JP2870628B2 - New microorganism-produced polysaccharide and its use - 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 KF-3 produced by a novel microorganism, and a flocculant, a water absorbing agent, a thickener and a flocculation method using the same. The polysaccharide KF-3 of the present invention can be widely used as a biodegradable flocculant or the like for improving the environment.

[0002]

2. Description of the Related Art As a flocculant, a polymer flocculant (eg, polyacrylamide), an inorganic flocculant (eg, a sulfate band), a microorganism-produced flocculant and the like are generally known. These are widely used in today's society in actual wastewater and sewage treatment plants in various industries, and their usage is expected to increase with their use in the future. However, the polymer flocculants and inorganic flocculants currently used are excellent in terms of economy, etc., but when released into the environment after treatment, problems in terms of pollution and safety have been pointed out. I have. Therefore, a microorganism-producing coagulant that can solve the problem has biodegradability due to the production of microorganisms, and is safe and has no danger of secondary pollution. Is expected.

On the other hand, water absorbents are generally used as sanitary products, disposable diapers, water retentive agents used in simple toilets and seedlings, and as moisturizers that have recently attracted attention in cosmetics and the like. The former is released into the environment as waste after use, and the latter is in contact with the human body, so that a safe and biodegradable water-absorbing agent is friendly to the environment and the human body, so its development is expected.

As a thickening agent, guar gum or xanthan gum produced by a bacterium belonging to the genus Xanthomonas, which is a kind of plant pathogen, is frequently used for food. Furthermore,
Thickeners are used in a wide variety of applications, such as those used in petroleum drilling, etc., and with the expansion of their uses, development of various types of thickeners with various properties, especially the environmentally friendly biodegradable type The development of adhesives is expected.

[0005]

Under these circumstances, the present invention provides a novel polysaccharide produced by a microorganism and has excellent biodegradability, is safe in a natural environment, and is free from secondary pollution. It is an object of the present invention to provide a coagulant, a water-absorbing agent, and a thickener that are free from fear. In order to make such an object possible, various types of microorganisms were collected from soils in the Okinawa region and the like, and the agglutinating ability of each microorganism culture solution was measured using the agglutinating activity measurement method described in the present invention. The bacteria provided in the invention were selected and obtained.

[0006]

The present invention has the following construction. 1. Polysaccharide KF-3 having the following physicochemical properties. (1) Color reaction: Ninhydrin reaction-Xanthoprotein reaction-Phenol sulfate method + Anthrone sulfate method + Carbazole sulfate method + Elson-Morgan reaction-(2) Color of substance: white (3) Solubility in solvent: distillation Poorly soluble in water Soluble in alkali Insoluble in organic solvents such as methanol, ethanol, acetone, ether, chloroform and hexane. (4) Ultraviolet absorption spectrum: Absorbance gradually increases from 350 nm and sharply increases from around 240 nm. (5) Infrared absorption spectrum: 3550cm ^-1 , 2950cm ^-1 , 1730
cm ^-1, absorption pattern is seen in 1650 cm ^-1 and 800~1200cm ^-1. 2. A method for producing a polysaccharide KF-3 as described above, comprising culturing a microorganism belonging to the genus Enterobacter and having a polysaccharide KF-3 producing ability, and collecting the polysaccharide KF-3 from the culture. 3. The method for producing a polysaccharide KF-3 as described above, wherein the microorganism belonging to the genus Enterobacter is Enterobacter agglomerans. 4. The polysaccharide according to the above, wherein the Enterobacter agglomerans is Enterobacter agglomerans KF-3 strain.
Manufacturing method of KF-3. 5. A flocculant containing polysaccharide KF-3 as a main component. 6. A method for aggregating suspended substances using polysaccharide KF-3. 7. Water absorbing agent containing polysaccharide KF-3 as a main component. 8. A thickener containing the polysaccharide KF-3 as a main component.

[0007] The strain used in the present invention belongs to the genus Enterobacter and may be any microorganism having a polysaccharide KF-3 producing ability. A representative strain thereof is Enterobacter agglomerans KF-3. Can be This strain has the following mycological properties. 1) Gram stain: Negative 2) Morphology: Bacillus 3) Size: width 0.8-1.0, length 1.8-2.2μm 4) Motility: positive 5) Aerobic undergrowth: positive 6) Anaerobic undergrowth: positive 7 ) Catalase: positive 8) Oxidase: negative 9) OF test: F (fermentation) 10) Arginine hydrolysis: negative 11) PPA test: negative 12) Reduction of nitrate: positive 13) Deamination of phenylalanine: negative 14) ONPG test: positive 15) VP reaction test: positive 16) indole production of: negative 17) H ₂ S production of: negative 18) utilization of citric acid: positive 19) urease production: negative 20) DN Ace production: negative 21) glucose 22) IPA test: negative 23) Lysine decarboxylation: negative 24) Ornithine decarboxylation: negative 25) Acylamidase production: negative 26) Gelatin liquefaction: positive 27) Yellow pigment formation: positive 28) Malonic acid Use: Positive 29) Monosaccharide Use (production of acid): D-glucose, D-arabinose, L-arabinose, D-arabitol, D-xylose, D-ribose, D-galactose, L-rhamnose, D-mannose, D-fructose, D-mannitol Utilizes glycerol and inositol to produce acids. It does not produce acid for darcitol, D-adnitol, L-sorbose, D-sorbitol, xylitol, meso-erythitol. 30) Use of oligosaccharides and polysaccharides (acid generation): Use maltose, D-cellobiose, melibiose, trehalose, sucrose, and salicin to generate acids. Lactose,
It does not generate acid for α-methyl-D-glucoside, meriditose, raffinose, inulin and glycogen. 31) Escalin hydrolysis: positive 32) Growth at 44 ° C: negative 30) Decomposition of Tween80: negative 34) Decomposition of Tween60: negative 35) Decomposition of Tween40: negative 36) GC content (mol%) in DNA: Barge Manual Systematic Tech Bacteriology, a bacterial taxonomy identification for more than 56.26% mycological properties
Volume 1 (Bergey's Manual of Systematic Bacteriology
Volume 1, 1984).
This bacterium was identified as Enterobacter agglomerans in agreement with Biogroup 1 of Anaerogenic strains of Enterobacter agglomerans described on the page.

[0008] In comparison with the type strain (ATCC 27155), the strain used in the present invention and the type strain match in the above-mentioned mycological properties. This strain of Enterobacter agglomerans KF-3 has been deposited with the Institute of Biotechnology, Industrial Science and Technology as FERM P-13681. Culture conditions using the Enterobacter agglomerans KF-3 strain of the present invention and nutrient sources of a medium used for production of the polysaccharide KF-3 are as follows. Any carbon source can be used as long as this strain can be assimilated, but monosaccharides such as maltose, glycerol, glucose, and fructose, and oligosaccharides are preferably used. In addition, inorganic nitrogen sources such as ammonium chloride and ammonium sulfate, organic nitrogen sources such as yeast extract, peptone, and malt extract, and other inorganic salts such as potassium phosphate, magnesium sulfate, and sodium chloride can be used as components of the culture medium. Can be cultured under appropriate conditions.

[0009] Culture is preferably liquid culture, pH 4.0 to 9.0,
Preferably pH 6.8-7.2, temperature 15-40 ° C, preferably 30-
It is carried out at a temperature of 36 ° C., and is usually carried out by aeration and shaking culture. The culturing is carried out for 20 hours to 7 days after inoculation, depending on the type of the carbon source and the like. The culture solution becomes turbid with the growth of the bacterium, and has viscous and spinnable properties due to the production of a sticky polysaccharide substance. The viscosity of the culture solution reaches 10 ² to 10 ³ (25 ° C.) as measured by a rotational viscometer at the end of the culture.

[0010] By culturing, a culture solution having an aggregating activity, a water absorbing activity and a thickening activity is obtained. Centrifuge this culture or use SDS (sodium dodecyl sulfate)
The culture solution from which the cells have been removed is acidified, and twice the amount of ethanol or acetone is added, and the precipitated cohesive, water-absorbing and thickening substances are collected by centrifugation and the water is removed by drying under reduced pressure, etc. Polysaccharide KF-3 is obtained as a cultured product. However, the coagulant, the water-absorbing agent and the thickener of the present invention can use the culture itself as it is, without using the processed culture product separated and purified in this way.

[0011]

Next, the present invention will be described in more detail by way of examples. However, the technical scope of the present invention is not limited by these examples. In addition, the aggregation activity measurement method, the water absorption activity measurement method, and the viscosity activity measurement method are as follows. <Aggregation activity measurement method> This activity measurement method was performed as follows. That is, 85 ml of a 5000 ppm kaolin suspension is collected in a 100 ml measuring cylinder, 3 ml of 10% calcium chloride is added, and 0.1 ml of a culture solution (or a culture-treated product) is distilled with distilled water for 5 ml.
After diluting and adding to l, the volume was increased to 100 ml. It was inverted and stirred, and then allowed to stand for 5 minutes, and the absorbance of the supernatant was measured at 550 nm using a spectrophotometer. Using the values obtained by measuring the respective absorbances, an agglutination activity value was calculated by the following formula, and this value was used as an index of agglutination precipitation. Aggregation activity value = (1 / test solution OD ₅₅₀ of the supernatant) - Note (1 / control solution OD ₅₅₀ of the supernatant), the control fluid culture medium to OD ₅₅₀ of (or culture treated)
The kaolin suspension before adding was used. <Method of measuring water absorption activity> This activity measurement method was performed as follows. That is, a container containing about 20 ml is prepared using a coffee filter, and a sample such as a dried culture product having a substantially constant weight is placed therein. Next, after soaking in pure water for 2 hours, it is allowed to stand still to remove excess water. The water-removed sample is placed in a weighing beaker (50 ml) having a constant weight, and the weight after water absorption (water absorption + dry sample amount) is accurately measured. After this, about 2 at 105 ° C
Drying was carried out for a period of time to completely evaporate the water, and the exact weight (dry sample amount) before water absorption was measured. For the weight after water absorption, the amount of water absorbed by the coffee filter is determined using only the coffee filter as a control, and a measured amount obtained by subtracting from the weight after water absorption is used.

After each weight was measured in this way, the water absorption (g) per 1 g of the dried sample was calculated by the following equation, and this value was used as an index of the water absorption. Water absorption = (weight after water absorption (g)-weight before water absorption (g)) /
(Weight before water absorption (g)) <Viscosity activity measurement method> This activity measurement method was performed using a B-type rotational viscometer (Bismetrone viscometer, Shibaura System Co., Ltd.). That is, about 400 ml of the culture solution was placed in a 500 ml beaker for 24 hours after inoculation, and the measurement was performed at a constant temperature of 25 ° C.
The conditions are as follows.

Rotor: Standard rotor No. 2 Number of revolutions: 60, 30 and 12 rpm Number of measurements: three times for each number of revolutions After measuring in this manner, the viscosity value was calculated by the following formula, and the viscosity was calculated. The index was used. Viscosity value (cP) = Viscometer indicated value × Standard viscosity conversion multiplier Example 1 Glycerol 2.0 g, K ₂ HPO ₄ 0.8 g, KH ₂ PO ₄ 0.4 g, NH ₄ C
_{l 0.01g, MgSO 4 · 7H 2} O0.04g, NaCl 0.02g, yeast extract
0.1 g was dissolved in 200 ml of distilled water, and the medium was adjusted to pH 7.0 ± 0.2. This was placed in a 500 ml Erlenmeyer flask, sterilized in an autoclave (120 ° C., 20 minutes), and the Enterobacter agglomerans KF-3 strain (FERM P-13681) was transplanted to the Erlenmeyer flask in an amount of one loop of platinum, and the mixture was placed at 30 ° C. Rotational shaking culture (rotation speed: 180 rpm) was performed. The time-dependent changes in the amount of bacterial cells and the aggregation activity during the main culture were examined. The amount of bacterial cells was determined by measuring the turbidity of the culture and measuring the absorbance at a wavelength of 660 nm using a spectrophotometer. Agglutination activity was measured based on the above-mentioned agglutination activity measurement method. Table 1 shows the results. The growth of the cells reached a logarithmic growth phase within one day of culture, and became almost steady after 24 hours of culture.

[0014]

[Table 1]

The results in Table 1 are shown below in detail. The absorbance of the kaolin suspension (control solution) supernatant is 1.734. After the addition of the culture solution for 20 hours, the absorbance of the kaolin suspension (control solution) supernatant is 0.092. The agglutination activity (from the above formula) is 10.29. It is. Based on the above, this Enterobacter agglomerans KF-3 strain (FE
RM P-13681)
Has a high agglutinating activity.

The cells were lysed by adding SDS to the culture, and the bacteria were removed. Thereafter, twice the amount of ethanol or acetone was added on the acidic side, and the precipitated flocculant was collected by centrifugation, and dried under reduced pressure to obtain a white culture product. By these operations, about 1.32 g (dry weight) of the culture product was obtained per 200 ml of the culture solution by using the above-mentioned medium.

Example 2 In addition to the culture product obtained in Example 1, maltose and glucose + fructose (1: 1 v / v) were used as carbon sources, respectively.
Instead, the other medium composition, culture,
Cell treatment (lysis) and precipitation with ethanol were performed to obtain each culture product. The three cultures were finely pulverized in an agate, and about 0.1 g of the sample was used to measure the amount of water absorption according to the method for measuring water absorption activity described above. Table 2 shows the results
It is as shown.

From the table below, it was confirmed that the culture product obtained from the Enterobacter agglomerans KF-3 strain (FERM P-13681) has water-absorbing activity.

[0019]

[Table 2]

Example 3 Using the culture solution obtained in Example 1, the viscosity was measured in accordance with the above-described method for measuring the viscosity activity. Table 3 shows the results. As shown in Table 3, this Enterobacter agglomerans KF-3
It was shown that the culture solution (or culture-treated product) obtained from the strain (FERM P-13681) had a thickening effect.

[0021]

[Table 3]

Example 4 The cultivated product obtained in Example 1 was dissolved in a 0.1N sodium hydroxide aqueous solution, and then the cultivated product was neutralized with hydrochloric acid. Analysis was performed on chemical and physical properties such as reactions. The results are as follows. (1) Color reaction: Ninhydrin reaction-Xanthoprotein reaction-Phenol sulfate method + Anthrone sulfate method + Carbazole sulfate method + Elson-Morgan reaction-(2) Color of substance: White (3) Solubility in solvent: Distilled water Insoluble in (neutral) (gelling) soluble in alkali Insoluble in organic solvents such as methanol, ethanol, acetone, ether, chloroform and hexane (4) Ultraviolet absorption spectrum: shown in FIG.

No absorption at 280 nm specific to proteins (peptides) and 260 nm specific to nucleic acids was observed, but the absorbance gradually increased from 350 nm and increased rapidly from around 240 nm. (5) Infrared absorption spectrum: shown in FIG. There is an absorption pattern of OH which seems to be derived from carbohydrates around 3550 cm ^-1 and CH which seems to be derived from carbohydrates around 2950 cm ^-1
There are absorption pattern of CH _2, there is absorption pattern of carboxy ester near 1730 cm ^-1, there is absorption pattern of carboxyl groups seems to be derived from uronic acid in the vicinity of 1650 cm ^-1, polysaccharides specific around 800～1200Cm ^-1 The absorption pattern can be seen. From the above results, it was shown that the cultured product was an acidic polysaccharide containing uronic acid containing a neutral saccharide as a main component.

[0024]

【Example of use】

Use Example 1 For the following wastewater and the like, a flocculant containing a polysaccharide KF-3 as an active ingredient was used. Since the polysaccharide KF-3 has a broad aggregation spectrum, it exhibited an aggregation effect on inorganic substances, organic wastewater, microorganisms, and other substances.

[0025]

[Table 4]

Use Example 2 A cosmetic (facial cleansing foam) containing a humectant having the following composition was prepared using polysaccharide KF-3.

[0027]

[Table 5]

Example 3 An ice cream containing a thickener having the following composition was produced using the polysaccharide KF-3.

[0029]

[Table 6]

[0030]

According to the present invention, a novel microorganism-produced polysaccharide and a flocculant, a water-absorbing agent and a thickener containing the same as a main component can be provided. Since these can be produced in a short period of time, they are convenient in terms of cost, and since they are polysaccharides, they can be used in the natural environment and accumulated in the environment like conventional synthetic products. And is degraded by microorganisms. For this reason, the flocculant, the water-absorbing agent and the thickener of the present invention have the advantage that they are safe for the environment and there is no risk of secondary pollution.

[Brief description of the drawings]

FIG. 1 is a view showing an ultraviolet absorption spectrum of a cultured product.

FIG. 2 is a view showing an infrared absorption spectrum of a cultured product.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12P 19/04 C12P 19/04 C // (C12P 19/04 C12R 1:01) Examiner Hiroshi Taniguchi (58) Field surveyed ( Int.Cl. ⁶ , DB name) C08B 37/00 C12P 19/04

Claims (8)

(57) [Claims] 1. A polysaccharide KF- having the following physicochemical properties:
3. (1) Color reaction: Ninhydrin reaction-Xanthoprotein reaction-Phenol sulfate method + Anthrone sulfate method + Carbazole sulfate method + Elson-Morgan reaction-(2) Color of substance: white (3) Solubility in solvent: distillation Poorly soluble in water Soluble in alkali Insoluble in organic solvents such as methanol, ethanol, acetone, ether, chloroform and hexane. (4) Ultraviolet absorption spectrum: Absorbance gradually increases from 350 nm and sharply increases from around 240 nm. (5) Infrared absorption spectrum: 3550cm ^-1 , 2950cm ^-1 , 1730
cm ^-1, absorption pattern is seen in 1650 cm ^-1 and 800~1200cm ^-1. 2. A polysaccharide KF-3 belonging to the genus Enterobacter.
Cultivate a microorganism capable of producing, and from the culture, the polysaccharide KF-3
2. The polysaccharide KF-3 according to claim 1, wherein the polysaccharide is collected.
Manufacturing method. 3. The microorganism belonging to the genus Enterobacter,
3. The method for producing polysaccharide KF-3 according to claim 2, which is Enterobacter agglomerans. 4. An Enterobacter agglomerans,
The method for producing a polysaccharide KF-3 according to claim 3, which is an Enterobacter agglomerans KF-3 strain. 5. An aggregating agent containing the polysaccharide KF-3 as a main component. 6. A method for coagulating a suspended substance using a polysaccharide KF-3. 7. A water-absorbing agent containing polysaccharide KF-3 as a main component. 8. A thickener containing the polysaccharide KF-3 as a main component.