. Therefore, it is useful as a food additive, a formulation aid, or an industrial material. Further, according to the present invention, this BS-2 substance can be easily produced by culturing a microorganism.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display area A61K 9/08 B 7329-4C B01F 17/56 (C12P 19/04 C12R 1:22) 7804-4B

Claims (4)

[Claims] 1. A BS-2 substance which is a polysaccharide produced by a microorganism belonging to the genus Klebsiella and has the following properties: a. Appearance: White to light brown powder. b. Solubility: Soluble in water, sparingly soluble in methanol, ethyl acetate, chloroform and benzene, hydrolyzed by mineral acid. c. Viscosity: 1-100 centipoise (0.4% aqueous solution, temperature 30
℃, shear rate 79.6sec ^-1 ). d. Main constituent sugar components: mannose 17.6 to 27.9%, galactose 37.0 to 56.9%, glucose 0.0 to 6.8%, glucuronic acid 22.6 to 35.8%. e. Color reaction: phenol-sulfuric acid reaction: positive, carbazole-sulfuric acid reaction: positive, Maurish reaction: positive, ninhydrin reaction: positive. f. Molecular weight: 10 to 10 million. g. Elemental analysis value: C 30.0 to 40.0%, H 4.0 to 6.0%,
N 0.1-1.0% h. Infrared absorption spectrum (KBr tablet method, Hitachi 260-50
(Measured with mold) (cm ^-1 ): 3400,2930,2120,1610,1410,131
0,1210,1130,1070,890,820,790,730 2. The method according to claim 1, which has the following properties:
The listed BS-2 substances; a. Appearance: odorless white powder b. Solubility: Easily soluble in water, methanol, ethyl acetate, black
Poorly soluble in loform and benzene, hydrolyzed by mineral acid. c. Viscosity: 10-200 centipoise (0.4% aqueous solution, temperature 30
℃, shear rate 79.6sec^{ー 1} ). d. Main constituent sugar components: Mannose 18.0-27.0%, Garaku
Tose 40.0-50.0%, Glucose 0.0-6.0%, Glucuro
Acid 23.0-35.0%. e. Color reaction: Phenol-sulfuric acid reaction: Positive, carbazo
Sulfuric acid reaction: positive, Mauritian reaction: positive, ninhi
Drin reaction: Negative. f. Molecular weight: 10,000-10 million. g. Elemental analysis value: C 31.0 to 40.0%, H 4.0 to 6.0%,
N 0.0-0.2% h. Infrared absorption spectrum (KBr tablet method, Hitachi 260-50
Measured with mold) (cm^{ー 1}): 3400,2930,2120,1610,1410,131
0,1210,1130,1070,890,820,790,730 3. The BS-2 substance according to claim 1, wherein the microorganism belonging to the genus Klebsiella is Klebsiella oxytoca JCM 1665. 4. A method for producing a BS-2 substance, which comprises culturing a BS-2 substance-producing bacterium belonging to the genus Klebsiella and collecting the BS-2 substance from the culture.