JP2007267718A - METHOD FOR PRODUCING PURIFIED beta-D-GLUCAN - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a purified β-D-glucan, with which a β-D-glucan is separated from a microorganism culture solution containing a β-D-glucan and purified, and which is a method for obtaining a β-D-glucan having high transparency and to obtain a purified microorganism-derived β-D-glucan having high transparency. <P>SOLUTION: The method for producing a purified β-D-glucan comprises a first process for adjusting a microorganism culture solution containing a β-D-glucan or a ground microorganism solution, a second process for removing a microorganism or an insoluble admixture to give a supernatant and a third process for ultrafiltering the supernatant in alkalinity so as to remove the whole or a part of a low-molecular admixture from the β-D-glucan. The purified β-D-glucan having light transmittance of ≥60% at 660 nm in 0.2 wt.% of an aqueous solution at 25°C is obtained by the method. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

    The present invention relates to β-D-glucan which is a polysaccharide useful as a soft drink, a health food material, or a food thickener from a culture solution of microorganisms, particularly microorganisms belonging to the genus Aureobasidium sp. In particular, the present invention relates to a method for producing purified β-D-glucan, in which β-1,3-1,6-D-glucan is separated, purified efficiently, hygienically and simply.

   Conventionally, it is known that microorganisms represented by the genus Aureobasidium sp. Produce β-D-glucan (Agric. Biol. Chem. 47 (6), 1167-1172 (1983)). . β-D-glucan has been suggested to have an anticancer effect and an immune activation effect, and is useful as a health food material.

  In general, β-D-glucan has a single-helix or triple-helix structure in an aqueous solution due to its structure, and thus a β-glucan aqueous solution easily forms a gel. For this reason, the microorganism culture solution in which β-glucan is produced outside the cells is highly viscous and difficult to purify (Fragrance Journal, 5, 71-75 (1995)). As usual, the culture solution of microorganisms belonging to the genus Aureobasidium also contains β-1,3-1,6-D-glucan produced outside the cell body, so that the viscosity is high and the cell body is removed from the culture solution. However, it is very difficult to recover and purify β-1,3-1,6-D-glucan. For this reason, there are few reports on industrial methods for separating and purifying β-1,3-1,6-D-glucan from the culture solution of microorganisms belonging to the genus Aureobasidium.

  The inventors of the present invention disclosed in Patent Document 1 a culture solution mainly composed of β-1,3-1,6-D-glucan produced by a microorganism belonging to the genus Aureobasidium. Sp. In addition, the viscosity of the culture solution is decreased by adding an alkali at room temperature to a pH of 12 or more, and then an acid is added to adjust the pH from the neutral range to the vicinity of the acidic range. A method for purifying β-1,3-1,6-D-glucan, which is desalted by ultrafiltration after separating a 1,3-1,6-D-glucan-containing solution, is reported.

  The above purification method is an epoch-making method in that the culture solution containing β-1,3-1,6-D-glucan is reduced in viscosity so that cells can be easily removed by methods such as ultrafiltration. there were.

    However, the β-1,3-1,6-D-glucan aqueous solution separated and purified from the cells by the above method is not sufficiently transparent for use in soft drinks where transparency is important. Therefore, it is unsuitable for the use which fills a transparent container and makes it purified water.

In addition, β-1,3-1,6-D-glucan purified by the above method has room for improvement in long-term storage stability and thermal stability. For example, when a high concentration β-1,3-1,6-D-glucan aqueous solution of 0.5% (w / v) or more is used as a fluid food such as a soft drink, heat sterilization (pH 3. When the heat treatment is performed at 5 ° C. at 90 ° C., β-1,3-1,6-D-glucan partially aggregates or gels, and the aqueous solution may become non-uniform. For this reason, it is unsuitable for using for a high concentration drink.
JP 2004-321177 A

    The present invention relates to a method for producing purified β-D-glucan in which β-D-glucan is separated and purified from a microorganism culture solution or a microorganism disruption solution containing β-D-glucan, and has high transparency. It is an object to provide a method capable of obtaining the above. Another object of the present invention is to provide a purified β-D-glucan having high transparency derived from microorganisms.

    The present inventors have conducted research to solve the above-mentioned problems, and the purification obtained by performing the ultrafiltration step of the crude β-D-glucan solution under neutrality, which has been conventionally performed under neutrality. It has been found that the transparency of β-D-glucan is significantly improved.

    The present invention has been completed based on the above findings, and provides the following method for producing purified β-D-glucan and purified β-D-glucan.

    Item 1. a first step of adjusting the microorganism culture solution or microbial disruption solution containing β-D-glucan to pH 12 or higher, a second step of obtaining a supernatant by removing microorganisms or insoluble impurities, and limiting the supernatant under alkaline conditions And a third step of removing all or part of contaminants having a lower molecular weight than that of β-D-glucan by external filtration, and a method for producing purified β-D-glucan.

    Item 2. Item 2. The method according to Item 1, wherein the microorganism is an Aureobasidium microorganism.

    Item 3. Item 3. The method according to Item 2, wherein the microorganism belonging to the genus Aureobasidium is Aureobasidium pullulans.

    Item 4. Item 4. The Aureobasidium pullulans is Aureobasidium pullulans GM-NH-1A1 strain (FERM P-19285) or Aureobasidium pullulans GM-NH-1A2 strain (FERM P-19286) Method.

    Item 5. Item 5. The method according to any one of Items 1 to 4, wherein the β-D-glucan is β-1,3-1,6-D-glucan.

    Item 6. Item 6. The method according to any one of Items 1 to 5, wherein the ultrafiltration is carried out under an alkali having a pH of 10 or more.

    Item 7. Item 7. The method according to any one of Items 1 to 6, wherein a substance having a molecular weight of 5,000 or less is excluded by ultrafiltration.

    Item 8. Item 8. The method according to any one of Items 1 to 7, further comprising a step of sterilizing remaining bacteria.

    Item 9. The method according to any one of Items 1 to 8, further comprising a step of adjusting the pH of the β-D-glucan aqueous solution to 3.5 to 10.

    Item 10. Item 10. The method according to any one of Items 1 to 9, wherein the microorganism culture solution containing β-D-glucan is adjusted to pH 12 or higher in the first step, and the microorganism is removed in the second step to obtain a culture supernatant.

    Item 11. Item β-D-glucan obtained by the method according to any one of Items 1 to 10.

    Item 12. Β-D-glucan having a transmittance of light of 660 nm at 25 ° C. of a 0.2% (w / v) aqueous solution of 60% or more.

  The method of the present invention is characterized by ultrafiltration of a crude β-D-glucan aqueous solution using an alkaline aqueous solution, and the purified β-D-glucan obtained by the present method has a very high transparency. Therefore, the purified β-D-glucan obtained by the method of the present invention can be suitably used as a beverage filled in a transparent container.

  The purified β-D-glucan obtained by the method of the present invention is excellent in long-term storage stability. That is, the conventional purified β-D-glucan tended to increase in turbidity when stored for a long time, but the transparency of the aqueous solution of purified β-D-glucan obtained by the method of the present invention is maintained for a long time.

    The purified β-D-glucan obtained by the method of the present invention is excellent in thermal stability. That is, the conventional purified β-D-glucan sometimes gelled or aggregated by heat sterilization when a high-concentration aqueous solution was prepared, but the purified β-D-glucan aqueous solution obtained by the method of the present invention is Since gelation and aggregation hardly occur even after heat sterilization, it can be prepared in a high concentration aqueous solution. For example, the heat sterilization of beverages does not change the properties even when heat sterilized at 90 ° C. for 15 minutes at pH 4 or lower.

  Moreover, the number of miscellaneous bacteria contained in the β-D-glucan solution and endotoxin are reduced by ultrafiltration under alkaline conditions. For this reason, the sanitary condition at the time of refinement | purification is improved.

  In addition, when ultrafiltration is performed under neutral or acidic conditions, it is necessary to add an antifoaming agent of about 50 ppm (Ryodo-polyglycerester etc.), but in the method of the present invention, it is difficult to foam during ultrafiltration. There is no need to add an antifoaming agent. For this reason, the lifetime of the ultrafiltration membrane is increased accordingly. Moreover, highly safe purified β-D-glucan containing no antifoaming agent can be obtained.

  Further, by ultrafiltration using an alkaline aqueous solution, the ultrafiltration membrane is hardly clogged, and the membrane can be easily washed and regenerated.

Hereinafter, the present invention will be described in detail.
(I) Method for Producing Purified β-D-glucan The method for producing purified β-D-glucan of the present invention includes a first step of adjusting a microorganism culture solution or a microorganism disruption solution containing β-D-glucan to pH 12 or more. A second step of removing microorganisms or insoluble contaminants to obtain a supernatant, and ultrafiltration of the supernatant under alkalinity to remove all or a part of contaminants having a lower molecular weight than β-D-glucan. It is a method including three steps.
First step
<Microbe / β-D-glucan>
The microorganism is not particularly limited as long as it can produce β-D-glucan. Examples of β-D-glucan include β-1,3-D-glucan, β-1,6-D-glucan, β-1,3-1,6-D-glucan and the like. These β-D-glucans exhibit useful physiological activities.

  Examples of microorganisms that produce these β-D-glucans include Aurebasidium sp. Microorganisms and baker's yeast (S. cerevisiae). Among these, Aureobasidium microorganisms are preferred because they mainly produce β-1,3-1,6-D-glucan, which has been reported to have an anticancer action and an immune activation action. Examples of microorganisms belonging to the genus Aureobasidium include microorganisms such as Aurebasidium pullulans.

    In particular, aureobasidium pullulans is preferable in that the culture medium has a relatively low viscosity and high production of β-1,3-1,6-D-glucan. Aureobasidium sp. K-1 strain ( Aureobasidium pullulans GM-NH-GM1A1 strain (hereinafter sometimes referred to as “GM1A1 strain”) and Aureobasidium pullulans GM-NH-GM1A2 strain (hereinafter also referred to as “K-1 strain”) , Sometimes referred to as “GM1A2 strain”). These strains have been deposited as FERM P-19285 and FERM P-19286 at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary, respectively. The Aureobasidium genus K-1 strain is known to produce two types of β-1,3-1,6-D-glucan having a molecular weight of 2 million or more and about 1 million. In addition, β-glucan produced by K-1 strain is known to have a sulfoacetic acid group (Arg. Biol. Chem., 47, 1167-1172 (1983)), Science and Industry, 64, 131-135 ( 1990)).

The present invention can also be applied to the purification of mushroom-derived β-D-glucan. In this case, mushroom cells may be disrupted in, for example, a buffer solution to prepare a disrupted solution containing glucan and used for the first step.
<Culture of microorganisms>
Microbial culture methods for secreting β-D-glucan into the culture medium are known. For example, a method of culturing an aureobasidium microorganism to produce β-1,3-1,6-D-glucan is described in Biosci. Biotech. Biochem., 57 (8), 1348-1349, 1993; No. 6-340701 (Shin Nippon Oil); Japanese Patent Application No. 9-56391 (Nippon Petroleum); Japanese Patent Application Laid-Open No. 7-51081 (Nippon Synthetic Chemical Industry).

    Examples of carbon sources that can be used for cultivation of microorganisms belonging to the genus Aureobasidium include carbohydrates such as sucrose, glucose and fructose, peptone, yeast extract and the like. Examples of the nitrogen source include inorganic nitrogen sources such as ammonium sulfate, sodium nitrate, and potassium nitrate. In some cases, inorganic salts such as sodium chloride, potassium chloride, phosphate, magnesium salt and calcium salt, and trace metal salts such as iron, copper and manganese are appropriately used to increase the production amount of β-D-glucan. Adding vitamins and gluconic acid is also an effective method.

  Further, for example, when a microorganism belonging to the genus Aureobasidium. Sp is cultured in a medium in which ascorbic acid is added to a tourpec medium containing sucrose as a carbon source, a high concentration of β-1,3-1,6- It has been reported to produce D-glucan (Agric. Biol. Chem., 47, 1167-1172 (1983); Science and Industry, 64, 131-135 (1990); Japanese Patent Application No. 5-22229 ). What added ascorbic acid to the tour peck culture medium containing sucrose, and also added organic nutrients, such as a yeast extract and peptone as needed, can also be used conveniently.

Aureobasidium sp. Microorganisms are usually aerobically cultured by aeration and agitation. The culture temperature is preferably about 10 to 45 ° C, more preferably about 20 to 35 ° C. Moreover, about 3-7 are preferable and, as for pH of a culture solution, about 3.5-5 are more preferable. It is also preferable to control the culture solution pH within the above range using an alkali or an acid during the culture. Further, an antifoaming agent may be added to suppress foaming of the culture solution. If the culture time is about 1 to 10 days, a sufficient amount of β-D-glucan can be produced, but usually about 1 to 4 days. The culture time may be determined while measuring the production amount of β-glucan.
<Microbial disruption solution>
Baker's yeast or the like produces β-D-glucan such as β-1,3-1,6-D-glucan in the microbial cells. Therefore, the culture solution of baker's yeast or the like is collected as it is or once suspended and suspended in a buffer solution or the like, the suspension is disrupted using ultrasonic waves to release β-D-gluca outside the cells. You can do it. This microorganism disruption solution is used for the next step.
<Low viscosity>
Β-glucan polysaccharide containing β-1,3-1,6-D-glucan as a main component is 0.1% to several percent (w / v) The viscosity of the contained culture solution is several hundred to several thousand cP ([mPa · s]) when measured at 30 ° C. using a BM type rotational viscometer (manufactured by Toki Sangyo Co., Ltd.). It exhibits a very high viscosity. The same applies to the microorganism disruption liquid.

    In the first step, the pH of the culture solution or disrupted solution is adjusted to 12 or more, preferably 13 or more. If an alkali is added while stirring the culture solution or crushed solution, the pH can be increased. The type of alkali is not particularly limited as long as it is soluble in water. For example, when sodium hydroxide is used, if the final concentration in the culture solution is 0.5% (w / v) or more, preferably 1.25% (w / v) or more, The pH can be 12 or higher.

When the pH is set to 12 or more, the viscosity of the culture solution is suddenly or instantaneously decreased to about several cP ([mPa · s]) (Japanese Patent Laid-Open No. 2004-321177).
Any known alkali can be used without limitation. Known alkalis include alkali carbonate aqueous solutions such as calcium carbonate aqueous solution, sodium carbonate aqueous solution, potassium carbonate aqueous solution and ammonium carbonate aqueous solution; alkali hydroxide aqueous solution such as sodium hydroxide aqueous solution, potassium hydroxide aqueous solution and calcium hydroxide aqueous solution. An aqueous ammonia solution or the like. For edible use, an aqueous sodium hydroxide solution recognized as a food additive is preferred.
Second Step In the second step, insoluble contaminants such as microbial cells and microbial cells are removed from the alkaline culture solution or crushed liquid.

  Conventionally, it has been difficult to remove cells from a highly viscous culture solution. Therefore, the culture solution of the microorganism of the genus Aureobasidium currently marketed contains bacteria. On the other hand, the culture solution or microorganism disruption solution whose viscosity has been reduced by the alkali treatment tends to settle the cells and insoluble substances, and therefore these can be easily removed.

  Methods for separating and removing bacterial cells and insoluble impurities from the culture solution or crushed solution are well known. Any removal method may be employed in the method of the present invention, but industrially, removal of cells by a filter press or a continuous centrifugal separator is preferred. Thereby, a part or all of insoluble impurities other than the cells are usually removed. It is preferable to completely remove insoluble impurities together with the cells.

  In removing the cells, an acid may be added to the culture medium in advance to adjust the pH from the neutral to the vicinity of the acidic range (pH of about 3 to 9, preferably about 3.5 to 8), or an alkaline culture solution. The cells may be removed as they are. Even if the pH is adjusted from near neutral to near acidic, the polysaccharide does not gel, and a low viscosity is maintained.

The kind of acid for adjusting the pH is not particularly limited, and a known acid can be used. Known acids include hydrochloric acid, phosphoric acid, sulfuric acid, citric acid, malic acid, gluconic acid, amino acids and the like. For edible use, citric acid, malic acid and gluconic acid which are recognized as food additives are preferred.
The third step alkali treatment, sterilization or removal of insoluble contaminants can be used for food as they are, but in order to improve the stability and storage stability when sterilized by heating, It is desirable to remove soluble impurities such as metal ions. Conventionally, these contaminants have been removed by ultrafiltration under acidic to neutral pH.

    In the method of the present invention, the impurities are removed by ultrafiltration of the culture supernatant obtained by sterilization using an alkaline aqueous solution. The ultrafiltration may be performed using an ultrafiltration membrane having an excluded molecular weight of about 55,000, preferably about 10,000.

The pH of the dialysate is usually 10 or higher, preferably 12 or higher. The upper limit of pH is usually about 13.5. The dialysate is an aqueous calcium carbonate solution such as 0.005 to 5% by weight calcium carbonate aqueous solution, sodium carbonate aqueous solution, potassium carbonate aqueous solution or ammonium carbonate aqueous solution; sodium hydroxide aqueous solution, potassium hydroxide aqueous solution or calcium hydroxide aqueous solution. An aqueous alkali hydroxide solution such as: an aqueous ammonia solution may be used. A sodium hydroxide aqueous solution is particularly preferable.
The alkaline β-D-glucan aqueous solution obtained in the third step of the pH adjustment step can be used as it is or after drying.

Moreover, when using as a drink component, a food additive, etc., it is preferable to adjust pH to about 3.5-10. The pH-adjusted β-D-glucan aqueous solution can be used as it is or after drying.
Sterilization step When the β-D-glucan aqueous solution obtained in the third step is used for food, it is preferably sterilized by a method such as heat sterilization, filter sterilization, or ultraviolet sterilization. It is stipulated that heat sterilization for edible use should normally be performed at about 65 to 90 ° C. under a pH of about 3.5 to 10.

  When the pH adjustment is performed, sterilization may be performed before or after that.

Each process except the sterilization in the method of the present invention can be performed at a temperature of about 5 to 50 ° C., and it may be normally performed at room temperature or room temperature.
(II) Purified β-D-glucan The purified β-D-glucan aqueous solution obtained by the above method has a very high transparency, and a 0.2% (w / v) aqueous solution of 660 nm light at 25 ° C. The transmittance is 60% or more. Moreover, the viscosity of the purified β-D-glucan aqueous solution is very low.

Further, the primary structure is considered to be the same as that of natural β-D-glucan before purification. That is, the ¹ H NMR spectrum of a β-D-glucan solution using 1N sodium hydroxide heavy aqueous solution as a solvent has two signals of 4.7 ppm and 4.5 ppm.
EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples and test examples, but the present invention is not limited to these examples.
(I) Purification of β-D-glucan
Examples 1-3, Comparative Example 1
(1) Culture production of β-D-glucan After putting 100 ml of liquid medium having the composition shown in Table 1 into a 500 ml shoulder flask and autoclaving at 121 ° C. for 15 minutes, GM1A1 strain was obtained. One platinum ear was inoculated aseptically from a slant having the same medium composition, and a seed culture solution was prepared by aeration-stirring culture at 30 rpm at 30 ° C. for 24 hours.

    Next, 200 L of the medium having the same composition was placed in a 300 L culture apparatus (manufactured by Maruhishi Bioengineering), and the above seed culture solution was aseptically inoculated into those subjected to autoclaving at 121 ° C. for 15 minutes, Aeration stirring culture was performed at 200 rpm, 28 ° C., 40 L / min.

    The pH at the start of the culture was 3.5. Further, the pH after culturing for 120 hours was 4.30, the turbidity was 30 OD at OD 660 nm, and the polysaccharide concentration was 0.9% (w / v). The polysaccharide concentration was collected by sampling several mL of the culture solution, centrifuging and removing the cells, and then adding ethanol to the supernatant so that the final concentration was 66% (v / v) to precipitate the polysaccharide. Then, it melt | dissolved in ion-exchange water and quantified with the phenol sulfuric acid method.

(2) Alkali treatment When the culture solution obtained in Example 1 was measured with a BM type rotational viscometer, the viscosity was 1200 [mPa · s] at 30 ° C. When 25% (w / w) sodium hydroxide was added to the culture solution so that the final concentration was 2.4% (w / v) and stirred, the pH became 13.6, and the viscosity immediately decreased.

Subsequently, the culture solution was transferred to a 1000 L tank, diluted to 3 times with drinking water, and the viscosity was measured in the same manner. The viscosity was 10 [mPa · s].
(3) Sterilization Next, 1 wt% of diatomaceous earth was added to this culture solution, and the cells were removed using a Kamata filter press (manufactured by Kamata Machinery Co., Ltd.), and finally about 600 L of a culture filtrate was obtained. When the polysaccharide concentration was quantified by the phenol-sulfuric acid method, it was 0.3% (w / v), and the polysaccharide recovery rate was almost 100%.
(4) Dialysis of β-glucan aqueous solution 50% citric acid aqueous solution was added to the above β-glucan aqueous solution (culture filtrate) to adjust to pH 10 of Example 1, and pH 12 adjusted to Example 2, What was adjusted to pH 13 was Example 3, and what was adjusted to pH 7 was Comparative Example 1. Dialysis was performed using a UF membrane (spiral type polysulfone UF membrane module CF30-F4-PT manufactured by Nitto Denko Corporation, molecular weight cut 50,000), and finally, the solution was concentrated by hydrolysis 64 times. That is, other than glucan was diluted 64 times. Here, hydration added the drinking water adjusted to each pH using sodium hydroxide.

Moreover, in this process, when dialysis is performed in the conventional neutrality (Comparative Example 1 of pH 7), foaming is observed, and it is necessary to add an antifoaming agent (Rhodo-polyglycerester) of about 50 ppm. It was. In this method (Examples 1 to 3), there was no foaming and the antifoaming agent did not need to be blended.
(5) Sterilization treatment Subsequently, the β-glucan concentrate is passed through a filter having a pore size of 0.8 μm, and then held at 95 ° C. for 3 minutes using a heating unit for hot filling (manufactured by Nisaka Seisakusho). Sterilization treatment was performed to obtain a final purified β-glucan aqueous solution. The concentration of β-D-glucan in this aqueous solution was 0.22% (w / v) as measured by the phenol sulfuric acid method. Further, the total recovery rate of β-D-glucan from the culture broth was about 70% by weight.

(II) Measurement of various physical properties of β-D-glucan Various physical properties of the purified β-D-glucan obtained in Examples 1 to 3 and Comparative Example 1 were measured. These physical properties were measured for an aqueous β-D-glucan solution adjusted to pH 3.5 with citric acid. The results are shown in Table 2 below.
Sampling the polysaccharide concentration purified glucan solution, ethanol is added to the final concentration of 66% (w / v) to precipitate the polysaccharide, the precipitate is recovered, dissolved in ion-exchanged water, phenol sulfate Quantified by the method.

    Specifically, 100 (μl) of the purified glucan solution was sampled, and ethanol was added thereto so that the final concentration was 66% (w / v) to precipitate the polysaccharide. (If a polysaccharide is present, a white precipitate is seen.) The supernatant was discarded by centrifuging, and a 66% (w / v) aqueous ethanol solution (1 ml) was added thereto and stirred, and then the polysaccharide was washed. The supernatant was discarded through a centrifuge, and ion exchanged water 1 (ml) was added to dissolve the polysaccharide.

The test solution 50 (μl) and ion-exchanged water 450 (μl), 5 (% (w / v)) phenol solution 500 (μl) are put into a test tube and stirred, and then concentrated sulfuric acid 2.5 (ml) is added and stirred. did. The absorbance (Abs 490 nm) of the solution allowed to cool at room temperature for 30 minutes was measured and quantified from a calibration curve prepared using glucose (“Sugar Chemistry” (Asakura Shoten) Shinya Ryu, Nozomi Nanura, Toshio Kithata, Onishi (See Masaken).
Sampled glucan solution with a total sugar concentration was sampled and quantified by the phenol sulfate method.

Specifically, purified glucan solution 0.1 (ml) was sampled, and ion-exchanged water 0.9 (ml) was added thereto and stirred. This solution 50 (μl) and ion-exchanged water 450 (μl), 5 (% (w / v)) phenol solution 500 (μl) were stirred and mixed, and concentrated sulfuric acid 2.5 (ml) was added thereto and stirred. . The absorbance (Abs 490 nm) of the solution that was allowed to cool at room temperature for 30 minutes was measured, and quantified from a calibration curve prepared using glucose.
Weight average molecular weight (Mw), number average molecular weight (Mn), peak molecular weight (Mp)
The number average molecular weight Mn, the weight average molecular weight Mw, and the molecular weight Mp of the peak were measured with a WATERS GPC measuring apparatus (ALLIANANCE 2695) (TSK-GELL column x 2 manufactured by Tosoh Corporation, developing solvent 200 mM potassium phosphate aqueous solution). Shodex pullulan was used as a molecular weight marker.
1,3 / (1,3 + 1,6) bond ratio β-1,3 bond ratio relative to the sum of β-1,3-bond and β-1,6-bond (β-1,3 / (β-1 , 3 + β-1,6)) was determined from the integration ratio of ¹ H-NMR (JEOL 500MHZ) signal. ^{In the 1} H-NMR spectrum, two signals of a proton at C1 position associated with 4.7 ppm of β-1,3 bond and a proton at C1 position associated with 4.5 ppm of β-1,6 bond were observed.
The ratio of β-D-glucan in the glucan purity polysaccharide was determined from the integral ratio of ¹ H-NMR (500 MHz manufactured by JEOL Ltd.) signal. ^{In 1} H-NMR, a signal of 4.7 ppm and a signal of 4.5 ppm were observed, but peaks derived from other polysaccharides such as 5.2 ppm derived from pullulan were not observed. From this, it was judged that the β-D-glucan purity in the polysaccharide was 100% in any example.
The viscosity of the aqueous solution for viscosity measurement was measured using a BM viscometer manufactured by TOKIMEC.
Light transmittance Using a Hitachi U-110 spectrophotometer, the light transmittance (%) at 660 nm of a 0.2% (w / v) aqueous solution at 25 ° C. was measured.
General viable count before and after sterilization of general viable count was measured using 3M Petri film (for general viable count).
Endotoxin Concentration The endotoxin concentration was measured using the Limulus scalar-KY test Wako series from Wako Pure Chemical Industries.

    From Table 2, the molecular weight is about 50,000, the average molecular weight is about 100,000, the glucan purity is about 100%, the degree of branching is about 50, and β-1,3-1,6 having almost the same structure. It can be seen that -D-glucan is obtained.

    About the transmittance | permeability, in Examples 1-3, the transparency of 2 times or more of the comparative example 1 was shown. This is probably because impurities were efficiently removed during dialysis by dialysis under alkaline liquidity. Moreover, since there is no foaming, it is thought that a defoamer is not required is also a cause. Moreover, since the viscosity was high, it was considered that the solubility of β-1,3-1,6-D-glucan was increased, and this was also considered to contribute to transparency. The conventional one dissolves about 40 to 50% at 30 ° C., whereas in the case of alkali, about 50 to 60% dissolves.

  Moreover, in Examples 1-3, compared with the comparative example 1, the general viable count before sterilization and the endotoxin of the last aqueous solution are very low. The reason why the number of viable cells is low in Examples 1 to 3 is considered to be due to the suppression of the growth of viable cells under alkaline conditions. Moreover, it is thought that the amount of endotoxins is small because viable bacteria themselves are small and endotoxins are decomposed by alkali. From this, it can be said that the method of the present invention is a sanitary and safe purification method.

(III) Examination of thermal stability A 2% (w / v) aqueous solution of each purified β-D-glucan obtained in Comparative Example 1 and Example 2 was further added to a UF membrane (spiral polysulfone UF manufactured by Nitto Denko Corporation). A membrane module CF30-F4-PT (molecular weight cut 50,000) was dialyzed and concentrated to prepare a β-D-glucan aqueous solution having a concentration shown in Table 3 below.

    These β-D-glucan aqueous solutions were heated at 90 ° C. for 30 minutes, the light transmittance at 660 nm was measured, and the properties were visually observed. The physical properties were measured or observed after adjusting the pH to 3.5 with citric acid. The results are shown in Table 3. In Table 3, ○ of the thermal stability item indicates that the fluidity is the same as that before heating, Δ indicates the tendency of gelation, that is, a state where gel is observed in some places, and × indicates that the whole is gelled. The state without fluidity is shown. Moreover,-of the item of the transmittance | permeability and a viscosity shows a measurement impossibility.

    The β-D-glucan of Comparative Example 1 dialyzed at pH 7 started to gel when the concentration exceeded about 0.5% (w / v), and completely fluidized at 0.7% (w / v) or more. Lost to. In contrast, the β-D-glucan of Example 2 dialyzed at pH 12 was fluid even at 1% (w / v) and had good thermal stability. Moreover, even if it concentrated to 1.0% (w / v), high transparency was shown.

    In this way, a practical β-glucan aqueous solution having a high concentration of β-glucan, which is unconventional and having high transparency and high thermal stability, could be supplied.

  Based on the above, high viscosity β-1,3-1,6-D-glucan produced by microorganisms belonging to the genus Aureobasidium is reduced in viscosity by alkali treatment, the cells are removed, and then dialyzed under alkalinity. It was confirmed that β-1,3-1,6-D-glucan having high transparency and excellent thermal stability can be obtained by the method of performing the above. Furthermore, contamination with various bacteria could be prevented, and the number of live bacteria and endotoxin before sterilization could be kept low. In other words, it was confirmed that it is effective for producing a hygienic and safe product.

Claims (12)

    a first step of adjusting the microorganism culture solution or microbial disruption solution containing β-D-glucan to pH 12 or higher, a second step of obtaining a supernatant by removing microorganisms or insoluble impurities, and limiting the supernatant under alkaline conditions And a third step of removing all or part of contaminants having a lower molecular weight than that of β-D-glucan by external filtration, and a method for producing purified β-D-glucan.     The method according to claim 1, wherein the microorganism is an Aureobasidium microorganism.     The method according to claim 2, wherein the microorganism belonging to the genus Aureobasidium is Aureobasidium pullulans.     The Aureobasidium pullulans is an Aureobasidium pullulans GM-NH-1A1 strain (FERM P-19285) or an Aureobasidium pullulans GM-NH-1A2 strain (FERM P-19286). the method of.     The method according to any one of claims 1 to 4, wherein the β-D-glucan is β-1,3-1,6-D-glucan.   The method according to any one of claims 1 to 5, wherein the ultrafiltration is performed under an alkali having a pH of 10 or more.   The method according to any one of claims 1 to 6, wherein a substance having a molecular weight of 5,000 or less is excluded by ultrafiltration.   Furthermore, the method in any one of Claims 1-7 including the process of sterilizing the remaining miscellaneous bacteria.   Furthermore, the method in any one of Claims 1-8 including the process of adjusting pH of (beta) -D-glucan aqueous solution to 3.5-10.   The method according to any one of claims 1 to 9, wherein a microorganism culture solution containing β-D-glucan is adjusted to pH 12 or higher in the first step, and microorganisms are removed in the second step to obtain a culture supernatant.   A β-D-glucan obtained by the method according to claim 1. Β-D-glucan having a transmittance of light of 660 nm at 25 ° C. of a 0.2% (w / v) aqueous solution of 60% or more.