JP6353225B2 - Process for producing β-glucan-containing composition from basidiomycetes - Google Patents

Description

  The present invention relates to a method for producing a β-glucan-containing composition using basidiomycetes as a raw material.

  β-glucan is a polysaccharide in which many glucoses are linked by β-bonds, and is produced by plants, fungi, and the like. β-glucan is known to have an immunostimulatory action and is also used clinically. For example, Ganoderma is a kind of basidiomycete belonging to the family Ganoderma, and has been used for medicinal purposes since ancient times, and has many physiological activities such as immunostimulatory action, anticancer action and blood lowering action. Yes. Previous studies have revealed that one of the active bodies of physiological action exhibited by Ganoderma is β-glucan, a type of polysaccharide (see, for example, Non-Patent Document 1).

  In general, the solubility of polysaccharides in water is low, and β-glucan contained in natural products is a constituent component such as cell walls by combining with other components. You cannot get efficiency. Therefore, in order to efficiently obtain β-glucan using basidiomycetes as a raw material, it is necessary to carry out an extraction process by an appropriate method.

  For example, as a method for extracting β-glucan from ganoderma, for example, Japanese Patent Application Laid-Open Publication No. 2004-259542 discloses pressure hot water pressurized at a pressure equal to or higher than the saturated vapor pressure and heated to the extraction temperature. And a method for extracting β-glucan by contacting it with water. In Patent Document 2 below, ganoderma is soaked in hot water to contain water, extraction with pressurized hot water using alkaline water or the like, pressure heat treatment with saturated steam on the water-containing solid content after the extraction, A method for extracting β-glucan in combination with repressurized hot water extraction for hydrous solids subjected to hydrothermal treatment is disclosed. Further, in Patent Document 3 below, deer horned turf is crushed and then finely pulverized, followed by heating in a slightly alkaline buffer in the presence or absence of protease to extract β-glucan. A method is disclosed.

Japanese Journal of Agricultural Chemistry Vol.58 No.9 (1984) pp.871-880

JP 2008-138195 A Japanese Patent Laid-Open No. 11-243908 JP 2006-37016 A

  However, in the method of Patent Document 1, in order to obtain an excellent extraction efficiency, it is necessary to hydrolyze pressurized hot water in contact with ganoderma at a high pressure of 2.0 to 5.0 MPa. Example of Document 1), a special device for pressurizing the high pressure state was necessary. Further, in the method of Patent Document 2, the operation of repeating the pressurized hot water extraction, the pressurized heat treatment with saturated steam for the water-containing solid content after the extraction, and the repeated pressurized hot water extraction is complicated. . Further, in the method of Patent Document 3, the raw material needs to be crushed and subsequently finely pulverized, and the raw material pretreatment process is complicated. In addition, a special blasting device is required, and labor is required for safe driving.

  An object of the present invention is to provide a β-bacteria made from basidiomycetes as a raw material, which does not require a special apparatus and can obtain an extract having a high β-glucan content efficiently and safely by a simple operation in view of the above-described conventional technology. It is providing the manufacturing method of a glucan containing composition.

  In order to achieve the above object, the method for producing a β-glucan-containing composition of the present invention comprises subjecting a mycelium and / or fruiting body of a basidiomycete to an extraction solvent and pressurizing it, and then subjecting the pressurized product to an enzyme. Is added, the enzyme treatment is performed, and β-glucan is eluted in the liquid part of the enzyme-treated product.

  According to the method for producing a β-glucan-containing composition of the present invention, an extraction solvent is added to the mycelium and / or fruiting body of basidiomycete and subjected to pressure treatment, so that an enzyme is contained in the constituent components of the cell wall of basidiomycete. Since the enzyme is treated by adding an enzyme thereto, β-glucan is easily eluted in the liquid part of the enzyme-treated product. Therefore, an extract having a high β-glucan content can be obtained efficiently and safely with a simple operation without requiring a special apparatus.

  In the method for producing a β-glucan-containing composition of the present invention, the basidiomycete is preferably ganoderma.

  The pressure treatment is preferably performed at a temperature of 100 to 200 ° C. and a pressure of 0.1 to 1.0 MPa.

  The enzyme treatment is preferably performed using glucanase alone or an enzyme containing glucanase and chitinase.

  Moreover, it is preferable to pulverize the mycelium and / or fruit body of the basidiomycete prior to the pressure treatment.

  In addition, after the enzyme treatment, the extract is recovered by solid-liquid separation, and then the second extraction solvent is added to the obtained insoluble residue and stirred, and the extract is recovered again by solid-liquid separation. It is preferable.

  According to the method for producing a β-glucan-containing composition of the present invention, an extract having a high β-glucan content can be obtained efficiently and safely with a simple operation, using basidiomycetes as a raw material, without requiring a special device. it can.

  The basidiomycete used in the present invention is not limited as long as it contains β-glucan as a constituent component of the cell body. , Hanabiratake, Tamogitake, Meshimakobu, Sarnoshokutake, Agaricus, Eringi, Beechaharitake and various other examples. Ganoderma is particularly preferable.

  The basidiomycete may be a mycelium or a fruit body. In general, since the fruiting body is rich in β-glucan, the fruiting body is more preferable.

  The extraction solvent used in the present invention may be any solvent that can extract β-glucan, such as water, pH-adjusted buffer solution, or water such as ethanol, methanol, propanol, 1,3-butylene glycol. Examples thereof include a water-containing organic solvent obtained by mixing one or more alcohol solvents and ketone solvents such as acetone. In order to facilitate adjustment to suitable conditions for enzyme treatment described below, it is preferable not to contain enzyme inhibitors, and inorganic ions, salts, organic solvents, etc. do not contain them or do not contain high concentrations. It is preferable. Water is particularly preferable.

  The enzyme used in the present invention may be any enzyme that facilitates the extraction of β-glucan from basidiomycetes. For example, glucanase, chitinase, cellulase, hemicellulase, amylase, protease, lysozyme, glucosidase, N- Examples include acetylglucosaminidase, xylanase, mannosidase, xylosidase, galactosidase, lipase, esterase, peroxidase, laccase and the like. One enzyme may be used alone, or two or more enzymes may be used simultaneously or sequentially. In particular, it is preferable to use glucanase alone or a combination of glucanase and chitinase.

  The mycelium and / or fruiting body of the basidiomycete may be used in any form as a raw material, and there is no particular limitation. For example, the mycelium of basidiomycetes can be used after solid culture or liquid culture in an appropriate medium or growth under appropriate fermentation conditions, with or without separation of the mycelia. Moreover, the fruit body of basidiomycete can be used as it is collected or dried after being collected. From the viewpoint of storage stability and ease of handling, it is preferable to use a dried product as the raw material.

  The mycelium and / or fruiting body of the basidiomycete is preferably pulverized or powdered from the viewpoint of extraction efficiency. For example, it is preferable to use the one that passes 3.5 mesh (aperture 5.5 mm) using a standard sieve according to JIS standard, and the one that passes 6 mesh (aperture 3.35 mm) is used. It is more preferable. Further, it is most preferable to use a finely pulverized product as shown in the examples described later. In this case, for example, by using a standard sieve according to JIS standard, 90% by mass or more of the whole passes 16 mesh (aperture 1.0 mm), and 50% by mass or more of the whole passes 20 mesh (aperture 0. 85 mm) is preferably finely pulverized, 90% by mass or more of the whole passes 100 mesh (aperture 0.15 mm), and 50% by mass or more of the mesh is 150 mesh (aperture) It is more preferable to use a finely pulverized product that passes 0.1 mm). As a means for fine pulverization, mechanical pulverization is effective. Specifically, a hammer mill, a grinder, a cutter mill, a ball mill, a roll mill, a disk mill, a homomixer, a pin mill, a jet mill, and the like can be used.

  In the present invention, an extraction solvent is added to the mycelium and / or fruiting body of basidiomycete and subjected to pressure treatment. The ratio of the extraction solvent added to 100 parts by mass of the solid content of the basidiomycete mycelium and / or fruiting body is preferably 300 to 3000 parts by mass, and more preferably 500 to 2000 parts by mass. As pressurization conditions, it is preferable to carry out for 0.5 to 5 hours at a temperature of 100 to 200 ° C. and a pressure of 0.1 to 1.0 MPa, and at a temperature of 110 to 150 ° C. and a pressure of 0.11 to 0.5 MPa. More preferably, it is performed for 3 hours. The means for the pressure treatment is not particularly limited, and for example, a hermetic heating device, an autoclave, a kettle with a lid and a pot for frying can be used. In addition, before and / or after this pressurization process, you may heat-process at a normal pressure of about 40-100 degreeC.

  In the present invention, the enzyme treatment is performed by adding an enzyme to the pressure-treated product after the pressure treatment. The pressure-treated product may be used as it is after being pressure-treated, and an enzyme may be added thereto. Factors affecting the enzyme reaction, such as mycelia of basidiomycetes in the pressure-treated product and / or Alternatively, the enzyme may be added to the fruit body after the content concentration, pH, salt concentration, mineral concentration and the like are appropriately adjusted. The enzyme reaction conditions may be set as appropriate according to the type of enzyme used. Typically, the enzyme is added as an amount added to 100 parts by mass of the solid body of the mycelium and / or fruiting body of the basidiomycete used. Can be exemplified by adding 0.1 to 10.0 parts by mass, and reacting at a temperature of 30 to 60 ° C. and a pH of 3 to 10 for 1 to 48 hours. Stirring and shaking may be performed during the enzyme reaction.

  In the present invention, β-glucan is eluted in the liquid part of the enzyme-treated product. Here, β-glucan means a polysaccharide in which a large number of glucoses are linked by β1,3 bonds. However, this is not intended to exclude things involving structures other than the structure. For example, β-glucan having a side chain or a branch due to β1,4 bond or 1,6 bond of glucose, or the like, sulfate group, phosphate group, acetyl group, Those having a sugar chain or the like are also included. The elution of β-glucan means that β-glucan released from the mycelium and / or fruiting body of the basidiomycete used is recovered in the liquid part of the enzyme-treated product.

  And the part (component) which is not free | released from the mycelium and / or fruiting body of the basidiomycete used can be removed by carrying out solid-liquid separation by filtration, centrifugation, etc. The β-glucan-containing composition thus obtained preferably contains 10-60% by mass of β-glucan in the solid content, more preferably 15-50% by mass. It is most preferable to contain 20-40 mass%. The amount of β-glucan is the total amount of glucan obtained by degrading glucan in a sample to glucose with a degrading enzyme (for example, exo-1,3-β-glucanase and β-glucosidase) and quantifying the amount of glucose. From the above, it can be calculated by subtracting the amount of α-glucan obtained by degrading to glucose with an enzyme (for example, amyloglucosidase) that specifically degrades α-glucan in the sample and quantifying the amount of glucose. The amount of glucose produced is determined by degrading to glucose with an enzyme that specifically degrades α-glucan (for example, amyloglucosidase), recovering insoluble matter by 80% ethanol precipitation, and hydrolyzing it with sulfuric acid. The amount of β-glucan can be determined.

  Hereinafter, the present invention will be described in more detail with reference to examples. In addition, these Examples do not limit the scope of the present invention. In the following Comparative Examples and Examples, the β-glucan content was calculated using a β-glucan measurement kit “Mushroom and yeast beta-glucan assay kit” (trade name, Megazyme).

<Comparative Example 1>
Ganoderma fruiting bodies were pulverized with a mixer and sieved using 6 mesh (aperture 3.35 mm). To 5 g of this ground ganoderma fruit body, 50 ml of water was added and hydrothermally treated in a boiling water bath for 2 hours, and then the residue and the extract were separated. Thereafter, 35 ml of water was added to the residue, and the mixture was stirred for 10 minutes to homogenize, and then the residue and the cleaning solution were separated. After integrating these extracts and washings, Bx solid yield, dry yield, β-glucan content per dry yield, and β-glucan yield were calculated.

<Comparative example 2>
After adding 50 ml of water to 5 g of ground ganoderma fruit bodies prepared in the same manner as in Comparative Example 1, pressure treatment (2 hours after reaching 120 ° C., pressure 0.11 to 0.12 MPa), The residue and the extract were separated. Thereafter, 35 ml of water was added to the residue, and the mixture was stirred for 10 minutes to homogenize, and then the residue and the cleaning solution were separated. After integrating these extracts and washings, Bx solid yield, dry yield, β-glucan content per dry yield, and β-glucan yield were calculated.

<Comparative Example 3>
To 5 g of ground ganoderma fruit bodies prepared in the same manner as in Comparative Example 1, 50 ml of water was added, 1% β-glucanase of the ground ganoderma weight used and 1% chitinase of the same weight were added, 50 Treated at ° C for 16 hours. After boiling the reaction liquid for 10 minutes and deactivating the enzyme, the same pressure treatment as in Comparative Example 2 (2 hours after reaching 120 ° C., pressure 0.11 to 0.12 MPa) was performed. Thereafter, in the same manner as in Comparative Example 1, an extract and a washing solution were obtained, and after these extracts and the washing solution were integrated, Bx solid yield, dry yield, β-glucan content per dry yield, and β-glucan yield Was calculated.

<Example 1>
50 ml of water was added to 5 g of ground ganoderma fruit bodies prepared in the same manner as in Comparative Example 1, and pressure treatment (2 hours after reaching 120 ° C., pressure 0.11 to 0.12 MPa) was performed. Thereafter, β-glucanase of 1% by weight of the ground ganoderma used was added and treated at 50 ° C. for 16 hours. After boiling the reaction solution for 10 minutes and deactivating the enzyme, an extract and a washing solution were obtained in the same manner as in Comparative Example 1. After these extracts and the washing solution were integrated, Bx solid yield, dry yield, drying The β-glucan content per yield and the β-glucan yield were calculated.

<Example 2>
50 ml of water was added to 5 g of ground ganoderma fruit bodies prepared in the same manner as in Comparative Example 1, and pressure treatment (2 hours after reaching 120 ° C., pressure 0.11 to 0.12 MPa) was performed. Thereafter, 1% β-glucanase and 1% chitinase of the same weight of ground ganoderma used were added and treated at 50 ° C. for 16 hours. After boiling the reaction solution for 10 minutes and deactivating the enzyme, an extract and a washing solution were obtained in the same manner as in Comparative Example 1. After these extracts and the washing solution were integrated, Bx solid yield, dry yield, drying The β-glucan content per yield and the β-glucan yield were calculated.

<Example 3>
The reishi fruit body was finely pulverized with a jet mill apparatus (manufactured by Nippon Pneumatic Industry Co., Ltd.) to prepare a fine powder having a median diameter of 4.3 μm. Except for the use of this fine particle ganoderma, an extract and a washing solution were obtained in the same manner as in Example 2, and after the extraction solution and the washing solution were integrated, Bx solid yield, dry yield, β-per dry yield The glucan content and β-glucan yield were calculated.

  The results of Comparative Examples 1 to 3 and Examples 1 to 3 are shown in Table 1.

  As shown in Table 1, from comparison between Comparative Example 1 and Comparative Example 2, by performing pressure treatment on the ground ganoderma fruit body, Bx solid yield, dry yield, dry yield compared with hydrothermal treatment The per β-glucan content and the β-glucan yield increased by about 1.6 times, 1.6 times, 1.3 times, and 2.1 times, respectively.

  As shown in Table 1, from the comparison between Comparative Example 2 and Example 1, after pressure-treating the ground ganoderma fruit body, by making glucanase act, compared to the case where glucanase is not acted, Bx solid yield, The dry yield, β-glucan content per dry yield, and β-glucan yield were about 1.2 times, 0.9 times, 1.4 times, and 1.3 times, respectively. A concomitant increase in yield was observed.

  As shown in Table 1, the comparison between Example 1 and Example 2 shows that after crushing the ground ganoderma fruit body, by using chitinase in combination with glucanase, Bx solids compared to the case of glucanase alone. Yield, dry yield, β-glucan content per dry yield, and β-glucan yield were about 1.0 times, 1.4 times, 1.0 times, and 1.4 times, respectively. Increase in yield was observed. In comparison with Comparative Example 2 in which the enzyme was not allowed to act, the Bx solid yield, the dry yield, the β-glucan content per dry yield, and the β-glucan yield were about 1.2 times and 1.3 times, respectively. Times, 1.3 times, and 1.8 times, respectively, in comparison with Comparative Example 1 with only hot water treatment, about 1.9 times, 2.1 times, 1.8 times, and 3.6 times, respectively. Met.

  As shown in Table 1, from the comparison between Comparative Example 3 and Example 2, glucanase and chitinase acted after pressurizing the ground ganoderma fruit body to act before pressurizing. Bx solid yield, dry yield, β-glucan content per dry yield, and β-glucan yield are about 1.1 times, 1.4 times, 1.4 times, and 1.9, respectively. Doubled, an increase in dry yield and yield with increasing β-glucan content was observed.

As shown in Table 1, from the comparison between Example 2 and Example 3, Bx solid yield, dry yield, per dry yield compared to coarse crush by performing fine grinding treatment on Ganoderma turf The β-glucan content and β-glucan yield were about 1.3 times, 0.9 times, 1.2 times, and 1.1 times, respectively, and the β-glucan content reached 28%, the maximum among the test examples. did. In comparison with Comparative Example 1 in which only hydrothermal treatment was performed, the Bx solid yield, the dry yield, the β-glucan content per dry yield, and the β-glucan yield were about 2.5 times and 1.9 times, respectively. 2.1 times and 4.0 times.

Claims (5)

After heating and pressurizing treatment by adding an extraction solvent to the mycelium and / or child entities of the basidiomycetes, by adding an enzyme comprising glucanase and chitinase enzyme treatment in the heating and pressing treatment was liquid enzyme treated parts to β- glucan was eluted method of the composition obtained, characterized in Rukoto β- glucan-containing composition containing 10 to 60 wt% of the β- glucan in solids.   The method for producing a β-glucan-containing composition according to claim 1, wherein the basidiomycete is ganoderma. The method for producing a β-glucan-containing composition according to claim 1 or 2, wherein the heat and pressure treatment is performed at a temperature of 100 to 200 ° C and a pressure of 0.1 to 1.0 MPa. Prior to the heat and pressure treatment, the mycelium and / or fruiting body of the basidiomycete passed through 16 mesh (aperture 1.0 mm) of 90% by mass or more using a standard sieve according to JIS standards. The β-glucan-containing composition according to any one of claims 1 to 3 , which is finely pulverized so that 50% by mass or more of the whole has a particle size that passes 20 mesh (aperture 0.85 mm) . Production method. After the enzyme treatment, the extract is recovered by solid-liquid separation, and then the second extraction solvent is added to the obtained insoluble residue and stirred, and the extract is recovered again by solid-liquid separation. Item 5. A method for producing a β-glucan-containing composition according to any one of Items 1 to 4 .