JP2007267720A - METHOD FOR PRODUCING PURIFIED beta-1,3-1,6-D-GLUCAN - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for highly producing a β-1,3-1,6-D-glucan by culturing Aureobasidium pullulans. <P>SOLUTION: The method for producing a β-1,3-1,6-D-glucan comprises a process for adjusting a medium to pH 2.7-6 by using gluconic acid and/or ascorbic acid and a process for culturing Aureobasidium pullulans in the obtained medium to produce a β-1,3-1,6-D-glucan. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for producing β-1,3-1,6-D-glucan by culturing Aureobasidium pullulans.

  β-glucan (β-1,3-D-glucan, β-1,6-D-glucan, or β-1,3-1,6-D-glucan) is abundant in mushrooms (the body of basidiomycetes) It is a component that is contained, and is contained not only in fruiting bodies but also in cultured mycelium. Β-glucan is also known to have immunostimulatory activity and antitumor activity. For example, β-glucan extracted from Suehirotake, Kawaratake and Shiitake is sold as a pharmaceutical such as an anticancer agent (Japanese Patent Laid-Open Nos. 61-146192 and 62-201901).

  In addition, it is known that microorganisms belonging to the genus Aureobasidium, which are incomplete bacteria, secrete and produce β-glucan outside the cells. It has been reported that this β-glucan has an immunostimulatory activity and can be used as a functional food or an intestinal regulating agent that performs functions such as intestinal bifidobacteria growth, constipation prevention, and immune enhancement (Japanese Patent Laid-Open No. 62-201901). JP, JP-A-6-34071, JP-A-5-308987).

  Since the production amount of β-1,3-1,6-D-glucan produced by microorganisms belonging to the genus Aureobasidium varies depending on the culture conditions, it is required to find conditions that allow efficient production.

  For example, Patent Document 1 discloses that a microorganism of the genus Aureobasidium is prepared by adjusting the pH of a Czapek medium supplemented with sucrose and ascorbic acid to 4 to 7, cultured at 10 to 50 ° C., and β-1,3-1, A method for secreting 6-D-glucan into a medium is disclosed.

  Patent Document 2 cultivates microorganisms belonging to the genus Aureobasidium using a liquid medium controlled to pH 6.8 to 8.5, and has an uncolored β-1 having a predetermined hyperbranching and molecular weight. , 3-1,6-D-glucan is disclosed.

  In addition, Patent Document 3 shows the result of examining the carbon source in the medium components in order to culture the Aureobasidium pullulans Foundation for Fermentation Research Institute deposit No. IFO4466 to produce highly branched β-glucan. Disclosure. According to this document, the carbon source of the medium in which β-glucan is highly produced is xylose, not sucrose or fructose.

  Patent Document 4 discloses that β-1,3-glucan is obtained by culturing a microorganism belonging to the genus Aureobasidium by aerobic fermentation in an aqueous medium containing glucose and fructose or sucrose as a carbon source. Have been disclosed to be produced efficiently. In this method, since the aqueous medium contains a specific amount of yeast extract, the production amount of β-1,3-glucan increases.

  In addition, Non-Patent Document 1 discloses that when a highly branched β-1,3-glucan is produced in the K-1 strain belonging to the genus Aureobasidium, by adding an ascorbic acid-related compound to the Czapec medium, the highly branched β- It discloses that production of 1,3-glucan is promoted. This document teaches that the amount of glucan produced hardly changes when D-gluconic acid is added to the medium.

However, according to the culture methods described in the above documents, β-1,3-1,6-D-glucan cannot be produced at a sufficiently high level for practical use by culturing aureobasidium pullulans.
JP 2004-321177 A JP-A-9-56391 JP-A-6-340701 JP-A-7-51081 Biosci. Biotech. Biochem., 57 (8), 1348-1349, (1993)

  The present invention relates to a method for producing β-1,3-1,6-D-glucan capable of culturing aureobasidium pullulans to produce β-1,3-1,6-D-glucan at a high level. It is an issue to provide.

  In order to solve the above-mentioned problems, the present inventor conducted research and used a medium whose pH was adjusted to 3 to 5 with gluconic acid and / or ascorbic acid when culturing aureobasidium pullulans. The inventors have found that the amount of β-1,3-1,6-D-glucan secreted into the medium is extremely high.

  The present invention has been completed based on the above findings, and provides the following method for producing β-1,3-1,6-D-glucan.

  Item 1. A step of adjusting the medium pH to 2.7-6 using gluconic acid and culturing aureobasidium pullulans in the obtained medium to produce β-1,3-1,6-D-glucan The manufacturing method of (beta) -1,3-1,6-D-glucan including a process.

  Item 2. Item 2. The method according to Item 1, wherein the pH is adjusted to 2.7 to 6 by adjusting the gluconic acid concentration in the medium to 0.2 to 1.2% (w / v).

  Item 3. Item 3. The method according to Item 1 or 2, wherein the medium pH is adjusted to 2.7 to 6 using ascorbic acid in addition to gluconic acid.

  Item 4. Item 4. The method according to Item 3, wherein the concentration of ascorbic acid in the medium is 0.1% (w / v) or less.

  Item 5. Item 5. The method according to any one of Items 1 to 4, wherein the acid in the medium at the start of culture is substantially only gluconic acid or further ascorbic acid.

  Item 6. Item 6. The method according to any one of Items 1 to 5, wherein fructose is contained at a concentration of 1 to 10% (w / v) in the medium at the start of culture.

  Item 7. Item 7. The method according to Item 6, wherein the carbon source in the medium at the start of the culture is substantially only fructose except for gluconic acid or further ascorbic acid.

  Item 8. Item 8. The method according to any one of Items 1 to 7, wherein the Aureobasidium pullulans is Aureobasidium pullulans GM-NH-1A1 strain (FERM P-19285).

  Item 9. Item 9. The method according to any one of Items 1 to 8, further comprising a step of recovering the produced β-1,3-1,6-D-glucan.

  Item 10. Β-1,3-1,6-D-glucan obtained by culturing aureobasidium pullulans in a medium containing 0.2 to 1.2% (w / v) gluconic acid and pH 2.7 to 6 The manufacturing method of (beta) -1,3-1,6-D-glucan including the process of producing.

  Item 11. Item 11. The method according to Item 10, wherein ascorbic acid is contained in the medium at a concentration of 0.1% (w / v) or less.

  Item 12. Item 12. The method according to Item 10 or 11, wherein the acid in the medium at the start of the culture is substantially only gluconic acid or further ascorbic acid.

  Item 13. Item 13. The method according to any one of Items 10 to 12, wherein fructose is contained in the medium at a total concentration of 1 to 10% (w / v).

  Item 14. Item 14. The method according to Item 13, wherein the carbon source in the medium is substantially only fructose except gluconic acid or further ascorbic acid.

  Item 15. Item 15. The method according to any one of Items 10 to 14, wherein the Aureobasidium pullulans is Aureobasidium pullulans GM-NH-1A1 strain (FERM P-19285).

  Item 16. Item 16. The method according to any one of Items 10 to 15, further comprising a step of recovering the produced β-1,3-1,6-D-glucan.

  Item 17. A step of adjusting the medium pH to 2.7-6 using ascorbic acid and culturing aureobasidium pullulans in the obtained medium to produce β-1,3-1,6-D-glucan The manufacturing method of (beta) -1,3-1,6-D-glucan including a process.

  Item 18. Item 18. The method according to Item 17, wherein the Aureobasidium pullulans is Aureobasidium pullulans GM-NH-1A1 strain (FERM P-19285).

  In the method of the present invention, when culturing aureobasidium pullulans to produce β-1,3-1,6-D-glucan, the medium pH is adjusted using gluconic acid and / or ascorbic acid as a pH adjusting agent. By adjusting to 2.7-6, the yield of glucan becomes very high.

  Further, when gluconic acid is used as a pH adjuster and the carbon source excluding gluconic acid is fructose, the yield of glucan is further increased. Further, when ascorbic acid is used as a pH adjuster and the carbon source excluding ascorbic acid is sucrose, the yield of glucan is further increased.

Hereinafter, the present invention will be described in detail.
(I) First Method The first method for producing β-1,3-1,6-D-glucan of the present invention comprises gluconic acid and is ole in a medium having a pH of about 2.7-6. It is a method comprising a step of culturing Obacidium pullulans to produce β-1,3-1,6-D-glucan.

Preferably, the step of adjusting the medium pH to 2.7 to 6 using gluconic acid, and culturing aureobasidium pullulans in the obtained medium to form β-1,3-1,6-D-glucan It is a method including the process of producing.
The microorganism microorganism may be an aureobasidium pullulans, and any strain having a large production amount of β-1,3-1,6-D-glucan may be selected and used. Among them, Aureobasidium pullulans GM-NH-GM1A1 strain (mutated as FERM P-19285 at the National Institute of Advanced Industrial Science and Technology (AIST)), which is a mutant strain of the Aureobasidium genus K-1 strain, and GM-NH -GM1A2 strain (deposited as FERM P-19286 at the National Institute of Advanced Industrial Science and Technology as the FERM P-19286) is a strain that produces a large amount of β-1,3-1,6-D-glucan and produces pullulan It is preferable because it is difficult. In particular, the GM-NH-1A1 strain is more preferable.
Medium Composition In the method of the present invention, a medium adjusted to about pH 2.7 to 6 using gluconic acid is used. It is preferable to adjust to about pH 3-5, and it is more preferable to adjust to about pH 3-4.

  The concentration of gluconic acid for pH adjustment is preferably about 0.2 to 1.2% (w / v), and more preferably about 0.2 to 0.5% (w / v). Moreover, it is preferable that the acid contained in a culture medium is substantially only gluconic acid. Thereby, β-1,3-1,6-D-glucan production amount is remarkably increased.

  In addition to the above-mentioned concentration range of gluconic acid, ascorbic acid is used at a concentration of 0.1% (w / v) or less and a medium whose pH is adjusted to about 2.7 to 6 is used. , 3-1,6-D-glucan production is significantly increased. Also in this case, it is preferable that the acid contained in the medium is substantially only gluconic acid and ascorbic acid.

  The medium component only needs to contain carbon and nitrogen necessary for the growth of microorganisms. 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, calcium salt, and trace metal salts such as iron, copper, manganese and vitamins are used as appropriate to increase the production of β-glucan. It is also an effective method to add a kind.

  Examples of the carbon source include carbohydrates such as fructose, glucose and sucrose, and organic nutrient sources such as peptone and yeast extract. Among these, fructose and sucrose are preferable, and fructose is more preferable in that the production amount of β-1,3-1,6-D-glucan is high. The carbon source contained in the medium is preferably substantially only fructose or sucrose, more preferably only fructose, except for gluconic acid added as a pH adjuster, and optionally ascorbic acid.

  The carbohydrate concentration (particularly fructose concentration) in the medium is preferably about 1 to 10% (w / v), more preferably about 3 to 5% (w / v). If it is the said range, while the production amount of (beta) -1,3-1,6-D-glucan will become sufficient, the production of pullulan will be suppressed and (beta) -1,3-1,6-D- in polysaccharide will be suppressed. The glucan ratio is high and the purification is easy.

As a suitable medium, in the Tuapec medium, the carbon source is only fructose or sucrose, 0.2 to 1.2% (w / v) gluconic acid, or even 0.1% (w / v) or less. Examples include a medium supplemented with ascorbic acid.
Cultivation method The liquid medium described above is inoculated with a seed culture solution of Aureobasidium pullulans or cells scraped from a solid medium, and aerated at a temperature of about 10 to 45 ° C, preferably about 20 to 35 ° C. Aerobic culture may be performed by stirring. The composition and pH of the seed culture solution are not particularly limited.

  In the method of the present invention, batch culture in which a carbon source is added to the liquid medium described above at the start of culture may be performed, or fed-batch culture in which a substrate is divided and added may be performed. Continuous culture can also be employed.

  In the case of batch culture, the culture time is preferably about 24 to 240 hours, more preferably about 72 to 168 hours. If it is the said range, the production amount of (beta) -1,3-1,6-D-glucan will become high.

  In general, in the fed-batch culture method, the number of additions and the addition amount of the substrate are judged from the acid, alkali titration, or the substrate (carbon source) concentration in the reaction solution to titrate the change in pH due to the progress of the reaction. This is a method of adding a substrate intermittently or intermittently (Kasai et al., Journal of Biotechnology, 1997, 75, 255-275). When fed-batch culture is performed in the present invention, the carbon source may be added continuously or intermittently. The carbon source is generally added after being dissolved in a sterilized medium, water, or buffer. This carbon source does not contain gluconic acid or ascorbic acid added as a pH adjuster. In particular, it is preferable to maintain the fructose or sucrose concentration at about 1 to 5% (w / v). In the case of fed-batch culture, the culture time is preferably about 24 to 240 hours, more preferably about 72 to 168 hours. If it is the said range, the production amount of (beta) -1,3-1,6-D-glucan will become high.

  In general, continuous culture is performed by, for example, determining the amount of substrate added from the acid or alkali titration, or the concentration of the substrate (carbon source) in the reaction solution, which titrates pH fluctuations due to the progress of the reaction. This is a method of supplying a culture medium containing a culture medium containing a purified product that is continuously discharged (Japanese Patent Laid-Open No. 2004-041076). When continuous culture is performed in the present invention, a carbon source may be intermittently added, and a culture solution containing β-1,3-1,6-D-glucan may be continuously collected together with the cells. The definition of the carbon source and the fructose and sucrose concentrations are as described for the fed-batch culture.

In any of the culture methods, gluconic acid or ascorbic acid added as a pH adjuster is consumed with the growth of the bacteria, and the medium pH increases. Therefore, during culture, the pH of the medium is controlled to about 2.7-6 by adding gluconic acid to the medium and keeping the concentration in the range of about 0.2-1.2% (w / v). It is preferable. In some cases, ascorbic acid may be used in addition to gluconic acid, and the culture medium pH during culture may be maintained at about 2.7-6. Ascorbic acid is preferably used in a range where the concentration in the medium is 0.1% (w / v) or less.
Recovery of β-1,3-1,6-D-glucan The culture solution thus obtained contains β-1,3-1,6-D-glucan secreted and produced from the cells. The culture fluid is highly viscous due to the viscosity of glucan. Therefore, usually, a culture solution containing β-1,3-1,6-D-glucan together with the cells may be collected.

Β-1,3-1,6-D-glucan is added to the supernatant obtained by centrifuging this culture solution at about 15,000 to 20,000 g for about 1 to 20 minutes, for example, by adding an organic solvent. Can be obtained as a precipitate.
(I) Second Method The second method for producing β-1,3-1,6-D-glucan of the present invention comprises ascorbic acid, and is administered in a medium having a pH of about 2.7-6. It is a method comprising a step of culturing Obacidium pullulans to produce β-1,3-1,6-D-glucan.

  Preferably, the step of adjusting the medium pH to about 2.7-6 using ascorbic acid, and culturing aureobasidium pullulans in the obtained medium to form β-1,3-1,6-D- It is a method including a step of producing glucan.

  It is more preferable to adjust the medium pH to about 2.7-6. The ascorbic acid concentration in the medium is preferably about 0.2 to 1% (w / v). Moreover, it is preferable that the acid contained in a culture medium is substantially only ascorbic acid. By using ascorbic acid, the transparency of the medium, and hence the transparency of the glucan produced, is increased. The carbon source excluding ascorbic acid is not particularly limited, but sucrose is preferable. Other points of the medium composition are as described in the first method.

Further, the microorganism to be used, the culture method, and the recovery of glucan are as described in the first method.
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.

Examination of appropriate pH After adjusting 5 ml of liquid medium having the composition shown in Table 1 below to pH 2 to 10 using hydrochloric acid or sodium hydroxide, it is put in a test tube and pressurized steam at 121 ° C. for 15 minutes. Sterilization was performed. Here, 100 μl of a culture solution of Aureobasidium pullulans GM-NH-1A1 strain (FERM P-19285) previously cultured in a test tube for 72 hours is inoculated and stirred at a speed of 130 rpm. The culture was then carried out with shaking at 27 ° C. for 48 hours.

<Measurement of bacterial mass>
The absorbance at 660 nm of the culture solution after 48 hours was measured using a spectrophotometer (HITACHI U-1100 Spectrophotometer) to evaluate the amount of cells.
<Polysaccharide concentration measurement>
1 ml of the culture solution is sampled for 48 hours, and the cells are removed by centrifugation at 15,000 g for 10 minutes. Ethanol was added to 100 μl of this sterilization solution 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, Namiura Nozomi, Kitahata Toshio, Onishi (See Masaken).
< Identification of polysaccharide by ¹ H-NMR>
A culture solution containing 30 mg of polysaccharide was centrifuged at 15,000 g for 10 minutes at 4 ° C. to obtain a supernatant. Ethanol was added to the supernatant to a final concentration of 66% (v / v) to precipitate the polysaccharide. The resulting polysaccharide was dissolved by adding 2 ml of ionic water and 1 ml of 1N NaOH, and freeze-dried overnight. A solution obtained by dissolving this in ¹ to 2 ml of heavy water is subjected to ¹ H-NMR (500 MHz made by JEOL), so that the ratio of β-D-glucan in the polysaccharide can be determined from the integral ratio of the ¹ H-NMR signal. I asked for it. ^{In 1} H-NMR, a signal of 4.7 ppm and a signal of 4.5 ppm were observed, but a peak derived from another polysaccharide, such as 5.2 ppm derived from pullulan, was not observed. From this, it was judged that the β-D-glucan purity in the polysaccharide was 100%.

  The results of polysaccharide concentration and bacterial turbidity are shown in Table 2 below. The polysaccharide concentration was expressed as a relative value with respect to 100 as the polysaccharide concentration at pH 4 where the production amount was highest.

  A polysaccharide concentration of 1.959 mg / ml was obtained in the liquid medium adjusted to pH 4. Also at pH 3 and pH 5, polysaccharide concentrations of 1.006 mg / ml and 1.240 mg / ml were obtained, respectively. From this, the preferred medium pH when culturing Aureobasidium pullulans GM-NH-1A1 strain to produce β-1,3-1,6-D-glucan is 3-5, The optimum pH was 4. At pH 4, the amount of polysaccharide production was greater than the amount of bacterial cells.

Examination of carbon source In the composition of Table 1, as a carbon source, 3.3% (w / v) sucrose, 3.3% (w / v) glucose, 3.3% (w / v) fructose, and 1. A liquid medium having the same composition as in Table 1 was prepared except that a mixture of 15% (w / v) glucose and 1.15% (w / v) fructose was used.

  After adjusting 100 ml of each of these media to pH 3.5, they were placed in a Sakaguchi flask and autoclaved at 121 ° C. for 15 minutes. Here, 2 ml of a culture solution of Aureobasidium pullulans GM-NH-1A1 strain previously cultured for 72 hours in a Sakaguchi flask was inoculated aseptically and cultured at 27 ° C. for 96 hours while stirring at a speed of 130 rpm. .

  The polysaccharide concentration and bacterial turbidity were measured as described above. The results are shown in Table 3 below. The polysaccharide concentration was expressed as a relative value with respect to 100 when the amount of fructose used was the highest.

  4.60 mg / ml polysaccharide was obtained in liquid medium using fructose as carbon source. Moreover, the polysaccharide concentration was higher than the amount of cells. From this, it can be seen that β-1,3-1,6-D-glucan can be produced at high yield by using fructose as a carbon source.

Confirmation of carbon source Using each of fructose and sucrose as carbon sources, the amount of polysaccharide produced after 3 to 6 hours of culture time was measured. Specifically, a liquid medium having the same composition as in Table 1 was prepared except that ascorbic acid was excluded from the composition shown in Table 1 and sucrose and fructose were used as carbon sources, respectively. Furthermore, 1 L of the medium adjusted to pH 3.5 was placed in a 2 L culture apparatus (manufactured by Maruhishi Bioengineering), and autoclaved at 121 ° C. for 15 minutes. Here, 10 ml of a culture solution of Aureobasidium pullulans GM-NH-1A1 strain previously cultured in a Sakaguchi flask for 72 hours was inoculated aseptically at 27 ° C. while aeration and stirring at 400 rpm at 0.3 L / min. For 6 days. The results of measuring the polysaccharide concentration and the bacterial turbidity over time are shown in Table 4 below.

  By culturing for 6 days, a polysaccharide of 11.02 mg / ml was obtained in a liquid medium using fructose as a carbon source. In addition, 6.596 mg / ml of polysaccharide was obtained in a liquid medium using sucrose as a carbon source. Similarly, 100 ml culture using a Sakaguchi flask and 1 L culture using an aeration stirrer jar can be obtained by using β-1,3-1,6-D-glucan by using fructose rather than sucrose as a carbon source. It can be seen that high production is possible.

Examination of pH adjuster In the composition of Table 1, a medium was prepared in which ascorbic acid was removed and the carbon source was replaced with 5% (w / v) fructose. This liquid medium was divided into five 100 ml portions and adjusted to pH 3.5 using ascorbic acid, gluconic acid, phosphoric acid, citric acid, and hydrochloric acid, respectively. These liquid media whose pH was adjusted were put into a Sakaguchi flask and autoclaved at 121 ° C. for 15 minutes. Here, 2 ml of a culture solution of Aureobasidium pullulans GM-NH-1A1 strain previously cultured in a Sakaguchi flask for 72 hours was inoculated aseptically and cultured at 27 ° C. for 144 hours while stirring at a speed of 130 rpm. .

  The results of the polysaccharide concentration after culture and the turbidity of the culture solution are shown in Table 5 below. The polysaccharide concentration was shown as a relative value with respect to 100 when the highest gluconic acid was used.

  The highest polysaccharide concentration of 4.505 mg / ml was obtained in a liquid medium adjusted to pH 3.5 with gluconic acid. From this, it is judged that β-1,3-1,6-D-glucan can be produced at high production by using gluconic acid as a pH adjuster.

Confirmation of pH adjuster In the composition of Table 1, a medium was prepared in which ascorbic acid was removed and the carbon source was replaced with 5% (w / v) fructose. This liquid medium was divided into two and adjusted to pH 3.5 using gluconic acid and ascorbic acid, respectively. 1 L of these liquid media was put into a 2 L culture device (manufactured by Maruhishi Bioengineering), and autoclaved at 121 ° C. for 15 minutes. Here, 10 ml of inoculated culture medium of Aureobasidium pullulans GM-NH-1A1 strain previously cultured for 72 hours in a Sakaguchi flask was inoculated, and cultured at 27 ° C. while aerated and stirred at 0.3 L / min at 400 rpm. Cultured for 7 days.

  The results of measuring the polysaccharide concentration and the turbidity of the culture solution over time are shown in Table 6 below.

  A polysaccharide concentration of 14.05 mg / ml was obtained after 7 days of culture in a liquid medium using gluconic acid as a pH adjuster. In contrast, a polysaccharide concentration of 5.81 mg / ml was obtained in a liquid medium using ascorbic acid as a pH adjuster. From this, it was confirmed that β-1,3-1,6-D-glucan can be produced more highly by using gluconic acid than by using ascorbic acid as a pH adjuster.

Study of pH adjuster In the composition of Table 1, the carbon source was replaced with 5% (w / v) fructose, and the ascorbic acid concentration was 0, 0.05, 0.1, 0.3, and 0.6, respectively. % (W / v) liquid medium was prepared. These were adjusted to pH 3.5 using gluconic acid. The pH of the liquid medium with an ascorbic acid concentration of 0.6% (w / v) was 3.5, and gluconic acid was not added.

  100 ml of each medium was placed in a Sakaguchi flask and autoclaved at 121 ° C. for 15 minutes. Here, 2 ml of a culture solution of Aureobasidium pullulans GM-NH-1A1 strain previously cultured in a Sakaguchi flask for 72 hours was inoculated aseptically and cultured at 27 ° C. for 6 days while stirring at 130 rpm.

  The results of polysaccharide concentration and turbidity of the culture solution are shown in Table 7 below. The polysaccharide concentration was expressed as a relative value with respect to 100 when the ascorbic acid concentration was 0.05% (w / v).

  In liquid media supplemented with 0, 0.05, and 0.1% (w / v) ascorbic acid, polysaccharide concentrations of 9.3 mg / ml, 9.4 mg / ml, and 9.1 mg / ml were obtained, respectively. Therefore, aureobasidium pullulans GM-NH-1A1 was cultured using a liquid medium adjusted to pH by adding 0-0.1% (w / v) of ascorbic acid in addition to gluconic acid. It can be seen that β-1,3-1,6-D-glucan can also be produced at high production.

Confirmation of pH adjuster In the composition of Table 1, a medium in which the carbon source was replaced with 5% (w / v) sucrose was prepared. This medium is divided into three parts, each adjusted to pH 3.5 with 0.6% (w / v) ascorbic acid, adjusted to pH 3.5 with gluconic acid, adjusted to pH 6 with gluconic acid and further added with ascorbic acid And adjusted to pH 3.5.

  1 L of each liquid medium was placed in a 2 L culture apparatus (manufactured by Maruhishi Bio-Engine) and autoclaved at 121 ° C. for 15 minutes. Here, 10 ml of a culture solution of Aureobasidium pullulans GM-NH-1A1 strain previously cultured in a Sakaguchi flask for 72 hours was inoculated aseptically at 27 ° C. with aeration and stirring at 400 L at 0.3 L / min. For 5 days.

  The results of polysaccharide concentration and culture turbidity are shown in Table 8 below.

  The highest polysaccharide concentration of 12.94 mg / ml was obtained in the medium adjusted to pH 3.5 with both ascorbic acid and gluconic acid. From this, it is β-1,3-1 to use a medium whose pH is adjusted using both gluconic acid and ascorbic acid rather than a medium whose pH is adjusted using gluconic acid or ascorbic acid alone. , 6-D-glucan can be produced at high production.

Examination of gluconic acid concentration In the composition of Table 1, a medium was prepared in which ascorbic acid was removed and the carbon source was replaced with 5% (w / v) fructose. Divide the liquid medium into 8 portions of 100 ml each, and use a 50% (w / v) gluconic acid solution (food additive gluconic acid manufactured by Fuso Chemical Industry Co., Ltd.) at pH 2.5, 3, 3.5, 4, Adjusted to 5, 6, 7, and 8.3. The liquid medium at pH 8.3 did not add gluconic acid.

  100 ml of each of these media was placed in a Sakaguchi flask, autoclaved at 121 ° C. for 15 minutes, and then cultured in advance for 72 hours in a Sakaguchi flask. A culture medium of Aureobasidium pullulans GM-NH-1A1 strain Aseptically, 2 ml of each was inoculated and cultured at 27 ° C. for 7 days while stirring at a speed of 130 rpm.

  Table 9 below shows the polysaccharide concentration and the turbidity of the bacterial solution on the third and seventh days of culture. The polysaccharide concentration was expressed as a relative value with respect to the highest concentration as 100. Table 9 shows the final concentration of gluconic acid in the culture solution.

  On the third day of culture, the polysaccharide concentration was high at pH 3.5 to 4 (gluconic acid concentration 0.25 to 0.42 (% (w / v)), and pH 3.5 (gluconic acid concentration 0.42 (% (w Polysaccharide concentration of 2.743 mg / ml was obtained, and pH 7-3.5 (gluconic acid concentration 0.42-1.1 (% (w / v) The polysaccharide concentration was high at pH 3 (gluconic acid concentration 1.1 (% (w / v)), and a polysaccharide concentration of 5.616 mg / ml was obtained. No significant polysaccharide production is seen when the concentration drops sharply and exceeds pH 4.

  From this, when adjusting the medium pH with gluconic acid, β-1,3-1,6-D can be obtained by adjusting the pH to 3 to 4 (gluconic acid concentration 0.2 to 1.2% (w / v)). -It can be seen that glucan can be produced at high production.

Claims (8)

  A step of adjusting the medium pH to 2.7-6 using gluconic acid and culturing aureobasidium pullulans in the obtained medium to produce β-1,3-1,6-D-glucan The manufacturing method of (beta) -1,3-1,6-D-glucan including a process.   The method according to claim 1, wherein the medium pH is adjusted to 2.7 to 6 using ascorbic acid in addition to gluconic acid.   The method according to claim 1 or 2, wherein the acid in the medium at the start of the culture is substantially gluconic acid, or further only ascorbic acid.   The method according to any one of claims 1 to 3, wherein the carbon source in the medium at the start of the culture is substantially only fructose except gluconic acid or further ascorbic acid.   The method according to any one of claims 1 to 4, wherein the Aureobasidium pullulans is Aureobasidium pullulans GM-NH-1A1 strain (FERM P-19285).   The method according to any one of claims 1 to 5, further comprising a step of recovering the produced β-1,3-1,6-D-glucan.   A step of adjusting the medium pH to 2.7-6 using ascorbic acid and culturing aureobasidium pullulans in the obtained medium to produce β-1,3-1,6-D-glucan The manufacturing method of (beta) -1,3-1,6-D-glucan including a process.   The method according to claim 7, wherein the Aureobasidium pullulans is Aureobasidium pullulans GM-NH-1A1 strain (FERM P-19285).