Method for producing β-1,3 / 1,6-glucan from aureobasidium pullulans n-163 strain

Abstract

The problem of the present invention is to provide: a method for stably producing β-1,3 / 1,6-glucan at a high yield from the Aureobasidium pullulans N-163 strain; and β-1,3 / 1,6-glucan produced by said method. This problem is solved by the discovery of the ability to obtain β-1,3 / 1,6-glucan stably at a high yield by using water that contains a large amount of silicon in the form of silicic acid in a step in which the Aureobasidium pullulans N-163 strain (accession number: NITE BP-03377) is cultured.　

Description

Method for producing β-1,3-1,6 glucan from Aureobasidium pullulans strain N-163

[0001] The present invention relates to a method for producing β-1,3-1,6 glucan from Aureobasidium pullulans strain N-163, the β-1,3-1,6 glucan obtained by the production method, and a composition containing the same.

[0002] β-glucan is a general term for polymers in which glucose units are linked by β-glucosidic bonds. Known examples include linear β-1,3-glucans with a β-1,3-linkage backbone and no side chains, β-1,3-1,6-glucans with a certain proportion of β-1,6-branched structures, and β-1,3-1,4-glucans with partial β-1,4-linkages. Among these, β-1,3-1,6-glucans are primarily produced by basidiomycetes such as mushrooms and fungi such as yeast and mold. Because their ingestion has been shown to stimulate the immune system, they have attracted attention as polysaccharides with physiological activities such as antitumor activity (Non-Patent Documents 1 and 2). However, β-1,3-1,6-glucans contained in basidiomycetes, for example, have poor extraction efficiency, and the extraction process can cause changes in the β-1,3-1,6-glucan bonds. Furthermore, the extracts are not water-soluble. In contrast, by using a strain that secretes water-soluble β-1,3-1,6-glucan extracellularly, such as a strain of the genus Aureobasidium, it has become possible to produce water-soluble β-1,3-1,6-glucan that can be used without an extraction process. However, because a culture method suitable for producing β-1,3-1,6-glucan had not yet been established, the yield of the resulting β-1,3-1,6-glucan was inconsistent. Furthermore, problems arose, such as the formation of a large amount of pellets that remained as impurities in the culture product and browning of the culture product due to melanin pigments.

[0003] To address these problems, Patent Document 1 describes the production of β-1,3-1,6 glucan using an Aureobasidium sp. strain (AFO-202 strain). The document also describes that β-1,3-1,6 glucan can be stably obtained by culturing chlamydospores induced in a nitrogen-depleted medium in a liquid medium containing crushed rice bran supplemented with vitamin E, glucose, and vitamin C, and adjusted to a pH of 5.0 to 6.0. Furthermore, the document describes that the culture product obtained from the strain has various functionalities, such as enhanced DNA synthesis, inhibition of leukemia cell proliferation, production of cytokines such as IL-8 and IL-12, and induction of NK activity.

[0004] Methods for producing β-glucan using the M-3 and APNN-M163 strains, which are black yeast-like fungi (Aureobasidium pullulans) similar to the AFO-202 strain, have also been reported. For example, Patent Document 2 describes the Aureobasidium pullulans M-3 strain, a highly productive β-glucan strain, and a method for producing β-glucan, including culturing the strain in a liquid medium. Patent Document 3 also describes the Aureobasidium pullulans APNN-M163 strain (Accession Number: NITE P-03377) (referred to herein as "N-163 strain") and a method for producing β-1,3-1,6-glucan, including culturing the strain. Furthermore, the same document describes that the β-1,3-1,6-glucan obtained by the strain has a high inhibitory effect on MMP-9 production.

[0005] Japanese Patent No. 4468654 Japanese Patent Application Laid-Open No. 2020-031580 Japanese Patent Application Laid-Open No. 2022-180249

[0006] "Fundamentals and Applications of Beta-Glucan," edited by Naohito Ohno, September 2010, CMC Publishing (ISBN-10: 4781302769, ISBN-13: 9784781302768); "Chemistry and Biochemistry of Mushrooms," edited by Taku Mizuno and Masayoshi Kawai, January 1992, Academic Press (ISBN-10: 4762256838, ISBN-13: 978-4762256837); and "Crystal Structure Analysis of Native Cellulose by Solid-State NMR," Chemistry and Biology, 1994, No. 32, pp. 361-366.

[0007] As the functionality of β-1,3-1,6 glucan has become clearer, there is currently a strong demand for more stable and higher yield production of β-1,3-1,6 glucan, which has excellent anticancer and immunostimulatory effects and is obtained from Aureobasidium pullulans N-163 strain. Therefore, the problem to be solved by the present invention is to provide a method for stably producing β-1,3-1,6 glucan from Aureobasidium pullulans N-163 strain in a stable and higher yield.

[0008] The present inventors have conducted extensive research to solve the above problems and have found that, when producing β-1,3-1,6 glucan, a stable, high yield of β-1,3-1,6 glucan can be obtained by using specific water in the process of culturing Aureobasidium pullulans N-163 strain (accession number: NITE BP-03377). Based on this finding, the inventors have continued their research and have completed the present invention.

[0009] That is, the present invention relates to the following: [1] A method for producing β-1,3-1,6 glucan, wherein water containing 3 mg / L or more of silicic acid silicon is used in the step of culturing Aureobasidium pullulans strain N-163 (accession number: NITE BP-03377). [2] A method for producing β-1,3-1,6 glucan in a higher yield than when water containing 3 mg / L or more of silicic acid silicon is not used, wherein water containing 3 mg / L or more of silicic acid silicon is used in the step of culturing Aureobasidium pullulans strain N-163 (accession number: NITE BP-03377). [3] The method according to [1] or [2] above, wherein the water further contains 0.2 mg / L or more of nitrate nitrogen. [4] The method according to any one of [1] to [3] above, wherein the culture is carried out for 85 to 90 hours. [5] A culture product containing β-1,3-1,6 glucan obtained in the culturing step in the method according to any one of [1] to [4] above. [6] A β-1,3-1,6-glucan produced by the method according to any one of [1] to [4] above. [7] The β-1,3-1,6 glucan according to [6] above, which contains β-1,3 and β-1,6 bonds, and the ratio of (β-1,6 bond) / (β-1,3 bond) is 1 or more and 3 or less. [8] The β-1,3-1,6 glucan according to [6] or [7] above, which contains β-1,4 bonds in addition to β-1,3 and β-1,6 bonds, and the ratio of β-1,4 bonds to the sum of the former two bonds ((β-1,4 bond) / {(β-1,3 bond) + (β-1,6 bond)}) is 0.2 or more and 0.6 or less. [9] A method for increasing the yield of β-1,3-1,6 glucan, wherein water containing 3 mg / L or more of silicic acid silicon is used in the step of culturing Aureobasidium pullulans N-163 strain (accession number: NITE BP-03377).

[0010] According to the method of the present invention, β-1,3-1,6 glucan can be stably provided in high yield from Aureobasidium pullulans strain N-163.

[0011] FIG. 1A shows a photograph of the culture product obtained by the method of the present invention (Production Example 1). The yield of β-1,3-1,6 glucan according to the present invention was 7.2 g / L. FIG. 1B shows a photograph of the culture product produced by the conventional method (Comparative Production Example 1). The content of β-1,3-1,6 glucan contained in each culture product was 2.1 g / L for β-1,3-1,6 glucan produced by the conventional method. FIG. 2 shows the solid phase of β-1,3-1,6 glucan obtained by purifying the culture product obtained by the culture step of the method of the present invention. １３ C-NMR measurement results.

[0012] FIG. 3 shows the effect of the solid phase of the control (curdlan) on the quantification of the (1,6)-linked glucose / (1,3)-linked glucose ratio of β-1,3-1,6 glucan. １３ The results of C-NMR measurements after waveform separation using a Lorentz function are shown. The thick line indicates the original spectrum, the thin line indicates the separated waveform, and the upper thin line indicates the difference spectrum. Figure 4 shows the results of the measurement of the (1,6)-linked glucose / (1,3)-linked glucose ratio of β-1,3-1,6 glucan using a control (cellulose) solid. １３ The results of C-NMR measurements after waveform separation using a Lorentz function are shown. The thick line shows the original spectrum, the thin line shows the separated waveform, and the upper thin line shows the difference spectrum. Figure 5 shows the solid state of purified β-1,3-1,6 glucan obtained by the method of the present invention. １３ The C-NMR measurement results are shown after waveform separation using a Lorentzian function. The thick line indicates the original spectrum, the thin line indicates the separated waveform, and the upper thin line indicates the difference spectrum.

[0013] In one aspect, the present invention relates to a method for producing β-1,3-1,6 glucan, in which water containing 3 mg / L or more of silicic silicon is used in the step of culturing Aureobasidium pullulans strain N-163 (accession number: NITE BP-03377). In another aspect, the present invention relates to a method for producing β-1,3-1,6 glucan, in which water containing 3 mg / L or more of silicic silicon is used in the step of culturing Aureobasidium pullulans strain N-163 (accession number: NITE BP-03377), with a higher yield than when water containing 3 mg / L or more of silicic silicon is not used. In yet another aspect, the present invention relates to a method for increasing the yield of β-1,3-1,6 glucan, characterized in that water containing 3 mg / L or more of silicic acid silicon is used in the step of culturing Aureobasidium pullulans N-163 strain (accession number: NITE BP-03377).

[0014] The liquid medium used in the culture step of the present invention is water containing a certain amount or more of inorganic nutrients. Examples of inorganic nutrients include, but are not limited to, silicic acid silicon, nitrate nitrogen, etc. In a preferred embodiment, water containing silicic acid silicon is used as the liquid medium used in the present invention. The content of silicic acid silicon is 3 mg / L or more, preferably 3.5 mg / L or more, more preferably 4.0 mg / L or more, and even more preferably 4.5 mg / L or more. The silicic acid silicon is orthosilicic acid (H ４ SiO ４ ), metasilicic acid (H ２ SiO ３ ) and metadisilicate (H ２ Si ２ O ５) and the like. Nitrate nitrogen is an example of an inorganic nutrient salt contained in the water used in the method of the present invention. In one embodiment, water containing nitrate nitrogen is used as the liquid medium used in the present invention. The nitrate nitrogen content is 0.2 mg / L or more, preferably 0.3 mg / L or more, and more preferably 0.4 mg / L or more. In another embodiment, water containing silicic silicon and nitrate nitrogen is used as the liquid medium used in the present invention. The concentrations of silicic silicon and / or nitrate nitrogen in the liquid medium used in the present invention are comparable to those in the water used in the present invention. In a preferred embodiment, the sodium and its compounds in the water used in the method of the present invention have a concentration of 10 mg / L or less, preferably 5 mg / L or less, more preferably 4 mg / L or less, and even more preferably 3 mg / L or less, and the chloride ion concentration is 50 mg / L or less, preferably 25 mg / L or less, more preferably 10 mg / L or less, even more preferably 5 mg / L or less, and particularly preferably 3 mg / L or less. The concentration of sodium and its compounds is calculated not only for ionized sodium but also for compounds that form sodium salts (for example, sodium chloride, sodium nitrate).

[0015] In one embodiment, the liquid medium contains bran, 0.5 to 2.0% by weight of glucose, and / or 0.2 to 0.5% by weight of vitamin C in the water. Preferably, the pH of the liquid medium is adjusted to about 5.0 to 6.0. In one embodiment, the Aureobasidium pullulans N-163 strain (Accession Number: NITE BP-03377) is cultured for 72 to 90 hours. Preferably, the Aureobasidium pullulans N-163 strain is cultured for 85 to 90 hours, more preferably for 88 hours. In one embodiment, the Aureobasidium pullulans N-163 strain is cultured while aerating at 10 to 100 volumes of medium per hour. Preferably, the Aureobasidium pullulans N-163 strain is cultured while aerating at 11 to 42 volumes of medium per hour. In one embodiment, the Aureobasidium pullulans N-163 strain is first cultured in a small tank and then transferred to a large tank (e.g., a tank with a capacity of 16 tonnes) for culture. In such an embodiment, the Aureobasidium pullulans N-163 strain is cultured in the small tank while being aerated, for example, at about 11 volumes of medium per hour, and in the large tank while being aerated, for example, at about 35 to 42 volumes of medium per hour. In one embodiment, the Aureobasidium pullulans N-163 strain is cultured at 20 to 28°C. Preferably, the Aureobasidium pullulans N-163 strain is cultured at 24 to 28°C. In one embodiment, the culturing step of the present invention refers to a step of inoculating chlamydospores induced in a nitrogen-depleted medium into the liquid medium, followed by agitation culture at 24 to 28°C for 88 hours while aerating the liquid medium at a rate of 11 to 42 times the volume of the medium per hour.

[0016] In the method of the present invention, Aureobasidium pullulans N-163 strain (accession number: NITE BP-03377) is cultivated using water containing a certain amount or more of inorganic nutrients, thereby obtaining a milky white viscous (or gel-like) culture product.

[0017] The β-1,3-1,6 glucan obtained by the method of the present invention is derived from the APNN-M163 strain, which is a strain belonging to the black yeast-like fungus Aureobasidium pullulans. The Aureobasidium pullulans APNN-M163 strain has been deposited at the following depository institution. (i) Name and address of depository institution Name: National Institute of Technology and Evaluation Patent Microorganism Depositary Center Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (ii) Date of deposit: February 9, 2021 (iii) Accession number: NITE P-03377 Although the domestic deposit was made on February 9, 2021, the Aureobasidium pullulans APNN-M163 strain was transferred to international deposition on September 14, 2021 at the National Institute of Technology and Evaluation Patent Microorganism Depositary Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818) under the accession number NITE BP-03377.

[0018] In another aspect, the present invention relates to a β-1,3-1,6 glucan produced by the method of the present invention, and a composition comprising the same. In one embodiment, such a composition is a milky white viscous culture product containing β-1,3-1,6 glucan obtained by the culturing step of the method of the present invention. In one embodiment, the β-1,3-1,6 glucan produced by the method of the present invention has a (β-1,6 bond) / (β-1,3 bond) ratio of 1 or more and 3 or less. The β-1,3-1,6 glucan produced by the method of the present invention further contains a β-1,4 bond. In one embodiment, the β-1,3-1,6 glucan produced by the method of the present invention has a ratio of β-1,4 bond to the sum of β-1,3 bond and β-1,6 bond ((β-1,4 bond) / {(β-1,3 bond) + (β-1,6 bond)}) of 0.2 or more and 0.6 or less. The structure of the β-1,3-1,6 glucan produced by the method of the present invention differs from the structure of the β-1,3-1,6-glucan described in Patent Document 3 in the ratio of (β-1,6 bond) / (β-1,3 bond) and / or the proportion of ((β-1,4 bond) / {(β-1,3 bond)+(β-1,6 bond)}).

[0019] The yield of β-1,3-1,6 glucan obtained by the method of the present invention is higher than that obtained by conventional methods, i.e., when water not containing a certain amount of inorganic nutrients is used. For example, the yield of β-1,3-1,6 glucan obtained by the method of the present invention is at least 1.5 times higher, more preferably at least 2 times higher, than that obtained when using the above water. In one aspect, the yield of β-1,3-1,6 glucan obtained when using the above water is 3 g / L or less, whereas the yield of β-1,3-1,6 glucan obtained by the method of the present invention is 5 g / L or more, preferably 6 g / L or more, more preferably 7 g / L or more.

[0020] Next, the present invention will be explained in more detail by way of examples, but it should be understood that the present invention is not limited to the examples shown below.

[0021] <Production Example 1> Aureobasidium sp. strain (N-163 strain) cultured on a PDA plate medium at 25°C for 5 days was inoculated into a liquid medium containing 8,000 L of production water, 0.2 wt% bran, 1.5 wt% glucose, and 0.44 wt% ascorbic acid, and adjusted to a pH of 5.1 to 5.44 using sodium hydroxide (5% aqueous solution) or hydrochloric acid (25% aqueous solution). The medium was cultured at 24 to 28°C and 180 rpm with aeration for 88 hours to obtain a culture product. The production water used contained 4.7 mg / L of silicic acid silicon and 0.4 mg / L of nitrate nitrogen. The resulting culture product was a milky white viscous liquid or gel. A photograph of the culture product obtained in Production Example 1 is shown in Figure 1A.

[0022] Comparative Production Example 1 A culture product was obtained in the same manner as in Production Example 1, except that water containing less than 0.1 mg / L of silicic acid silicon and less than 0.1 mg / L of nitrate nitrogen was used as the production water. The obtained culture product was in the form of a milky white viscous liquid or gel, similar to the culture product obtained in Production Example 1. A photograph of the culture product obtained in Comparative Production Example 1 is shown in Figure 1B.

[0023] Example 1 Pure water was added to the culture products obtained in Production Example 1 and Comparative Production Example 1, and the mixture was stirred for 24 hours to obtain a uniform dilution (2 wt %) of the culture product. The absorbance at 400 to 900 nm of the diluted culture product was measured using a UV-visible spectrophotometer (JASCO Corporation, V730). The culture product obtained in Production Example 1 had a higher overall absorbance and a whiter liquid color than that obtained in Comparative Production Example 1. The absorbance at 830 nm for the culture product of Production Example 1 was 1.94592, while that of the culture product of Comparative Production Example 1 was 1.84366. Furthermore, the absorbance at 640 nm of the diluted culture product obtained in Production Example 1 diluted to 2 wt % with pure water was 0.084322, and the transmittance at 830 nm was 87%.

[0024] Furthermore, the amount of β-1,3-1,6 glucan contained in the culture products obtained in Production Example 1 and Comparative Production Example 1 was measured according to the following method. <Method for Purifying and Measuring β-1,3-1,6 Glucan in Culture Product> 2.30 g of culture broth was diluted 10-fold with distilled water, thoroughly stirred, and then centrifuged at 18,000 rpm at 4°C for 20 minutes to remove insoluble solids, yielding a supernatant. 1 ml of benzalkonium chloride solution was added to 7.00 g of the supernatant and stirred, followed by addition of 20 ml of distilled water and stirring, followed by centrifugation at 18,000 rpm at 4°C for 20 minutes. The supernatant was decanted, and the precipitate was dried. 14.84 g of 1.5 M NaOH was added to the dried product, stirred, and then shaken overnight at 125 rpm at room temperature to prepare a sample for β-glucan measurement. This sample was used to measure the amount of β-1,3-1,6 glucan using the phenol-sulfuric acid method. As a result, the amount of β-1,3-1,6 glucan in the culture product obtained in Production Example 1 was 7.2 g / L, and the amount of β-1,3-1,6 glucan in the culture product obtained in Comparative Production Example 1 was 2.1 g / L. This indicates that the culture product obtained in Production Example 1 contains about 3.5 times as much β-1,3-1,6 glucan as the culture product obtained in Comparative Production Example 1.

[0025] Example 2 The culture product obtained by the present invention was purified by the method described above, and the resulting powder was then subjected to solidification. １３ C-Nuclear magnetic resonance apparatus (solid state) １３Measurement was performed using C-NMR under the following conditions: (Solid １３ C-NMR measurement) The sample is placed in a sample tube as is, and the solid １３ Measurement was performed using a C-NMR device (JEOL, JNM-ECX400). <Measurement conditions> Sample tube diameter: 4 mm, Sample tube rotation speed: 10 kHz, Measurement mode: CP / MAS, Measurement temperature: 20.5°C, Resonance frequency: 100 MHz, Number of scans: 256, Contact time: 1.0 ms, Relaxation time: 5.0 s, Analysis conditions: trapezoid3 (0%, 8%, 8%), Zerofill (16, TRUE), Manual phase correction. １３ The results of C-NMR measurement are shown in Figure 2. The measurement results supported that the purified product was a β-1,3-1,6 glucan.

[0026] Example 3 The ratio of (1,6)-linked glucose to (1,3)-linked glucose in β-1,3-1,6 glucan and the ratio of (1,4)-linked glucose to ((1,3)-linked glucose + (1,6)-linked glucose) were determined by CP / MAS solid-state spectroscopy under the same conditions as in Example 2, using the β-1,3-1,6 glucan obtained in Production Example 1 and curdlan (Fujifilm Wako Pure Chemical Industries, Ltd.) and cellulose (Fujifilm Wako Pure Chemical Industries, Ltd.) as controls (reference substances). １３C-NMR was measured. Next, waveform separation of the C-1 to C-6 carbon peaks of the obtained data was performed using NMR analysis software Delta (manufactured by JEOL Ltd.), and the area value of each carbon peak was calculated. Specifically, a correction factor X was calculated from the area ratio of the C-1 and C-6 carbon peaks of the reference material, curdlan, and then the (1,6)-linked glucose / (1,3)-linked glucose ratio of the sample was calculated. Alternatively, the reference material was changed to cellulose, and a correction factor Y was calculated from the area ratio of the C-1 and C-6 carbon peaks, and then the (1,6)-linked glucose / (1,4)-linked glucose ratio in the sample was calculated. <Calculation of the proportions of (1,3)-linked glucose and (1,6)-linked glucose> The following formula was used. (1,6)-linked glucose / (1,3)-linked glucose ratio = (A / B) x X - 1, where A: C-1 carbon peak area in the measurement sample, B: C-6 carbon peak area in the measurement sample, and X: correction factor (curdlan). The correction factor X was calculated using the following formula: X = B ０ / A ０ A ０ B: C-1 carbon peak area value in standard substance (curdlan) ０ : C-6 carbon peak area value in standard substance (curdlan)

[0027] Solid reference material (curdlan) １３ The results of C-NMR measurement after waveform separation using a Lorentz function are shown in Figure 3. The area values ​​of each carbon peak after waveform separation of the reference material (curdlan) are as shown in the table below (Table 1). The C-1 carbon peak area value (A ０ ) and C-6 carbon peak area value (B ０ ) the correction factor X was calculated to be 0.96.

[0028] <Calculation of the ratio of (1,6)-linked glucose and (1,4)-linked glucose> The following formula was used: (1,6)-linked glucose / (1,4)-linked glucose ratio = (A / B) x Y-1 A: C-1 carbon peak area in the measurement sample B: C-6 carbon peak area in the measurement sample Y: Correction factor (cellulose) The correction factor Y was calculated using the following formula: Y = B １ / A １A １ : C-1 carbon peak area value in standard material (cellulose) B １ : C-6 carbon peak area value in standard material (cellulose)

[0029] Solid reference material (cellulose) １３ The results of C-NMR measurement after waveform separation using a Lorentzian function are shown in Figure 4. The area values ​​of each carbon peak after waveform separation of the reference material (cellulose) are as shown in the table below (Table 2). Regarding the C-6 carbon peak of cellulose, in this result, it was observed as two peaks, C-6 and C-6', but Non-Patent Document 3 shows that the C-6 carbon peak splits depending on the type of natural cellulose used. Therefore, the C-1 carbon peak area value (A １ ) and the total area of ​​the C-6 carbon peak and the C-6' carbon peak (B １ ) and the correction factor Y was calculated to be 0.97.

[0030] Solid β-1,3-1,6 glucan obtained in Production Example 1 １３ The results of C-NMR measurement (FIG. 2) after waveform separation using a Lorentz function are shown in FIG. 5. The area values ​​of each carbon peak after waveform separation for the β-1,3-1,6 glucan obtained in Production Example 1 were as shown in Table 2 below.

[0031] (1,3)-linked glucose, (1,6)-linked glucose From the above results, the ratio of (1,3)-linked glucose to (1,6)-linked glucose (1,6-linked glucose / 1,3-linked glucose) contained in the β-1,3-1,6 glucan obtained in Production Example 1 was calculated to be 1.56. Furthermore, the β-1,3-1,6 glucan obtained in Production Example 1 contains 1,4-linked glucose, and the ratio of this bond to the sum of 1,3-linked glucose and 1,6-linked glucose (1,4-linked glucose) / (1,3-linked glucose+1,6-linked glucose) was calculated to be 0.37.

[0032] In other words, the ratio of glucosidic bonds contained in the β-1,3-1,6 glucan obtained by Production Example 1 was (1,6) bond:(1,3) bond:(1,4) bond=44%:29%:27%4, indicating that the β-1,3-1,6-glucan obtained by Production Example 1 has a structure containing a relatively high proportion of 1,6 bond and 1,4 bond in addition to 1,3 bond. The β-1,3-1,6 glucan produced by the method of the present invention is expected to have excellent anticancer and immunostimulatory effects.

[0033] [Rule 26 Amendment 01.07.2025]

Claims

1. A method for producing β-1,3-1,6 glucan, wherein water containing 3 mg / L or more of silicic acid silicon is used in the step of culturing Aureobasidium pullulans N-163 strain (accession number: NITE BP-03377).

2. A method for producing β-1,3-1,6 glucan in a higher yield than when water containing 3 mg / L or more of silicic acid silicon is not used in the step of culturing Aureobasidium pullulans N-163 strain (accession number: NITE BP-03377).

3. The method according to claim 1 or 2, wherein the water further contains 0.2 mg / L or more of nitrate nitrogen.

4. The method according to claim 1 or 2, wherein the culture is carried out for 85 to 90 hours.

5. A culture product containing β-1,3-1,6 glucan obtained by the culturing step in the method according to claim 1 or 2.

6. β-1,3-1,6 glucan produced by the method according to claim 1 or 2.

7. A β-1,3-1,6 glucan according to claim 6, in which the ratio of β-1,3 bonds to β-1,6 bonds ((β-1,6 bonds) / (β-1,3 bonds)) is 1 or more and 3 or less.

8. A β-1,3-1,6 glucan according to claim 6, which contains β-1,4 bonds in addition to β-1,3 and β-1,6 bonds, and the ratio of β-1,4 bonds to the sum of the former two ((β-1,4 bonds) / {(β-1,3 bonds)+(β-1,6 bonds)}) is 0.2 or more and 0.6 or less.

9. A method for increasing the yield of β-1,3-1,6 glucan, wherein water containing 3 mg / L or more of silicic acid silicon is used in the step of culturing Aureobasidium pullulans N-163 strain (accession number: NITE BP-03377).

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