JPH09296002A - Microorganism-derived polysaccharide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a polysaccharide which is composed of fucose and mannose, and can be used as a thickener having advantages in that it is easily soluble in water and has excellent stability, by culturing Alcaligenes latus B-16 strain and subjecting the resultant culture to separation and purification. SOLUTION: Alcaligenes latus B-16 strain (FERM-BP-2015) belonging to Alcaligenes latus is cultured in a medium so that a polysaccharide being composed of fucose and mannose is produced and accumulated. Subsequently, the resultant culture is mixed with water and then is rendered to be alkaline by adding an alkaline agent. The obtained mixture is passed through a column packed with an ion exchange resin, so that proteins, nucleic acids, low molecular weight components of polysaccharides and the like are adsorbed on the ion exchange resin. Polysaccharides adsorbed on the ion exchange resin are eluted and the eluted solution is condensed to precipitate a polysaccharide, followed by drying. The obtained polysaccharide has a molar ratio of fucose to mannose of 1:(0.8-1.2), and can be advantageously used as a stabilizing agent for an abrasive, an emulsion stabilizer for a latex, and a thickener for a paint, a dressing and the like.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel polysaccharide derived from a microorganism and a thickener containing the same as an active ingredient.

[0002]

2. Description of the Related Art Polysaccharides are carbohydrates in which a plurality of monosaccharides are linked by glycosidic bonds, and many are known, including naturally occurring ones and naturally occurring ones chemically modified. Monosaccharides are generally soluble in water,
Although there are many types of polysaccharides and their properties vary, they are generally difficult to dissolve in water, and when they are dissolved, they often dissolve in a colloidal state. However, even if dissolved in water or dissolved in colloid, it becomes a highly viscous solution,
It has very distinctive behavior and has high industrial utility value.
Therefore, many polysaccharides that have been chemically modified and processed to dissolve in water are used. For example, cellulose is insoluble in water, but methyl cellulose obtained by reacting this with methyl chloride, ethylene oxide, propylene oxide, etc., becomes soluble in water, and is used as one of the very general industrial materials. ing. However, in the case of producing a chemically modified semi-synthetic product, in many cases, the reaction does not proceed ideally and partial alignment of hydrophilic groups and hydrophobic groups occurs, resulting in a water-insoluble portion, which continues. May cause powder. From this point of view, it is desired to have various properties as a polysaccharide and be soluble in water.

[0003]

Under such a background, the present invention is to cultivate a specific microorganism and to separate and collect a novel polysaccharide from the culture. It is intended to provide a highly stable thickener which is very easily dissolved in water as an active ingredient.

[0004]

[Means for Solving the Problems] The inventors of the present invention have cultivated a specific microorganism and focused on a specific component of the culture. The present inventors have found that they can be highly soluble and highly stable rheology improvers or thickeners, and have completed the present invention. That is, the present invention is a polysaccharide whose constituent monosaccharides are substantially fucose and mannose, and a thickener using this polysaccharide.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION In the polysaccharide of the present invention, the constituent monosaccharides are substantially fucose and mannose, preferably the composition ratio of fucose and mannose is 1: (0.8 to 1.2),
More preferably, most of the constituent monosaccharides, fucose, and mannose are alternately bonded. The polysaccharide of the present invention preferably has a molecular weight of 1 × 10 ^{2 to} 1 × 10 ⁷ .
If it is smaller than 1x10 ² , the thickening effect is small, while on the other hand, it is 1x1
0 ⁷ becomes larger and a gel, both unfavorable as a thickener.

The polysaccharide of the present invention is an Alcaligenes laetus B-16 strain (FERM-BP) belonging to Alcaligenes latus.
-2015) can be advantageously extracted from the output of the strain. The mycological properties and culturing method of Alcaligenes laetus B-16 strain are described in Japanese Patent Publication No. Heisei 6-37521. Regarding the polysaccharides produced from Alcaligenes reitus strain B-16, the above-mentioned patent publications, further JP-A-4-200389 and JP-A-5-3 have already been described.
Although reported in No. 01904, the polysaccharide of the present invention is produced simultaneously with the polysaccharides described in these publications, but is a completely different kind. That is, the polysaccharide (comprising glucose, rhamnose, glucuronic acid, fucose, and mannose) described in the above publication is separately separated and purified from the unfractionated fraction.

When the polysaccharide of the present invention is used as a thickener, it is usually used as an aqueous solution of about 0.01 to 10% by weight. An aqueous solution having a concentration of less than 0.01% by weight requires a large amount to achieve the intended purpose and is not efficient. An aqueous solution having a concentration of more than 10% by weight has insufficient solubility of the polysaccharide and is substantially soluble. There is a problem that it cannot be done, and further, the viscosity of the solution is too high, which makes it impossible to handle. The polysaccharide of the present invention is easily dissolved in water, and the viscosity of the aqueous solution thereof is hardly affected by pH, metal salts, etc. and is very stable. Therefore, when it is used as a thickener, it is easily soluble in water and does not leave a powdery insoluble component, and the viscosity of the resulting aqueous solution does not change depending on the environment, thus stabilizing the process and improving product quality. Can greatly contribute to

The application field of the thickener of the present invention is not particularly limited, but as a suspension stabilizer for fine particles such as abrasives, waxes and pigments, latex, agricultural chemicals, ink, paint, lubricating oil, processing It is applied to substantially water-based systems as an emulsion stabilizer for oils or as a thickener for paints and dressings. The amount to be added at the time of application is not limited, and should be determined according to the place of application and purpose of application. At this time, it does not prevent the combined use of other types of thickeners.

[0009] Polysaccharides are separated and purified from their culture products to obtain the desired polysaccharides, but in the case of polysaccharides of low purity, other polysaccharides and oligosaccharides are often mixed. . When using the polysaccharide of the present invention as a thickener,
In some fields, high purity is not required depending on the purpose of use and target product, and in this case, a polysaccharide other than the polysaccharide of the present invention may be mixed. The application of the thickener of the present invention does not prevent the mixture of such other polysaccharides.

[0010]

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

[0011]

Embodiment 1 Culturing conditions According to the method of Example 1 of JP-A-5-301904, Alcaligenes reitus B-16 strain (FERM BP-
No. 2015) was cultured. The composition of the culture medium is as follows. <Composition of culture medium> The following components were dissolved in ion-exchanged water to make 1 L as a whole. Glucose [Kanto Chemical Co., Ltd. reagent] 15 g KH ₂ PO ₄ [Kanto Chemical Co., Ltd. reagent] 4.5 g K ₂ HPO ₄ [Kanto Chemical Co., Ltd. reagent] 1.5 g NaCL [Kanto Chemical Ltd. reagents 0.1 g MgSO ₄ · 7H ₂ O [Kanto Chemical Co., Ltd. reagents 0.2 g urea [Kanto Chemical Co., Ltd. reagents 1.0 g yeast extract [Oxoid (OXOID) 0.5 g pH = 7 100 mL of this culture medium was sterilized with a 0.2 μm sterilized membrane filter and then placed in a 300 mL sterilized Erlenmeyer flask, and Alcaligenes lettus B-16 strain was treated with 1 platinum. After inoculation with ears, the cells were cultured with shaking at 30 ° C. for 7 days.

2. Separation / Purification The polysaccharide produced and accumulated by the culture was separated and purified by the following method. 400 mL of water was added to 100 mL of the culture obtained by culturing Alcaligenes reitus B-16 strain, and the pH was adjusted to 12 with a 1N NaOH aqueous solution. Then use this solution as an ion exchange resin: Diaion HP
The mixture was passed through a column filled with 100 mL of A-75 [manufactured by Nippon Nermizu Co., Ltd.]. (The water flow rate was 8 times or less the volume of the ion exchange resin). By this treatment, the ion exchange resin is
Nucleic acids and low molecular weight components of polysaccharides are adsorbed. At this time, the high-molecular weight component of the polysaccharide is contained in the water that has passed through without being adsorbed by the ion exchange resin column. This high molecular weight component is the polysaccharide described in the above publication (JP-A-5-301904).

300 mL of pure water in the ion exchange resin after passing water
After thoroughly washing through the column, add 0.1M NaCL solution to 3 times.
Water was passed through 0 mL to elute the polysaccharide adsorbed on the ion exchange resin column. Separate eluate from ion exchange resin: AG50
Desalination was carried out by passing through W-X8 (H type) [Bio-Rad] and AG1-X4 (Ac-OH type) [Bio-Rad]. After the desalted solution was concentrated by a rotary evaporator, ethanol was added to precipitate the polysaccharide. It was dried at room temperature under reduced pressure to obtain the polysaccharide of the present invention.

3. Molecular weight measurement Gel permeation chromatography (G
The average molecular weight was determined by PC) analysis. Table 1 shows the obtained results. <Conditions for GPC analysis> High performance liquid chromatography (HPLC) device [manufactured by Waters Co.] Column: Ultrahydrogen Gel 1000, Ultrahydrogen Gel 2000, and Guard Column 3
This use [all are manufactured by Waters Co., Ltd.] Mobile phase: 0.1N-NaNO ₃ flow rate: 0.5 mL / min Column temperature: 45 ° C. Detector: Differential refractometer standard sample: Pullulan [Shodex (Shodex)
manufactured by dex)] as standard molecular weights of 8.5 × 10 ⁵ , 4.8 × 10 ⁴ ,
Table 1 below shows the results of three kinds of 5.8 × 10 ³ GPC analysis.

[0015]

[Table 1] From these results, the molecular weight of this polysaccharide is 1 × 10 ⁴
It was found to be ˜5 × 10 ⁴ .

4. Determination of constituent monosaccharides After hydrolysis of the polysaccharide of the present invention under the following conditions, HPL
Analyzed at C. Conditions for hydrolysis: 14.0 mg of the polysaccharide of the present invention was dissolved in 4 mL of ion-exchanged water, 1 mL of concentrated sulfuric acid was added, the tube was sealed in an ampoule, and heated at 100 ° C for 2 hours. After cooling, this treatment liquid was neutralized with barium carbonate to remove precipitates, and then concentrated.

<HPLC measurement conditions> Column: SH-1011 [manufactured by Shoredex] Column temperature: 50 ° C Mobile phase: 0.01N-H ₂ SO ₄ Flow rate: 0.5 mL / min Standard sample: Glucuronic acid, Glucose, mannose, rhamnose and fucose were used. Standard peak (min): Glucuronic acid; 38.35, Glucose; 42.05, Mannose; 44.30, Rhamnose; 45.98, Fucose; 49.77 The results are shown in FIG. From this result, it was found that the constituent monosaccharides of the polysaccharide of the present invention were fucose and mannose.

5. Separation of partially hydrolyzed product and determination of constituent monosaccharide About 100 mg of the polysaccharide of the present invention was put in 5 mL of 0.25N-trifluoroacetic acid, and heated at 80 ° C. for 30 minutes for hydrolysis. This hydrolyzed liquid is used as an ion exchange resin: DEAE
-Neutralized with Sephadex (carbonic acid type) (manufactured by Biorat), filtered, and the filtrate was freeze-dried to prepare a partial hydrolyzate. This hydrolyzate was converted to Asahipak
It was analyzed by HPLC using a column of NH2P-50 (4.6 x 250 mm; manufactured by Showdex). The results are shown in FIG. No. 6 as the partial hydrolyzate of the polysaccharide of the present invention. It was possible to fractionate the oligosaccharide components 1 to No8.

<HPLC measurement conditions> Column: Asahipak NH2P-50 (4.6 x 250 mm) Temperature: 30 ° C Solvent: A mixture of acetonitrile: water (volume ratio 6: 4) Flow rate: 1.0 mL / min Isolated No. Each of the oligosaccharide components 1 to No. 8 was hydrolyzed under the same conditions and method as described in 4, and the constituent monosaccharides were determined. The composition of the isolated oligosaccharide component is shown in Table 2. From this result, it was revealed that all oligosaccharides were composed of fucose and mannose in almost equimolar amounts.

[0020]

[Table 2]

6. Analysis of Constituent Monosaccharides by Nuclear Magnetic Resonance Spectra (NMR) The polysaccharide of the present invention contains 85% hydrous dimethyl sulfoxide (D).
MSO), and using a nuclear magnetic resonance spectrum analyzer [BRUKER ARX500, 500 Mz for ¹ H, 125 MHz for ¹³ C],
It was measured at 0 ° C. The chemical shift is based on trimethylsulfoxide (TMS) δ 2.49 (
¹ H) and δ39.5 ( ¹³ C). One-dimensional N
In case of MR, measurement time is 2.1 with 30 pulse at ¹ H.
Second, the waiting time was 2.0 seconds, and for ¹³ C, 30 ° pulse, the measuring time was 0.5 seconds, and the waiting time was 0.1 seconds.
The results are shown in Table 3 and FIGS. 3 and 4. ¹³ C one-dimensional NM of 3
From the measurement of R, 12 signals were clearly observed, and it was found that there was no sugar other than fucose and mannose. Further, from the measurement of ¹ H one-dimensional NMR in FIG. 4,
The area ratio of the signals of the anomeric proton (H-1) of fucose and mannose was calculated to be 0.995: 1.000.
Therefore, it was found that the constituent monosaccharide was fucose: mannose = 1: 1.

Further, the ¹³ C one-dimensional NMR signal at the anomeric position of fucose and mannose has one sharp peak, and therefore it was found that the structure was such that fucose and mannose were alternately bonded.

< ¹ H-NMR spectrum measurement conditions> Solvent: 85% water-containing DMSO-d6 ¹ H: 500 MHz Temperature: 80 ° C. Pulse: 30 ° Measurement time: 2.1 seconds Waiting time: 2.0 seconds Chemical shift: The signal of DMSO is δ2.
49 ( ¹ H)

<Measurement conditions for ¹³ C-NMR spectrum> Solvent: 85% water-containing DMSO-d6 ¹³ C: 125 MHz Temperature: 80 ° C. Pulse: 30 ° Measurement time: 0.5 sec Waiting time: 0.1 sec Chemical shift: DMSO signal is δ3 from TMS
9.5 ( ¹³ C)

[Table 3]

[0025]

Embodiment 2 1. Polysaccharides used for comparison HPMC: Hydroxypropyl methylcellulose [Metroose 90SH-15000 (trade name), manufactured by Shin-Etsu Chemical Co., Ltd.] HEC: Hydroxyethyl cellulose [HEC Daicel SP-800 (trade name), Daicel Chemical Industry Co., Ltd.]
Made] Alginic acid: [made by Kibun Foods Co., Ltd.] Curdlan: [made by Wako Pure Chemical Industries, Ltd.] Xanthan gum: [made by Kelco] Locust bean gum: [made by Sansho Co., Ltd.]

2. Effect of pH on stability of aqueous solution An aqueous solution of 1% by weight of the polysaccharide of the present invention or a comparative polysaccharide was prepared, and hydrochloric acid and acetic acid or sodium hydroxide and sodium phosphate were added to adjust the pH.
The viscosity of each aqueous solution was measured using a B-type viscometer at 30 ° C. and 30 rpm. The results are shown in Table 4 below. The aqueous solution of the polysaccharide of the present invention had a constant viscosity and was stable at any pH.

[0027]

[Table 4]

3. Effect of metal salt on stability of aqueous solution The polysaccharide of the present invention or a comparative polysaccharide was dissolved in ion-exchanged water to prepare a 1 wt% aqueous solution. After adding a predetermined amount of sodium chloride or aluminum sulfate, the viscosity of the aqueous solution was measured with a B-type viscometer at 30 ° C. and 30 rpm. The results are shown in Table 5 below. The aqueous solution of the polysaccharide of the present invention showed stable viscosity without being affected by the presence of sodium chloride or aluminum sulfate.

[0029]

[Table 5]

4. Comparison of ease of dissolution Each of the polysaccharide of the present invention, or the comparative polysaccharide,
The particle size was adjusted to 100 to 200 mesh, 1 g thereof was added to a beaker containing 1000 mL of ion-exchanged water, and the mixture was stirred with a stirring device at 300 rpm and 30 ° C. for 10 minutes. The solution of each polysaccharide was suction-filtered with No6 filter paper (diameter 150 mm) whose constant weight had been measured. After drying this filter paper at 105 ° C for 2 hours, put it in a desiccator 1
Cool to room temperature for hours. After measuring the weight of this dried filter paper, the dissolution rate of each polysaccharide was determined by the following formula.

Next, 1 g of each of the polysaccharides of the present invention and comparative polysaccharides was placed in 1000 ml of ion-exchanged water, and the mixture was stirred with a stirring device at 300 rpm and 60 ° C. for 120 minutes, and then according to the above-mentioned method. The dissolution rate was determined. As shown in Table 6 below, it was confirmed that the polysaccharide of the present invention was completely dissolved in a short time even at a water temperature of about room temperature.

[Table 6]

[0032]

INDUSTRIAL APPLICABILITY The polysaccharide of the present invention is a novel polysaccharide and becomes a highly stable thickener which is very soluble in water. It can be widely applied in various industrial fields.

[Brief description of drawings]

FIG. 1A is a HPLC chart of a hydrolyzate of a standard substance, and B is HPL of a hydrolyzate of a polysaccharide of the present invention.
It is a C chart.

FIG. 2 shows a reverse-phase HPLC elution pattern of the hydrolyzate of the polysaccharide of the present invention.

FIG. 3 shows a ¹ H-NMR spectrum of the polysaccharide of the present invention.

FIG. 4 shows a ¹³ C-NMR spectrum of the polysaccharide of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Ryuichiro Kurane 1-3 East Higashi Tsukuba, Ibaraki Prefectural Institute of Industrial Science and Technology, Institute of Biotechnology (72) Inventor Yasuhiro Nobata Yokkaichi, Mie 6-6-9 No. Hakuto Co., Ltd. Yokkaichi Research Institute (72) Inventor Zenji Yamaguchi Yokkaichi City, Mie Prefecture 6-69 Another name Hakuto Co., Ltd. Yokkaichi Research Institute (72) Inventor Shuji Anazawa 4-19-18 Minamioizumi, Nerima-ku, Tokyo (72) Inventor Ichiro Matsuura 3-27 Nakadai, Itabashi-ku, Tokyo K-212

Claims (5)

[Claims] 1. A polysaccharide whose constituent monosaccharide consists essentially of fucose and mannose. 2. The composition ratio of fucose and mannose is
The polysaccharide according to claim 1, which is 1: (0.8 to 1.2). 3. The polysaccharide according to claim 1, which has a molecular weight of 1 × 10 ^{2 to} 1 × 10 ⁷ . 4. Alcaligenes laitas (Alcal
igenes latus) Alcaligenes
The method for producing a polysaccharide according to any one of claims 1 to 3, which comprises culturing Lactus B-16 strain (FERM-BP-2015), and separating and collecting from the culture. 5. A thickener containing the polysaccharide according to any one of claims 1 to 3 as an active ingredient.