JP4441304B2 - Method for preparing water-soluble low-viscosity β-D-glucan-containing culture solution - Google Patents

Description

  The present invention relates to a water-soluble low-viscosity β-D-glucan useful as a food additive, a health food material, etc. from a culture solution mainly composed of a water-soluble β-D-glucan produced by microorganisms outside the cell. It relates to a preparation method.

β-glucan (β-1,3-D-glucan, β-1,6-D-glucan and β-1,3-1,6-D-glucan) is a mushroom (the basidiomycete fruit body) that inhabits nature. Recently, it is becoming clear that it is contained not only in their fruit bodies but also in cultured mycelium. These β-glucans are known to have antitumor activity. For example, each β-glucan extracted from Suehirotake, Kawaratake and Shiitake is already manufactured and sold as a pharmaceutical such as an anticancer agent.
On the other hand, it is known that a microorganism belonging to the genus Aureobasidium, which is an incomplete bacterium, also produces β-glucan.
However, in general, β-glucan aqueous solution has a single-helix or triple-helix structure from its structure, so that a gel is easily formed, and the culture solution is highly viscous, so that purification is extremely difficult (Non-Patent Document 1). reference). For example, β-glucan-containing cultures produced by microorganisms belonging to the genus Aureobasidium are also highly viscous, and there have been few reports on industrial separation, recovery and purification of bacterial cells and water-soluble β-glucan from the cultures. It is currently used with bacteria.

In addition, β-glucan contained in fruit bodies such as mushrooms is very difficult to dissolve in water and is only solubilized in an alkaline solution. Recently, after insoluble β-glucan such as mushrooms are extracted with hot water or alkali, A patent application has also been filed for a method in which high-pressure treatment (300-800 kgf / cm ² ) is carried out in the presence of a dispersing agent such as an emulsifier to form colloidal ultrafine particles (Patent Document 1).
Other,

1) According to Patent Document 2, Aureobasidium. Sp. FERM-P. A method is disclosed in which a culture solution of No. 4257 strain is sterilized by heating and then filtered or centrifuged to obtain a culture solution that can be used as food and drink.

2) According to Patent Document 3, hot water or a dilute alkaline aqueous solution is contacted with filtered or centrifuged cells or cell walls of microorganisms belonging to the genus Aureobasidium, and water-soluble β-1,3-1,6-D- A method for eluting and collecting glucan is disclosed.

3) According to Patent Document 4, an alkaline heat treatment (0.2 N or more, in an aqueous dispersion of an insoluble β-glucan produced by a microorganism or a culture solution (glucan or cell wall component produced in a cell) in the presence of a hydrophilic organic solvent. In general, a purification method is disclosed in which a mixed solution in which insoluble β-glucan is precipitated is neutralized by performing 0.2-1N, preferably 0.3-0.5N alkali).

4) According to Patent Document 5, peroxide and hydroxide are added to a suspension of cell bodies (particularly cell wall components) containing insoluble β-glucan such as mushrooms and microorganisms to adjust the pH to 10-12.5, and water. A method for recovering and purifying insoluble β-glucan is disclosed.

5) According to Patent Document 6, the pH is adjusted to 9-10 by adding caustic to a culture solution containing water-soluble β-1,3-1,6-D-glucan produced by microorganisms belonging to the genus Aureobasidium. And the method of spray-drying the produced alkali salt is disclosed.
International Publication No. 02/087603 Pamphlet JP-A-61-146192 Japanese Patent Publication No. 6-92441 JP-A-5-308987 Japanese Patent Laid-Open No. 9-322795 Japanese Patent Laid-Open No. 10-276739 Fragrance Journal, 5, 71-75 (1995) Agric. Biol. Chem., 47, 1167-1172 (1983) K. Ogawa, Carbohydrate Research, 67, 527-535 (1978) Supervised by Tadayuki Imanaka, Development of microbe utilization, 1012-1015, NTS (2002) Hamada et al., Science and Industry, 64, 131-135 (1990)

  The object of the present invention is to reduce the viscosity of a culture solution mainly composed of water-soluble β-D-glucan produced by microorganisms, particularly microorganisms belonging to the genus Aureobasidium, and to reduce the viscosity of water-soluble β-D. -Concerning glucan.

The present inventors fermented microorganisms, and β-D-glucan contained in the obtained culture broth, particularly water-soluble β-D-glucan contained in the culture broth of microorganisms belonging to the genus Aureobasidium, in a highly alkaline aqueous solution. As a result of the treatment, it was found that a β-D-glucan-containing culture solution that is water-soluble and low in viscosity can be obtained, and the present invention has been completed.
That is, the present invention relates to a water-soluble β-D-glucan, for example, a β-1,3-D-glucan, a microorganism having β-1,3-1,6-D-glucan as a main component, for example, Aureobasidium spp. A water-soluble low-viscosity β-D-, characterized by adding an alkali or an aqueous solution thereof to a culture solution of a microorganism belonging to the genus Aureobasidium such as pullulans and adjusting the pH concentration to lower the viscosity of the culture solution The present invention relates to a method for preparing a culture solution containing glucan.

The present invention also relates to a method for purifying water-soluble low-viscosity β-D-glucan by separating impurities containing microorganisms from a water-soluble low-viscosity β-D-glucan-containing culture solution obtained by the above method.
The present invention is adjusted to weak alkaline to acidic by adding an acid to the water-soluble low-viscosity β-D-glucan-containing culture solution obtained by the above method or the water-soluble low-viscosity β-D-glucan-containing aqueous solution obtained by the above method. The present invention relates to a method for preparing a water-soluble low-viscosity β-D-glucan-containing culture solution or a water-soluble low-viscosity β-D-glucan-containing aqueous solution.
The present invention relates to a food containing a water-soluble low-viscosity β-D-glucan obtained by the above method or an aqueous solution thereof.
The present invention also relates to a water-soluble low-viscosity β-D-glucan produced by adding an alkaline aqueous solution to a microorganism culture solution mainly composed of water-soluble β-D-glucan and adjusting the pH to 12 or more.
The present invention also relates to a dry water-soluble low-viscosity β-D-glucan obtained by drying a water-soluble low-viscosity β-D-glucan-containing aqueous solution obtained by the above method.

  As described above, the high viscosity water-soluble β-D-glucan produced by microorganisms belonging to the genus Aureobasidium has been successfully reduced by high alkali treatment. As a result, it was possible to remove the bacterial cells, which was difficult with the conventional culture solution, and to be pulverized by spray drying.

The microorganism used in the present invention is not particularly limited as long as it can produce water-soluble β-D-glucan, but is preferably an Aureobasidium microorganism, particularly an Aureobasidium pullulans.
Mutant strains GM-NH-1A1 or GM-NH-1A2 obtained by mutation treatment from Aureobasidium sp. K-1 strain (see Non-Patent Document 2) also produce low-molecular glucans. Particularly preferred bacteria. The original strain Aureobasidium sp. K-1 produces several kinds of high molecular weight β-glucan (over 2 million and about 1 million β-glucan), and the β-glucan-containing culture solution Has a high viscosity and is used as a food additive as a thickener. In addition, β-glucan produced by Aureobasidium sp. K-1 strain is partially substituted with sulfoacetic acid groups, and the content varies depending on the culture method such as medium composition (added non-patent literature).

  As shown in the Examples, the mutant strains GM-NH-1A1 and GM-NH-1A2 have a high molecular weight β-glucan with a main peak of about 1 million and a low molecular weight with a main peak of about 100,000 to 200,000. In the strains that produce both β-glucans, the β-glucans produced by them were partially substituted with sulfoacetic acid groups as in the original strains, and their contents were all about 0.1%. Reference 2). The properties of these strains are as shown in the table below. From the morphological properties of each strain and the base sequence of 28s rDNA, it was confirmed that these strains belong to Aureobasidium pullulans. These strains are deposited as FERM P-19285 and FERM P-19286 at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center, respectively.

In order to cultivate microorganisms to produce water-soluble β-D-glucan, a known water-soluble β-D-glucan-producing bacterium or the above-mentioned deposited bacterium is used. It can be performed by the culture medium and culture method.
Various methods for producing water-soluble β-1,3-1,6-D-glucan by culturing microorganisms belonging to the genus Aureobasidium have also been reported. As a usable carbon source, sucrose is used. , Carbohydrates such as glucose and fructose, and organic nutrient sources such as peptone and yeast extract. Examples of the nitrogen source include inorganic nitrogen sources such as ammonium sulfate, sodium nitrate, and potassium nitrate. In some cases, in order to increase the production amount of β-glucan, as appropriate, inorganic salts such as sodium chloride, potassium chloride, phosphate, magnesium salt, calcium salt, trace metal salts such as iron, copper, manganese, etc. Adding vitamins is also an effective method.

For example, when a microorganism belonging to the genus Aureobasidium is cultured in a medium containing sodium ascorbate in a tourpec medium containing sucrose as a carbon source, a high concentration of water-soluble β-1,3-1,6-D-glucan is added. It has been reported to produce (see Non-Patent Document 2). However, the culture solution is not limited to this composition, and an organic nutrient source such as yeast extract or peptone may be added as necessary.
The conditions for aerobic culture of microorganisms belonging to the genus Aureobasidium in the above medium are a temperature of 10-50 ° C, preferably 20-35 ° C, and a pH of 4-7, preferably 4.5-6.5.
In order to effectively control the culture pH, it is also a good idea to control the pH of the culture solution with alkali or acid. Furthermore, an antifoaming agent may be added as appropriate for defoaming the culture solution. The culture time is usually preferably 1-10 days. If cultured for 1-4 days, water-soluble β-D-glucan can be produced. The culture time may be determined while measuring the production amount of β-D-glucan.

When a microorganism belonging to the genus Aureobasidium is cultured under aeration and agitation for 4-6 days under the above conditions, 0.1-β-glucan polysaccharide mainly composed of water-soluble β-1,3-1,6-D-glucan is contained in the culture solution. % To several% (w / v), and the viscosity of the culture broth is from several thousand cP [mPa · s] at 30 ° C to several hundred cP (by Toki Sangyo Co., Ltd.). [mPa · s]).
The viscosity can be drastically reduced by adding an alkali or an aqueous solution thereof to the culture solution obtained as described above at normal temperature and adjusting the pH to a value necessary for reducing the viscosity of the culture solution.

Examples of the alkali or an aqueous solution thereof used in the present invention include calcium carbonate aqueous solution, sodium carbonate aqueous solution, potassium carbonate aqueous solution, ammonium carbonate aqueous solution and the like alkali carbonate aqueous solution, sodium hydroxide aqueous solution, potassium hydroxide aqueous solution, calcium hydroxide aqueous solution and the like. There is no particular limitation as long as it is a commonly used water-soluble alkali such as an aqueous alkali hydroxide solution or an aqueous ammonia solution. However, it goes without saying that alkalis recognized as food additives are preferred for edible use.
The pH adjustment varies depending on the polysaccharide concentration in the culture medium of interest, but in the case of a relatively high concentration, an alkali or an aqueous solution thereof is used so that the pH is 11 or more, preferably 12 or more, more preferably 13 or more. Added. In the case of a low concentration, the viscosity can be lowered by processing at a lower pH.

As a method for separating insoluble substances such as bacterial cells from a water-soluble β-D-glucan culture solution with reduced viscosity after alkali treatment, there is no particular limitation as long as it is a method capable of separating insoluble substances from the culture solution, Centrifugation, total filtration using filter paper or filter cloth, filter press, and membrane filtration (ultrafiltration such as MF membrane) can be performed. In the case of total filtration with filter paper or filter cloth, it is one means to use a filter aid such as celite. When sterilizing by filtration, the viscosity of the culture solution is preferably adjusted to 100 cp or less, preferably 60 cp or less, more preferably 30 cp or less.
After alkali treatment, acid is added to the water-soluble β-D-glucan-containing culture solution with reduced viscosity, or to a water-soluble β-D-glucan-containing aqueous solution after purification such as sterilization. ) To acidic (pH 2) neutralization treatment.
In the present invention, the culture solution containing the water-soluble low-viscosity β-D-glucan obtained by the above method is reduced in viscosity by alkali treatment, and further, the cells are removed, neutralized, or desalted. The water-soluble, low-viscosity β-D-glucan that has been subjected to the above can be dried to produce dry water-soluble low-viscosity β-D-glucan. As a drying method, a known method such as a spray drying method or a freeze drying method can be employed. Moreover, in order to improve the workability of spray drying or increase the recovery rate, a sugar compound such as lactose, dextrin, cyclodextrin, cluster-dextrin, trehalose and the like can be appropriately blended as necessary.

According to the present invention, polysaccharides such as glucan are gelated even when the pH of a water-soluble β-D-glucan-containing culture solution or purified β-D-glucan-containing aqueous solution after alkali treatment is adjusted from near neutral to near acidic. The viscosity remains low.
As described above, the present invention reduces the viscosity of a culture solution containing high-viscosity water-soluble β-D-glucan by alkaline treatment, and after refining as necessary, the pH is subsequently adjusted to acidic (pH 2). It is useful as a health food material, particularly a health food beverage material. In particular, the method according to the present invention is a practical method because it can be used for hot pack sterilization (pH is 4 or less, 90 ° C. or less for several minutes) as it is.
In this case, the pH may be adjusted appropriately according to the purpose.
The acid used in the method of the present invention is not particularly limited as long as it can normally neutralize alkali such as hydrochloric acid, phosphoric acid, sulfuric acid, citric acid. However, for food, acids that are recognized as food additives, such as citric acid and malic acid, are used.

  The results of viscosity reduction experiments conducted using the two types of culture solutions shown in Examples 1 and 2 are shown below. The viscosity was measured after adjusting the alkali-treated culture solution to pH 4.9 with a 50% (w / v) aqueous citric acid solution.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these examples.

Example 1 Use of Aureobasidium pullulans GM-NH-1A1 1) Culture production of β-glucan-1
Put 60ml liquid medium with the following composition into a 300ml Erlenmeyer flask with baffle, autoclaved at 121 ° C for 15 minutes, and then sterilize GM-NH-1A1 strain from the slant with the same composition. A seed culture solution was prepared by inoculating 1 platinum ear and incubating at 130 rpm with aeration and stirring at 30 ° C. for 24 hours. Next, 3 L of the medium with the same composition was placed in a 5 L jar fermenter (manufactured by Mitsuwa Riken), autoclaved at 121 ° C for 15 minutes, and the seed culture solution obtained above was aseptically inoculated. Then, aeration and agitation culture at 400 rpm, 30 ° C. and 600 ml / min were performed. The pH was controlled at pH 4.6 to 5.0 using sodium hydroxide and hydrochloric acid. The turbidity after about 96 hours was 24 OD (660 nm), and the polysaccharide concentration was 0.6% (w / v). The polysaccharide concentration was determined by the phenol-sulfuric acid method after centrifuging and removing ethanol from the culture, adding ethanol to 66% (v / v), precipitating it, and dissolving in ion-exchanged water.

Subsequently, the wavelength shift from 480 nm to around 525 nm could be confirmed by the Congo-Red method, and it was proved that β-glucan was contained (see Non-Patent Documents 3 and 4). The shift difference to the maximum value at that time was Δ0.48 / 500 μg polysaccharide. Also, from the two-dimensional NMR ^{(13 C1} H COSY NMR) spectrum of the Honda sugar, ¹ H NMR spectra 4.7ppm correlating with ¹³ C NMR 104 ppm derived from the C1 carbon of D- glucose is constituent sugar and 4.4 Two signals near ppm were obtained. As a result, it was proved that β-glucan is β-1,3-1,6-D-glucan (see Non-Patent Document 4). From the integral ratio of the respective ¹ H NMR signals, the ratio of β-1,3-bond / β1,6-bond was found to be 1.17.
Further, ethanol was added to the culture supernatant from which the cells had been removed so that the final concentration was 66%, and β-glucan was collected by precipitation. Then, it melt | dissolved in ion-exchange water again, and insoluble each part was filtered and removed with the filter paper. Sodium chloride was added to the supernatant to a final concentration of 0.9%, and β-glucan was again collected with 66% ethanol. This β-glucan recovery and purification operation was repeated twice more, and the resulting β-glucan aqueous solution was dialyzed against ion-exchanged water and then freeze-dried to obtain β-glucan powder. From the compositional analysis results of this β-glucan, the S content was 239 mg / kg, and the substituted sulfoacetic acid content calculated from this was 0.09%. This could also be confirmed from the sulfoacetic acid content measured by the method described in Non-Patent Document 2.

2) Alkali treatment When 3 L of the culture solution obtained in 1) was measured with a BM type rotational viscometer, the viscosity was 1400 cP ([mPa · s]) at 30 ° C. When 25% (w / w) sodium hydroxide was added to this culture broth to a final concentration of 2.4% (w / v) and stirred, the viscosity decreased instantaneously. The pH at that time was 13.6. Subsequently, when the viscosity was measured after adjusting the pH to 4.9 with a 50% (w / v) aqueous citric acid solution, the viscosity at that time was 18 cP ([mPa · s]). Next, KC Flock (manufactured by Nippon Paper Industries Co., Ltd.) is added to the culture broth as a filter aid, and the cells are removed by suction filtration using filter paper (Advantech No. 2). About 3.5L was obtained. The polysaccharide concentration was 0.6% (w / v), and the recovery rate was almost 100%.

3) Desalination of β-D-glucan aqueous solution The above β-glucan aqueous solution (culture filtrate) is desalted with a UF membrane (molecular weight cut 50,000, manufactured by Spectrum), and finally the salt concentration is 30-40. Desalinated to about 1 / min. Finally, after the pH was adjusted to 3.5 with a 50% (w / v) citric acid aqueous solution, sterilization was performed at 95 ° C. for 3 minutes to obtain a 3.5 L β-D-glucan aqueous solution as a final product. The concentration of β-D-glucan at this time was 0.43% (w / v) as measured by the phenol sulfate method, and the yield from the culture was about 70%.
The obtained β-glucan aqueous solution was dialyzed against ion-exchanged water and then freeze-dried to obtain β-glucan powder. From the compositional analysis result of this β-glucan, the S content was 330 mg / kg, and the substituted sulfoacetic acid content calculated from this was 0.12%. This could also be confirmed from the sulfoacetic acid content measured by the method described in Non-Patent Document 2.
In addition, when Toyopearl HW65 manufactured by Tosoh Corporation (column size 75cm x φ1cm, excluded molecular weight 2.5 million (dextran)) was subjected to gel chromatography using 0.1M sodium hydroxide aqueous solution as the eluent, the molecular weight of the polysaccharide obtained Was found to consist of two types of low molecular fractions with an average molecular weight of 50,000 to 300,000 peaks and high molecular fractions of 500,000 to over 2.5 million. A pullulan manufactured by Shodex was used as a molecular weight marker.

Example 2 Use of Aureobasidium pullulans GM-NH-1A1 1) Culture production of β-D-glucan-2
60 ml of the liquid medium having the composition shown in Table 4 was placed in a 300 ml Erlenmeyer flask with baffle and autoclaved at 121 ° C for 15 minutes, and then GM-NH-1A1 strain was slanted with the same medium composition. One platinum ear inoculation was performed more aseptically, and a polysaccharide production experiment was conducted by aeration-stirring culture at 30 ° C. and 130 rpm for 96 hours. The turbidity after 96 hours was 35 OD (660 nm), and the polysaccharide concentration was 0.3% (w / v). The polysaccharide concentration was determined by the phenol-sulfuric acid method after centrifuging and removing ethanol from the culture, adding ethanol to 66% (v / v), precipitating it, and dissolving in ion-exchanged water.

2) The properties of this polysaccharide were examined in the same manner as in Example 1.
When the shift effect in Congo-Red was investigated, the maximum absorption was observed to shift from 480 nm to around 525 nm, and it was proved to contain β-D-glucan. The shift difference to the maximum value at that time was Δ0.45 / 500 μg polysaccharide. Two-dimensional NMR ( ¹³ C- ¹ H COSY NMR) analysis was also performed. From the result, it was proved that the obtained polysaccharide was β-1,3-1,6-D-glucan. From the integral ratio of each ¹ H signal, the ratio of β-1,3-bond / β1,6-bond was 1.11.
Subsequently, when 60 ml of the obtained culture broth was measured with a BM type rotational viscometer, the viscosity was 140 cP ([mPa · s]) at 30 ° C. When 25% (w / w) sodium hydroxide was added to this culture broth to a final concentration of 2.4% (w / v) and stirred, the viscosity decreased instantaneously and the pH was 13.4. Subsequently, the pH was adjusted to 4.9 with a 50% (w / v) aqueous citric acid solution. The viscosity at that time was 8.5 cP ([mPa · s]).

Example 3 The same procedure as in Example 1 was performed except that the strain used in the Aureobasidium pullulans GM-NH- 1A2 strain was changed from the GM-NH-1A1 strain to the GM-NH-1A2 strain. As a result, the produced polysaccharide concentration was 0.5% (w / v), and the viscosity at that time was 1300 cP ([mPa · s]). When sodium hydroxide was added to the obtained culture broth in the same manner as in Example 2 so that the final concentration was 2.4% (w / w), the pH was 13.6 and the viscosity was significantly reduced. Subsequently, when the viscosity was measured after adjusting the pH to 4.9 with a 50% (w / v) citric acid aqueous solution, the viscosity was 7 cP ([mPa · s]). The polysaccharide concentration at that time was 0.5% (w / v).

Thereafter, the properties of this polysaccharide were examined in the same manner as in Example 1.
When the shift effect in Congo-Red was examined, the maximum absorption was observed to shift from 480 nm to around 525 nm. The shift difference to the maximum value at that time was Δ0.45 / 500 μg polysaccharide. Two-dimensional NMR ( ¹³ C- ¹ H COSY NMR) analysis was also performed. From the results, it was proved that the obtained polysaccharide was β-1,3-1,6-D-glucan. From the integral ratio of each ¹ H signal, the ratio of β-1,3-bond / β1,6-bond was 1.23.
Furthermore, when the molecular weight was measured, it was found that the molecular weight was composed of two types, a low molecular fraction having an average molecular weight of 50,000 to 300,000 peaks and a high molecular fraction having 500,000 to 2.5 million or more.
In addition, the S content was 229 mg / kg from the compositional analysis result of this β-glucan, and the substituted sulfoacetic acid content calculated from this was 0.09%. This could also be confirmed from the sulfoacetic acid content measured by the method described in Non-Patent Document 2.

Example 4 The same procedure as in Example 1 was performed except that the strain used for the Aureobasidium sp. K-1 strain was changed from the GM-NH-1A1 strain to the K-1 strain. As a result, the produced polysaccharide concentration was 0.5% (w / v), and the viscosity at that time was 1800 cP ([mPa · s]). When sodium hydroxide was added to the obtained culture broth in the same manner as in Example 2 so that the final concentration was 2.4% (w / w), the pH was 13.5, and the viscosity was significantly reduced. Subsequently, when the viscosity was measured after adjusting the pH to 4.9 with a 50% (w / v) aqueous citric acid solution, the viscosity was 30 cP ([mPa · s]). The polysaccharide concentration at that time was 0.5% (w / v).

Thereafter, the properties of this polysaccharide were examined in the same manner as in Example 1.
When the shift effect in Congo-Red was examined, the maximum absorption was observed to shift from 480 nm to around 525 nm. The shift difference to the maximum value at that time was Δ0.52 / 500 μg polysaccharide. Two-dimensional NMR ( ¹³ C- ¹ H COSY NMR) analysis was also performed. From the results, it was proved that the obtained polysaccharide was β-1,3-1,6-D-glucan. From the integral ratio of each ¹ H signal, the ratio of β-1,3-bond / β1,6-bond was 1.42. Further, when the molecular weight was measured, it was found that the molecular weight was 100,000 to 3 million.
In addition, the S content was 6,800 mg / kg from the compositional analysis result of this β-glucan, and the substituted sulfoacetic acid content calculated from this was 2.7%. This could also be confirmed from the sulfoacetic acid content measured by the method described in Non-Patent Document 2.

Example 5 The method of Example 1 was used except that the strain of the medium composition was changed to the GM-NH-1A2 strain and sodium ascorbate in the medium of Table 4 was not added. As a result, the concentration of the produced polysaccharide was 0.6%, and the viscosity at that time was 1500 cP ([mPa · s]). When sodium hydroxide was added to the obtained culture broth in the same manner as in Example 2 so that the final concentration was 2.4% (w / w), the pH was 13.6, and the viscosity was significantly reduced. Subsequently, when the viscosity was measured after adjusting the pH to 4.9 with a 50% (w / v) aqueous citric acid solution, the viscosity was 7 cP ([mPa · s]). In the Congo-Red method, a maximum absorption shift from 480 nm to around 525 nm was observed. The shift difference to the maximum value at that time was Δ0.45 / 500 μg polysaccharide.
Further, two-dimensional NMR ( ¹³ C- ¹ H COZY NMR) analysis was performed in the same manner as in Example 1. As a result, it was proved that the obtained polysaccharide was β-1,3-1,6-D-glucan. From the integral ratio of each ¹ H signal, β-1,3-bond / β1,6- The binding ratio was 1.21.
Subsequently, the molecular weight was measured in the same manner as in Example 3. The molecular weight was composed of two types of a low molecular fraction having an average molecular weight peak of 50,000 to 300,000 and a high molecular fraction having 500,000 to 2.5 million or more. It has been found.

Example 6 Viscosity reduction test of water-soluble polymer β-glucan Aureobasidium sp. Water-soluble β-1,3-1,6-D glucan-containing culture solution A (trade name “Tenji no Shizuku”, Sold by Fujika Inc FS 2-7-39 Shimizu, Miyazaki-shi, Miyazaki) and B (trade name “Akfa-Zimax”, sold by Ikko Chemical Co., Ltd. 93-59 Hamaka, Satosho-cho, Asakuchi-gun, Okayama) A viscosity reduction experiment was conducted.
When the polysaccharide concentration of the product was measured, the culture solution A was 0.45% (w / v), and the viscosity at that time was 250 cP ([mPa · s]). As in Example 1-2), sodium hydroxide was added to the culture solution to a final concentration of 2.4% (w / w) (pH 13.4), followed by 50% (w / v) citric acid. When the viscosity was measured after adjusting the pH to 4.9 with an aqueous solution, the viscosity decreased to 10 cP ([mPa · s]). The polysaccharide concentration at that time was 0.46% (w / v).
The culture solution B had a polysaccharide concentration of 0.07% (w / v), and the viscosity at that time was 115 cP ([mPa · s]). As in Example 1-2), sodium hydroxide was added to the culture solution to a final concentration of 2.4% (w / w) (pH 13.4), followed by 50% (w / v) citric acid. When the viscosity was measured after adjusting the pH to 4.9 with an aqueous solution, the viscosity decreased to 20 cP ([mPa · s]).
Therefore, it was confirmed that the viscosity of the Aureobasidium sp. Water-soluble β-1,3-1,6-D glucan-containing culture broth was also reduced.

On the other hand, when the maximum absorption wavelength of the culture solution A was measured by the Congo-Red method in the same manner as in Example 1, a shift from 480 nm to around 525 nm was observed. The shift difference to the maximum value at that time was Δ0.41 / 500 μg polysaccharide. In addition, when two-dimensional NMR (13C-1H COZY NMR) analysis was performed, it was proved that the obtained polysaccharide was β-1,3-1,6-D-glucan. From the integration ratio of each 1H signal, The ratio of β-1,3 bond / β-1,6 bond was 1.33.
Further, when the molecular weight of the culture broth A was measured by the same method as in Example 1-3), it was found that the molecular weight consisted of one kind of polymer fraction having an average molecular weight of 500,000 to 2.5 million or more.

Example 7 Powdering (Spray-Dry Method)
After performing the viscosity reduction treatment with alkali in Example 1-2), β-glucan (including fungus bodies) adjusted to pH 6 with citric acid was applied to a spray drying apparatus R-3 (diameter 1200 mm, manufactured by Sakamoto Giken). , Height 800mm), spray drying with hot air inlet temperature 145 ° C, hot air outlet temperature 65 ° C, atomizer rotation speed 15000rpm, liquid feed speed 1000ml / h, β-1,3-1,6-D- About 50 g of glucan powder was obtained. This powder was slightly reddish brown and had a β-glucan purity of about 8%.

Example 8 Powdering (Ethanol Precipitation-Freeze Drying Method)
200 ml of the final product (bacteria-removed product) that had been subjected to alkali treatment, desalting and pH 3.5 adjustment in Example 1-2) was mixed with 800 ml of ethanol and allowed to stand overnight, and then the precipitate was centrifuged. It was collected. This precipitate was dried using a freeze drying apparatus manufactured by EYERA. The dried product was pulverized in a mortar to obtain about 0.6 g of powder. Although this powder showed a slight brown color, it was confirmed that the purity of β-glucan was almost 100%. Thus, the obtained dry powder is easily redispersed in water, warm water and alkaline water, and is useful as an additive for food and drink.

Claims (10)

It is characterized by adding an alkaline solution to the culture solution of microorganisms belonging to the genus Aureobasidium whose main component is water-soluble β-1,3-1,6-D-glucan, and adjusting the pH to 13 or more. For preparing a water-soluble low-viscosity β-1,3-1,6-D-glucan- containing culture solution. The method according to claim 1 , wherein the microorganism belonging to the genus Aureobasidium is Aureobasidium pullulans or Aureobasidium sp. Aureobasidium pullulans strain Aureobasidium pullulans GM-NH-1A1 (domestic deposit number FERM P-19285) or Aureobasidium pullulans GM-NH-1A2 strain (domestic deposit number) FERM P-19286). The method according to claim 1 or 2 . Water-soluble low-viscosity by separating insoluble matter containing microorganisms from a water - soluble low-viscosity β-1,3-1,6-D-glucan- containing culture solution obtained by the method according to any one of claims 1 to 3 A method for purifying an aqueous solution containing β-1,3-1,6-D-glucan . An acid is added to the water-soluble low-viscosity β-1,3-1,6-D-glucan-containing culture obtained by the method according to any one of claims 1 to 3 to adjust the culture to pH 2 to 10 For preparing a water-soluble low-viscosity β-1,3-1,6-D-glucan-containing culture solution. A water-soluble low-viscosity β- solution wherein the aqueous solution is adjusted to pH 2 to 10 by adding an acid to the water-soluble low-viscosity β-1,3-1,6-D-glucan-containing aqueous solution obtained by the method according to claim 4. A method for purifying an aqueous solution containing 1,3-1,6-D-glucan. A water-soluble low-viscosity β-1,3-1,6-D-glucan-containing culture solution is prepared by the method according to claims 1 to 3, and the culture solution is added to food. A method for producing a culture liquid-containing food containing a viscosity β-1,3-1,6-D-glucan. A water-soluble low-viscosity β comprising purifying a water-soluble low-viscosity β-1,3-1,6-D-glucan-containing aqueous solution by the method according to claim 4 and adding the aqueous solution to food. A method for producing an aqueous solution containing -1,3-1,6-D-glucan. Add an alkaline solution at room temperature to the culture solution of microorganisms belonging to the genus Aureobasidium, whose main component is water-soluble β-1,3-1,6-D-glucan, and adjust the pH to 13 or higher to achieve water-soluble low-viscosity β- A culture solution containing a water-soluble low-viscosity β-1,3-1,6-D-glucan obtained by obtaining a culture solution containing 1,3-1,6-D-glucan and adding the culture solution to food Manufacturing method of compound food. Add an alkaline solution at room temperature to the culture solution of microorganisms belonging to the genus Aureobasidium, whose main component is water-soluble β-1,3-1,6-D-glucan, and adjust the pH to 13 or higher to achieve water-soluble low-viscosity β- 1,3-1,6-D-glucan-containing culture broth is obtained, insoluble matter containing microorganisms is removed from the culture broth, and further adjusted to pH 2-10, water-soluble low viscosity β-1,3-1, A method for producing a water-containing low-viscosity β-1,3-1,6-D-glucan-containing aqueous solution characterized by obtaining an aqueous solution containing 6-D-glucan and then adding the aqueous solution to food.