Method for producing β-1,3-1,6-glucan from aureobasidium pullulans AFO-202

Abstract

The problem addressed is to provide a method for stably producing β-1,3-1,6-glucan at a high yield from Aureobasidium pullulans AFO-202 and to provide β-1,3-1,6-glucan produced by said method. It was discovered that β-1,3-1,6-glucan is obtained stably at a high yield by using water containing a large amount of SiO2-Si in a step in which Aureobasidium pullulans AFO-202 (FERM MP-19327) is cultured, and the above problem was solved.

Description

Method for producing β-1,3-1,6 glucan from Aureobasidium pullulans strain AFO-202

[0001] The present invention relates to a method for producing β-1,3-1,6 glucan from Aureobasidium pullulans AFO-202 strain, a 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-M-163 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-M-163 strain (accession number: NITE P-03377) 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 Center (ISBN-10: 4762256838, ISBN-13: 978-4762256837)

[0007] As the functionality of β-1,3-1,6 glucan has become clearer, there is currently a strong demand for the stable production of β-1,3-1,6 glucan from Aureobasidium pullulans AFO-202 strain, which exhibits excellent effects in various antitumor activities, in higher yields. 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 AFO-202 strain in higher yields.

[0008] The present inventors have conducted extensive research to solve the above-mentioned problems, and as a result 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 AFO-202 strain. 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, characterized in that water containing 3 mg / L or more of silicic acid silicon is used in the step of culturing Aureobasidium pullulans AFO-202 strain (FERM BP-19327). [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, characterized in that water containing 3 mg / L or more of silicic acid silicon is used in the step of culturing Aureobasidium pullulans AFO-202 strain (FERM BP-19327). [3] The method according to [1] or [2] above, characterized in that 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, characterized in that 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 of the method according to any one of [1] to [4] above. [6] 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 culturing step of Aureobasidium pullulans AFO-202 strain (FERM BP-19327).

[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 AFO-202 strain.

[0011] FIG. 1A shows a photograph of the culture product obtained by the method of the present invention (Production Example 1). 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 in each culture product was 6.1 g / L for the product obtained by the method of the present invention and 2.9 g / L for the product produced by the conventional method. FIG. 2 shows the solid content 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 results of Fourier transform infrared spectroscopy (FTIR) absorption spectroscopy 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. Fig. 4 shows the results of the solid-state analysis of the control (curdlan) in the quantification of the (1,6)-linked glucose / (1,3)-linked glucose ratio of β-1,3-1,6 glucan. １３ 4A shows the results of C-NMR measurements. FIG. 4A shows the results before waveform separation. FIG. 4B shows the results of measurements of FIG. 4A after waveform separation using a Lorentz function. The thick line shows the original spectrum, the thin line shows the separated waveform, the medium-thick line shows the composite waveform, and the upper dashed line shows the difference spectrum. FIG. 5 shows the results of C-NMR measurements of purified β-1,3-1,6 glucan obtained by the method of the present invention in the quantification of the (1,6)-linked glucose / (1,3)-linked glucose ratio of β-1,3-1,6 glucan. １３ The figures show the results of C-NMR measurements. Figure 5A shows the measurement results before waveform separation. Figure 5B shows the measurement results of Figure 5A after waveform separation using a Lorentzian function. The thick line indicates the original spectrum, the thin line indicates the separated waveform, the middle thick line indicates the composite waveform, and the upper dashed line indicates the difference spectrum.

[0013] In one aspect, the present invention relates to a method for producing β-1,3-1,6 glucan, characterized in that water containing 3 mg / L or more of silicic silicon is used in the step of culturing Aureobasidium pullulans AFO-202 strain (FERM BP-19327).In another aspect, the present invention relates to 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 silicon is not used, characterized in that water containing 3 mg / L or more of silicic silicon is used in the step of culturing Aureobasidium pullulans AFO-202 strain (FERM BP-19327). 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 AFO-202 strain (FERM BP-19327).

[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, Aureobasidium pullulans strain AFO-202 (FERM BP-19327) is cultured for 72 to 90 hours. Preferably, Aureobasidium pullulans strain AFO-202 (FERM BP-19327) is cultured for 85 to 90 hours, more preferably for 88 hours. In one embodiment, Aureobasidium pullulans strain AFO-202 (FERM BP-19327) is cultured while aerating at 10 to 100 times the volume of the medium per hour. Preferably, Aureobasidium pullulans strain AFO-202 (FERM BP-19327) is cultured while aerating at 11 to 42 volumes of medium per hour. In one embodiment, Aureobasidium pullulans strain AFO-202 (FERM BP-19327) 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, Aureobasidium pullulans strain AFO-202 (FERM BP-19327) is cultured in the small tank while aerating at, for example, about 11 volumes of medium per hour, and in the large tank while aerating at, for example, about 35 to 42 volumes of medium per hour. In one embodiment, Aureobasidium pullulans strain AFO-202 (FERM BP-19327) is cultured at 20 to 28° C. Preferably, Aureobasidium pullulans strain AFO-202 (FERM BP-19327) is cultured at 24 to 28° C. In one embodiment, the culture step in 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 at a rate of 11 to 42 volumes of the medium per hour.

[0016] In the method of the present invention, Aureobasidium pullulans AFO-202 strain (FERM BP-19327) is cultured using water containing a certain amount or more of inorganic nutrients, resulting in a culture product with a milky white viscous (or gel-like) color with a slight yellowish tint. This culture product has a higher degree of whiteness compared to culture products produced using water that does not contain a certain amount of inorganic nutrients. The degree of whiteness of the culture product can be confirmed, for example, by measuring the absorbance of the culture product at visible light in the range of 400 nm to 900 nm. In such measurements, a low overall absorbance and a small change in absorbance with wavelength indicate a liquid color closer to white. In one aspect, a culture product containing β-1,3-1,6 glucan obtained by the method of the present invention, when diluted to 2 wt % with pure water, exhibits an absorbance of 0.45 or less at 640 nm and a transmittance of 80% or more at 830 nm.

[0017] The β-1,3-1,6 glucan obtained by the method of the present invention is derived from the AFO-202 strain (Aureobasidium pullulans AFO-202 strain), a strain belonging to the black yeast-like fungus Aureobasidium pullulans. The Aureobasidium pullulans AFO-202 strain, also known as the FO-68 strain, was deposited at the Patent Microorganisms Depositary Center (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) on April 23, 2003, under the accession number FERM P-19327. The domestic deposit was transferred to an international deposit at the Patent Microorganisms Depositary Center on April 21, 2021, under the accession number FERM BP-19327.

[0018] In another aspect, the present invention relates to β-1,3-1,6 glucan produced by the method of the present invention and a composition containing the same. In one embodiment, the composition is a milky white viscous culture product with a slight yellowish tinge, containing β-1,3-1,6 glucan obtained by the culturing step of the method of the present invention.

[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 1.4 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 less than 4 g / L, whereas the yield of β-1,3-1,6 glucan obtained by the method of the present invention is 4 g / L or more, preferably 5 g / L or more, more preferably 6 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> A strain of Aureobasidium sp. (AFO-202 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 5.1-5.44 using sodium hydroxide (5% aqueous solution) or hydrochloric acid (25% aqueous solution). The medium was cultured at 24-28°C and 180 rpm with aeration for 88 hours, yielding a culture product. The production water used contained 4.7 mg / L of silicic acid and 0.4 mg / L of nitrate nitrogen. The resulting culture product was a pale yellowish, 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 had a stronger yellowness than 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 culture products were stirred for 24 hours to obtain uniform dilutions (2 wt %). The absorbance at 400 to 900 nm of these dilutions was measured using a UV-visible spectrophotometer (JASCO Corporation, V750). As a result, the culture product obtained in Production Example 1 had lower overall absorbance and less change in absorbance with wavelength compared to that obtained in Comparative Production Example 1, resulting in a liquid color closer to white. The absorbance at 640 nm for the culture product of Production Example 1 was 0.342, while that of the culture product of Comparative Production Example 1 was 0.868. Furthermore, the light transmittance at 830 nm for the culture product of Production Example 1 was 91%, while that of the culture product of Comparative Production Example 1 was 63%.

[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 6.1 g / L, and the amount of β-1,3-1,6 glucan in the culture product obtained in Comparative Production Example 1 was 2.9 g / L. This indicates that the culture product obtained in Production Example 1 contains more than twice the amount of 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) １３Measurements were performed using a C-NMR and a Fourier transform infrared spectrophotometer (FTIR) under the conditions shown below. １３ C-NMR measurement conditions) The sample was 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: 1024, Relaxation time: 2.5 s, Analysis conditions: trapezoid3 (0%, 15%, 15%), zerofill (16, TRUE) (FTIR measurement conditions) The sample was compressed together with KBr powder into a pellet and measured using an FTIR device (JASCO, FT / IR-4200ST). <Measurement conditions> KBr plate method, Resolution: 2 cm －１ Measurement range: 7800-350 cm －１ , Number of integrations: 32, Sample chamber temperature: 25°C, Analysis method: Smoothing processing performed

[0026] The culture product obtained in Production Example 1 was purified by the method described above to obtain a solid β-1,3-1,6 glucan. １３ The results of C-NMR and FTIR measurements are shown in Figures 2 and 3, respectively. Both measurement results supported that the purified product was β-1,3-1,6 glucan.

[0027] Example 3: Quantification of the (1,6)-linked glucose / (1,3)-linked glucose ratio in β-1,3-1,6 glucan The method for measuring the (1,6)-linked glucose / (1,3)-linked glucose ratio in β-1,3-1,6 glucan is described in detail, for example, in JP 2008-139066 A. The β-1,3-1,6 glucan obtained in Production Example 1 was subjected to solid-state chromatographic / mass spectrometry under the following measurement conditions. １３C-NMR was measured. Curdlan (Fujifilm Wako Pure Chemical Industries, Ltd.) was used as a control. <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). Using NMR analysis software Delta (JEOL Ltd.), waveform processing of the C-1 to C-6 carbon peaks of the obtained data was performed, and the area values ​​of each carbon peak were calculated. The following equation was used to calculate the proportions of (1,3)-linked glucose and (1,6)-linked glucose. (1,6)-linked glucose / (1,3)-linked glucose ratio = (A / B) x X-1, where A: C-1 carbon peak area value in the measurement sample, B: C-6 carbon peak area value in the measurement sample, and X: correction factor. The correction factor X was calculated using the following formula: X = B ０ / A ０ A ０ : C-1 carbon peak area value in control B ０ : C-6 carbon peak area value in the control

[0028] Control solid １３ The results of C-NMR measurement are shown in Figure 4A. The measurement results of Figure 4A after waveform separation using a Lorentzian function are shown in Figure 4B. The area values ​​of each carbon peak after waveform separation of the control were as shown in Table 1 below. Table 1. Area values ​​of each carbon peak in the control Therefore, the correction factor was calculated as X=1013.88 / 999.68=1.01.

[0029] Solid β-1,3-1,6 glucan obtained in Production Example 1 １３ The results of C-NMR measurement are shown in Figure 5A. The measurement results of Figure 5A after waveform separation using a Lorentz function are shown in Figure 5B. The area values ​​of each carbon peak after waveform separation of the β-1,3-1,6 glucan obtained in Production Example 1 are as shown in Table 2 below. Table 2. Area values ​​of each carbon peak in the β-1,3-1,6 glucan obtained in Production Example 1 Therefore, the (1,6)-linked glucose / (1,3)-linked glucose ratio of the β-1,3-1,6 glucan obtained in Production Example 1 was calculated to be (688.09 / 356.39) × 1.01 - 1 = 0.95. From the above, it was shown that the β-1,3-1,6-glucan obtained in Production Example 1 has a structure with a very high degree of branching in which glucoses having one glucose bonded via a 1 → 6 bond are successively linked via a 1 → 3 bond, and more specifically, the β-1,3-1,6 glucan obtained by the method of the present invention has the following structure with a degree of branching of 95%. Furthermore, since it has a similar structure, the β-1,3-1,6 glucan obtained by the method of the present invention is expected to exhibit excellent physiological activities such as antitumor activity, similar to the β-1,3-1,6 glucan described in Patent Document 1.

[0030] [Rule 26, 22.08.2024]

Claims

1. A method for producing 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 AFO-202 strain (FERM BP-19327).

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 is not used, the method being characterized by using water containing 3 mg / L or more of silicic acid in the step of culturing Aureobasidium pullulans AFO-202 strain (FERM BP-19327).

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

4. The method according to any one of claims 1 to 3, characterized in that the culture is carried out for 85 to 90 hours.

5. A culture product containing β-1,3-1,6 glucan obtained in the culture step of the method according to any one of claims 1 to 4.

6. 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 process of culturing Aureobasidium pullulans AFO-202 strain (FERM BP-19327).

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