JP2012055307A - Development of system for efficient ethanol production from biomass using mushroom - Google Patents

Abstract

PROBLEM TO BE SOLVED: To elucidate an efficient alcohol production method using a basidiomycete, a production method for an alcoholic beverage, a production method for alcohol-containing food products, or a spawn used therefor.SOLUTION: An alcohol production method producing alcohol from a carbon source by using a basidiomycete of genus Trametes, is used, or, an alcohol production method producing alcohol from a carbon source by using one or more basidiomycetes selected from the group consisting of Trametes hirsuta, Trametes versicolor and Trametes suaveolens, is used.

Description

  The present invention relates to an alcohol production method, an alcoholic beverage production method, an alcohol-containing food production method, or an inoculum used for them.

  Currently, about 90% of the world's energy consumption depends on fossil fuels such as oil, and it is in a place of energy depletion. In addition to the recent rise in crude oil prices, global environmental issues such as an increase in carbon dioxide and global warming are attracting attention, which may lead to future depletion of fossil fuels and high crude oil prices. Prior to this, new renewable energy alternatives to fossil fuels are being developed around the world. Under such circumstances, expectations for biofuels are increasing, with movements related to the development and introduction of biofuels such as bioethanol becoming active mainly in Brazil, the United States, and Asian countries. However, most of these are produced using food and feed crops such as sugarcane and corn as raw materials, and the aftermath of farmers' crop rotations, for example, has led to a rapid increase in the grain market. Therefore, in the medium to long term, the utilization of unused resources such as waste wood and herbs is important on a global scale with the aim of putting biofuels that do not compete with food and feed crops into practical use.

  On the other hand, a technique is known in which sugar is produced from biomass resources such as woody and herbaceous, and alcohol such as ethanol that is useful as a fuel is produced from the obtained sugar. For example, Patent Document 1 and Non-Patent Document 1 disclose that lignin is decomposed by white rot fungi having lignin-decomposition prior to saccharification when producing biofuel from woody biomass (or lignocellulosic plant material). A method for performing biological treatment (pretreatment) is disclosed.

  Research has also been conducted on producing alcohol from carbon sources using fungi. For example, Patent Documents 2 to 4 and Non-Patent Documents 2 to 6 include Agaricus blazei, Hypsizygus marmoreus, Tricholoma matsutake, Pleurotus ostreatus, Lentinellus cochleatus, and Enokitake. A method for producing alcohol from a carbon source using basidiomycetes such as (Flammulina velutipes) is disclosed.

JP 2008-6372 JP 2001-286276 A JP 2004-298109 A Japanese Unexamined Patent Publication No. 2006-223159

Bioenergy technology and application development, issued October 31, 2003, CMC Publishing Co., pp.165-171 Tokumitsu OKAMURA, Tomoko OGATA, Norie MINAMOTO, Tomomi TAKENO, Hiroko NODA, Shoko FUKUDA and Masahiro OHSUGI, "Characteristics of Wine Produced by Mushroom Fermentation", Bioscience, Biotechnology, and Biochemistry Vol. 65 (2001), No. 7 pp.1596 -1600 Tokumitsu Okamura-Matsui, Tomomi Tomoda, Shoko Fukuda and Masahiro Ohsugi, "Discovery of alcohol dehydrogenase from mushrooms and application to alcoholic beverages", Journal of Molecular Catalysis B: Enzymatic, Volume 23, Issues 2-6, 1 September 2003, pp. 133-144 Chemical Industry Daily, published on October 22, 2007, Chemical Industry Daily Inc. 'Use of whole crop sorghums as a raw material in consolidated bioprocessing bioethanol production using Flammulina velutipes.' Mizuno et al., Biosci Biotechnol Biochem. 2009 Jul; 73 (7): 1671-3. Epub 2009 Jul 7. 'Properties of ethanol fermentation by Flammulina velutipes.' Mizuno et al., Biosci Biotechnol Biochem. 2009 Oct; 73 (10): 2240-5. Epub 2009 Oct 7.

  However, in the production of alcohol from biomass such as conventional woody materials, the steps of hydrolyzing cellulose and hemicellulose, which are the main components of biomass, to produce sugar (saccharification), and the produced sugar to sugar by yeast and bacteria The two steps of producing alcohol by fermenting the alcohol were necessary. Therefore, in the whole alcohol production process from biomass, complicated work is required, and there is a problem that costs and work time increase.

  Moreover, in the process of saccharification mentioned above, since hydrolysis with sulfuric acid is required, it has been regarded as a problem in terms of processing cost and environment. Moreover, reaction control was required in order to suppress the excessive decomposition of sugars, such as produced | generated glucose.

  Furthermore, as a saccharification method other than using sulfuric acid, (1) pulverization, steaming (steaming explosion, steaming, hydrothermal decomposition, pressurized hot water treatment, etc.), energy rays (electron beams, γ rays, microwaves) (2) Chemical pretreatment that uses an acid (sulfuric acid, sulfurous acid, phosphoric acid), alkali (caustic soda, ammonia, etc.), etc. (3) Pretreatment by biological pretreatment using lignin-degrading bacteria such as white rot fungi, decomposes refractory components such as lignin, and then adds saccharifying enzyme such as cellulase to hydrolyze cellulose. There is an enzymatic saccharification method that breaks down into sugar.

  However, the above-described pretreatment method (1) requires expensive equipment such as a high-temperature / high-pressure apparatus for cooking and an irradiation apparatus for energy beams such as electron beams, γ-rays, and microwaves. In addition, the pretreatment process must be strictly managed. In the pretreatment method (2) described above, since chemicals such as acids and alkalis are used for saccharification or pretreatment of biomass, it is necessary to use equipment that can withstand such chemicals. There was a problem of high costs. Then, it is necessary to neutralize the acid or alkali used for saccharification or pretreatment and to treat a large amount of neutralized waste (such as calcium sulfate) generated by the treatment, or to remove the solvent used for the pretreatment. Therefore, it took time and cost, and there was an environmental problem. Furthermore, since the saccharifying enzyme is used in the pretreatment method (3) described above, it is expensive, and reaction control is necessary to suppress the excessive decomposition of sugar by the enzyme. Although this method does not cause inconveniences like other pretreatment methods, it is necessary to remove white rot fungi for the enzymatic saccharification step and the subsequent step of sugar fermentation such as yeast. There was a problem that it took. In the first place, white rot fungi are not introduced with the intention of producing alcohol, and actual alcohol production is carried out by fermentation of yeast or the like, so that the white rot fungus used in the above method (3) is used. There was no particular need to consider the alcohol-producing ability.

  Moreover, in the conventional alcohol production method using basidiomycetes, sugars classified as pentose such as xylose could hardly be fermented to alcohol. Therefore, the amount of ethanol obtained from biomass was not sufficient.

  The present invention has been made in view of the above circumstances, and an object thereof is to provide an efficient alcohol production method using a specific basidiomycete.

  ADVANTAGE OF THE INVENTION According to this invention, the alcohol production method which produces | generates alcohol from a carbon source using the basidiomycete of the genus Trametes is provided.

  It has been demonstrated that this production method can be used to efficiently produce alcohol in the examples described below. Therefore, if this production method is used, alcohol can be produced efficiently.

  Moreover, according to this invention, the production method of the alcoholic beverage which produces | generates the liquid which contains alcohol from a carbon source using the basidiomycete genus Basiophilus is provided.

  Here, it has been demonstrated that a liquid containing alcohol can be efficiently generated from a carbon source using a basidiomycete genus basidiomycete in Examples described later. Therefore, if this production method is used, a liquid containing alcohol can be produced efficiently.

  Moreover, according to this invention, the production method of the alcohol containing foodstuff which produces | generates the composition containing alcohol from a carbon source using the basidiomycete belonging to the genus White mushroom is provided.

  Here, it has been demonstrated that a composition containing alcohol can be efficiently generated from a carbon source using a basidiomycete genus basidiomycete in Examples described later. Therefore, if this production method is used, an alcohol-containing food can be produced efficiently.

  According to the present invention, there is provided an inoculum for producing alcohol from a carbon source using a basidiomycete genus basidiomycetes, comprising: Provided.

  This inoculum comprises basidiomycete hyphae that have been proven to be able to produce alcohol efficiently in the examples described below. Therefore, if this inoculum is used, alcohol can be produced efficiently.

  ADVANTAGE OF THE INVENTION According to this invention, efficient alcohol production from biomass etc. is realizable by the fungi excellent in alcohol production efficiency and the utilization property of a carbon source.

FIG. 1 (a) and FIG. 1 (b) are flowcharts for explaining a method of producing alcohol from a carbon source by a basidiomycete basidiomycete according to an embodiment. FIG. 2A, FIG. 2B, and FIG. 2C are conceptual diagrams schematically showing the structure of an inoculum using a basidiomycete genus basidiomycete according to the embodiment. FIG. 3 is a conceptual diagram for explaining a conventional ethanol production process from typical biomass. FIG. 4 is a conceptual diagram for explaining an example of a method for reusing unused biomass resources using the basidiomycetous basidiomycetes according to the embodiment. FIG. 5 is a conceptual diagram for explaining a cycle of reusing an unused biomass resource as a fuel alcohol using a basidiomycete basidiomycete according to an embodiment. FIG. 6 is an experimental protocol for explaining a method for culturing and recovering fungus basidiomycetes belonging to the genus White mushroom.

  As a result of intensive studies, the present inventors have performed a single step of producing a sugar by saccharifying a variety of carbon sources and a step of producing an alcohol by fermenting the sugar. The present invention was completed by finding that alcohol can be produced directly from biomass by confirming that it has high alcohol productivity.

  Hereinafter, embodiments of the present invention will be described in detail. In addition, in order to avoid the repetition complexity about the same content, description is abbreviate | omitted suitably.

  One embodiment of the present invention is an alcohol production method in which alcohol is produced from a carbon source using a basidiomycete genus basidiomycete. If this production method is appropriately used, alcohol can be produced efficiently as demonstrated in the examples described later. The basidiomycetes of the genus Shilotake mushroom are not particularly limited, but, for example, Aragekawaratake (Trametes hirsuta), Kawaratake (Trametes versicolor), Shiroamitake (Trametes suaveolens), Ouchirimentaitake (Trametes gibbosa), Rabbittake (Trametes trogiiBerkeient), ), Yakifukutake (Trametes pubescens), Tirimentake (Trametes elegans), and Trametes lactinea. Among them, Algae kawatake, Kawaratake, and white mushroom can produce alcohol from fructose, mannose, galactose, cellobiose, maltose, arabinose, xylose, sucrose, starch, CMC, wheat bran, rice straw, and rice straw after ball milling. This is demonstrated in the examples described below. This jellyfish, bamboo moth, and white mushroom are white rot fungi that generally have lignin-degrading ability and are distributed worldwide. In particular, it is demonstrated in the Examples described later that Alagekawaratake and Kawaratake can produce a high concentration of alcohol even from biomass such as wheat bran, rice straw, and rice straw after ball milling.

  Conventionally, in alcohol production methods using basidiomycetes, sugars classified as pentose such as xylose could hardly be fermented to alcohol. Therefore, the amount of ethanol obtained from biomass was not sufficient. On the other hand, basidiomycetous basidiomycetes can produce alcohol from such sugars, and thus can produce alcohol from biomass more efficiently. In particular, it is demonstrated in the examples described later that Aragekawaratake and Kawaratake can efficiently convert such sugars to ethanol. This was particularly noticeable in Kawatake. Xylose is known to exist widely in plants as a constituent of xylan, one of hemicelluloses. Although Pichia stipitis is known as a yeast capable of fermenting xylose, ethanol tolerance is low, and ethanol conversion efficiency and glucose fermentation efficiency are not sufficient. Moreover, since this yeast has no saccharification ability, it was necessary to provide a saccharification step separately or to carry out a method such as genetic modification in order to produce ethanol from biomass.

  In the present embodiment, one of the characteristics is that a basidiomycete belonging to the genus Shiroamitake is used for alcohol production. On the other hand, as shown below, nothing has been reported so far on the alcohol-producing ability of basidiomycetes belonging to the genus Shiroamitake. For example, basidiomycetes described in Patent Document 1 and Non-Patent Document 1 are used for biological pretreatment in the saccharification process described above, and are not used for directly generating alcohol. Therefore, in Patent Document 1 and Non-Patent Document 1, it is not necessary to consider the alcohol-producing ability of basidiomycetes, and naturally, the alcohol-producing ability is not even suggested. Also, Patent Documents 2 to 4 and Non-Patent Documents 2 to 6 do not describe anything about producing alcohol from a carbon source using a basidiomycete genus basidiomycete.

  That is, no alcohol-producing ability of the basidiomycetes belonging to the genus White mussel has been reported so far, and this embodiment can be said to be an excellent alcohol production method that has not existed so far. In addition, since the functions of the respective basidiomycetes are different for different eyes, families, or genera, it is difficult to conceive the alcohol production method of the present embodiment from these documents.

  In the present embodiment, the basidiomycetes belonging to the genus Shiroamitake are not toxic and can be wild, not genetically modified. Therefore, in the alcohol production method of the present embodiment, highly safe alcohol production can be realized.

  Further, basidiomycetes belonging to the genus Lactobacillus are not generally used for food. Moreover, there was no report that it was used effectively industrially, and it was hardly used until now. And the basidiomycetes belonging to the genus Lactobacillus grow relatively fast, are distributed worldwide and are relatively easy to obtain. Therefore, in the alcohol production method according to the present embodiment, it is unlikely that the basidiomycete basidiomycete used will at least directly compete with the industry, and is highly useful and advantageous as a resource.

  FIG. 1 is a flow chart for explaining a method for producing alcohol from a carbon source by a basidiomycete basidiomycete according to an embodiment. As will be described later, the present inventors have found that basidiomycetes belonging to the genus White mussel have a wide range of sugar utilization.

  Specifically, as shown in FIG. 1A, first, basidiomycetous basidiomycetes are inoculated into a woody or herbaceous biomass or a carbon source containing various sugars (S102). Next, the carbon source inoculated with the inoculum of the basidiomycete belonging to the genus Shiroamitake is cultured (S104). And the liquid containing the alcohol produced | generated from the carbon source by the basidiomycete genus basidiomycete is collect | recovered by methods, such as filtration (S106).

  When the carbon source includes woody biomass or the like, the carbon source is saccharified by basidiomycetous basidiomycetes (S108) as shown in FIG. Then, the saccharified carbon source is fermented with a basidiomycete basidiomycete to produce alcohol (S110).

  In addition, although the above-mentioned fermentation process may be anaerobic conditions and may be aerobic conditions, microaerobic conditions or anaerobic conditions are preferable from the surface of alcohol production efficiency.

  Moreover, the above-mentioned carbon source may contain saccharide | sugar. Furthermore, the above-described carbon source may contain one or more sugars selected from the group consisting of glucose, fructose, mannose, galactose, cellobiose, maltose, arabinose, xylose, sucrose, and lactose. As will be described later, the present inventors have found that basidiomycetes belonging to the genus White mussel have assimilation properties for these sugars. Furthermore, the above-mentioned carbon source may contain molasses, molasses, super white sugar, brown sugar, malt extract, starch and the like. The basidiomycetous basidiomycetes according to this embodiment may produce alcohol from a medium in which a plurality of carbon sources are mixed. The mass% or volume% of the individual carbon sources in the medium may be, for example, 1, 2, 3, 5, 10, 20, 50, 80, or 100%, within the range of those values or their It may be greater than or equal to the value.

  Or the above-mentioned carbon source may contain the material (biomass etc.) of cellulose, woody, or herbaceous. Examples of cellulose include plant cellulose obtained from cotton linter, wood pulp, dissolved pulp and the like, cellulose produced by microorganisms belonging to the genus Acetobacter, regenerated cellulose, microcrystalline cellulose, and the like. Examples of the wood-based material include waste wood such as building waste, lumber from sawmill, thinned wood, and forest land, wood, sawdust, paper such as waste paper and waste paper, and pulp. Examples of herbaceous materials include agricultural residues including bran such as wheat bran, straw such as rice straw and straw, and rice husks, bagasse, cut grass and weeds. As described later, the present inventors have found that basidiomycetous basidiomycetes have an alcohol-producing ability directly from wheat bran and rice straw as a carbon source and have a high alcohol yield. Moreover, it is suggested that it has a cellulose resolution in the Example mentioned later.

  Moreover, the above-mentioned carbon source may include raw garbage or waste beverage (hereinafter also referred to as “raw garbage”). The above-mentioned garbage etc. may be commercial or household garbage. Moreover, the garbage etc. which are discharged | emitted from a cafeteria, a hospital, a school, a hotel, a food factory, an agricultural livestock farm, etc. may be sufficient. Moreover, the garbage etc. which were prepared in the laboratory or the factory may be sufficient. Moreover, the garbage may contain a discarded food or a discarded beverage. In addition, in this specification, a discarded food means a discarded food, a food having no commercial value as food, or a food after the expiration date. In the present specification, the discarded beverage means a discarded beverage, a beverage having no commercial value for drinking, or a beverage after the expiration date. The discarded food may include, for example, discarded vegetables, discarded fruits, discarded meat, discarded fish, discarded cooked rice, discarded corn, discarded eggs, or discarded noodles. The discarded beverage may include, for example, a discarded milk-based beverage. Moreover, the garbage may be in a state after crushing treatment. The composition of raw garbage may include an inorganic substance such as sugar, lignin, protein, lipid, amino acid, organic acid, vitamins, or mineral.

  The above-mentioned carbon source may contain a food or a dairy beverage. The food may include processed food or fresh food. The food may contain, for example, vegetables, fruits, meat, fish, cooked rice, corn, eggs, or noodles. The milk-based beverage may contain milk, for example. The milk may be commercially available milk such as Meiji delicious milk (Meiji Co., Ltd.). The milk may be, for example, component unadjusted milk or adjusted (eg, low fat, no fat, high fat) milk. The food or dairy beverage may be within the expiration date or after the expiration date.

  Moreover, the alcohol produced | generated by the above-mentioned Bacillus subtilis genus basidiomycete may contain ethanol. As will be described later, the present inventors have found that basidiomycetes belonging to the genus White mushroom produce ethanol from a carbon source.

And you may produce alcoholic beverages or alcohol-containing foods using the alcohol produced | generated by the above-mentioned basidiomycete basidiomycetes. In addition to alcohol, these alcoholic beverages or alcohol-containing foods may contain various components produced by the basidiomycetes of the genus Shiroamitake. In addition, the alcohol-containing food may be solid, liquid, or gel.

  FIG. 2 is a conceptual diagram schematically showing a configuration of an inoculum using a basidiomycete belonging to the genus White mushroom according to the embodiment. An inoculum is a culture that is the basis for growing and increasing the fungi to be studied. An inoculum can be paraphrased as a material for alcohol production. FIG. 2 (a) shows an inoculum of a basidiomycete belonging to the genus Shiroamitake using sawdust as a carrier. In this inoculum 200, sawdust 208 is spread in a container 202 having a lid 204. In this sawdust, white mycelia 206a and 206b are carried. In addition, the mycelium of the basidiomycete belonging to the genus White mushroom does not need to form a mushroom as shown in FIG.

  FIG. 2 (b) shows an inoculum of the basidiomycete of the genus Shirotake using wood chips as a carrier. In this inoculum 300, wood chips 304a, 304b, 304c, 304d, 304e, and 304f having a cork stopper shape are accommodated in a container 302. These wood chips carry hyphae 306a, 306b, 306c, 306d, 306e, and 306f of basidiomycetes belonging to the genus White mushroom. In addition, the mycelium of the basidiomycete belonging to the genus White mushroom does not need to form a mushroom as shown in FIG.

  FIG. 2 (c) shows an inoculum of a basidiomycete belonging to the genus Shiroamitake using a liquid medium as a carrier. In the inoculum 400, a liquid medium 408 containing a carbon source such as various sugars is stored in a container 402 having a lid 404. These liquid media carry the mycelium 406 of basidiomycetous basidiomycetes. In addition, the mycelium of the basidiomycete belonging to the genus White mushroom does not need to form a mushroom as shown in FIG.

  The inoculum described above is an inoculum for producing alcohol from a carbon source using fungi, and includes a mycelium of a basidiomycete belonging to the genus Shiroamitake and a carrier that carries this mycelium. The hypha may be a stationary hypha. Specifically, this mycelium may be a mycelium 5 to 7 days after the start of culture.

Hereinafter, the effect of the alcohol production method according to the embodiment will be further described.
As described below, the basidiomycete belonging to the genus Shiroamitake has an alcohol-producing ability equal to or higher than that of mold and yeast, and thus can achieve excellent alcohol production efficiency.

  In addition, as mentioned below, basidiomycetes belonging to the genus Shiroamitake have an assimilation property for glucose, fructose, mannose, galactose, cellobiose, maltose, arabinose, xylose, and sucrose, and therefore, a wide variety of basidiomycetes that have been difficult for basidiomycetes Alcohol fermentation can be performed using a carbon source.

  Therefore, according to the alcohol production method according to the above-described embodiment, the production efficiency of the alcohol production method is improved because the basidiomycete of the genus Shiroamitake, which is excellent in the alcohol production efficiency and the assimilability of the carbon source, is used. The range of types can be expanded.

  FIG. 3 is a conceptual diagram for explaining a conventional ethanol production process from typical biomass. Conventionally, the step of saccharifying the carbon source and the step of fermenting the sugar have been performed by separate units. FIG. 4 is a conceptual diagram for explaining an example of a method for reusing unused biomass resources using the basidiomycetous basidiomycetes according to the present embodiment. Since this method includes a step of producing sugar by saccharifying the genus Basidium of the genus Shiroamitake, and a step of producing alcohol by fermenting the saccharide of the genus Shiroamitake genus Both the saccharification and fermentation steps can be performed without going through the hydrolysis step. For this reason, an unused biomass resource can be efficiently converted into ethanol by a saccharification / fermentation process using a basidiomycete genus basidiomycete. Moreover, it goes without saying that it leads to a reduction in environmental load, cost and labor. Moreover, saccharification and fermentation may be performed in the same container in parallel or simultaneously in the same container, and in this case, it is more efficient.

  The process of producing alcohol by performing saccharification and fermentation in parallel is generally referred to as consolidated bioprocessing (CBP). CBP is described, for example, in [Lynd et al., Curr Opin Biotechnol. 2005 Oct; 16 (5): 577-83.], [Van Zyl et al., Adv Biochem Eng Biotechnol. 2007; 108: 205-35.], [ Mizuno et al., Biosci Biotechnol Biochem. 2009 Jul; 73 (7): 1671-3. Epub 2009 Jul 7.]. And the basidiomycete belonging to the genus White mushroom according to the present embodiment can purify alcohol by CBP. In this CBP, enzymes and acids may be added to decompose lignin, cellulose, etc., but when using the above-mentioned basidiomycetous basidiomycetes, it is efficient without adding enzymes or acids. Can produce alcohol. In the present embodiment, it was considered that degradation of cellulose was important for alcohol production. However, in this embodiment, the use of the above-mentioned basidiomycete basidiomycetes having excellent lignin resolving power is an unpredictable high ethanol. This is thought to be one of the reasons why productivity has been achieved. Lignin is contained in woody or herbaceous materials.

  In this CBP, the basidiomycete basidiomycete according to the present embodiment may decompose lignin, cellulose, or hemicellulose, and further produce the alcohol by fermenting the produced monosaccharide or oligosaccharide. At this time, the monosaccharide may be hexose or pentose, and may be one or more sugars selected from the group consisting of glucose, fructose, mannose, galactose, arabinose, and xylose. The oligosaccharide may be one or more sugars selected from the group consisting of cellobiose, maltose, and sucrose. Such CBP is low in cost and low in environmental load. In addition, the principle similar to CBP is utilized also in the production process of brewing sake, and in that case, it is also called parallel double fermentation. In this parallel double fermentation, saccharification and fermentation are performed in parallel.

  Here, carbon sources obtained by acid saccharification of woody biomass such as wood and paper generally contain several percent of mannose in addition to glucose. Yeast suitably assimilate glucose, but has low assimilation ability for mannose. On the other hand, the basidiomycetes belonging to the genus Shiroamitake have the ability to assimilate mannose which is difficult to assimilate with yeast, and therefore can also assimilate mannose remaining after fermentation with yeast. Therefore, resource recycling efficiency and alcohol production efficiency can be increased by sugar-oxidizing woody biomass and performing alcoholic fermentation by combining yeast and basidiomycetous basidiomycetes. Natural yeast cannot directly convert biomass such as wheat bran into ethanol. In contrast, basidiomycetous basidiomycetes can produce ethanol directly from wheat bran and rice straw, as will be described later. Therefore, the alcohol production method according to the embodiment is rich in superiority and usefulness in that alcohol can be produced directly from biomass that cannot be used by natural yeast.

  FIG. 5 is a conceptual diagram for explaining a cycle of reusing unused biomass resources as alcohol for fuel using the basidiomycetous basidiomycetes according to the embodiment. In this cycle, unused biomass resources can be efficiently converted to alcohol by the saccharification / fermentation process using basidiomycetous basidiomycetes, so a resource circulation system can be constructed between biomass and fuel alcohol. . Therefore, it is possible to construct an energy supply system that can achieve both global environmental conservation and industrial development.

  In addition, according to the method for producing an alcoholic beverage according to the embodiment, the production efficiency of the method for producing an alcoholic beverage is improved by using a basidiomycete genus Basidiomycete, which is excellent in alcohol production efficiency and assimilation of a carbon source. The range of source types can be expanded.

  Furthermore, according to the method for producing an alcohol-containing food according to the embodiment, the production efficiency of the method for producing an alcohol-containing food is improved because the basidiomycete belonging to the genus Shiroamitake, which is excellent in alcohol production efficiency and assimilation of the carbon source, is used. Can widen the range of carbon source types.

  And, according to the inoculum according to the embodiment, the mycelia of the white basidiomycete belonging to the genus Shiroamitake, which are excellent in alcohol production efficiency and carbon source utilization, are supported on the carrier, so that alcohol is generated from the carbon source using fungi. Therefore, it can be suitably used as an inoculum.

  As mentioned above, although embodiment of this invention was described with reference to drawings, these are the illustrations of this invention, Various structures other than the above are also employable.

EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these. However,% displayed in the examples indicates mass percent unless otherwise specified.
[Used strain]
The strains in Table 1 were prepared. 28S rDNA nucleotide sequences of each strain in Table 1 were analyzed, and homology search was performed based on nucleotide sequence databases such as GenBank / DDBJ / EMBL. 100% homology with the 28S rDNA nucleotide sequences registered in the database. Showed the rate.

1) Medium used MYG medium or T medium was used as the medium used. Below, the basic composition of each culture medium is shown.
MYG medium Glucose *: 0.4%
Yeast extract: 0.4%
Malt extract: 1.0%
T medium Glucose *: 2.0%
Yeast extract: 1.0%
KH ₂ PO ₄ : 1.0%
(NH ₄ ) ₂ SO ₄ : 0.2%
MgSO ₄ .7H ₂ O: 0.05%
*) About glucose, it changed into the other carbon source as needed.

2) Cultivation and microbial cell recovery FIG. 6 is an experimental protocol for explaining the cultivation method of Kawaratake. First, in the pre-culture step, a 5 mm square mycelium fragment was cut out from Kawaratake's plate medium, added to 50 ml of MYG medium in a 500 ml Erlenmeyer flask, and the Erlenmeyer flask was aerobic using a cotton stopper or a silico stopper. The culture was performed at 28 ° C. for 5 to 7 days.

  Next, in the main culture step, 50 ml of T medium was added to a 500 ml Erlenmeyer flask, inoculated with pre-cultured mycelia of Kawaratake that was aseptically filtered and washed, and the Erlenmeyer flask was sealed with a silicon rubber stopper, and 28 ° C. Was subjected to stationary culture (culture under microaerobic conditions).

  Subsequently, in the culture solution collection step, collection was performed over time according to the following procedure. That is, the culture solution was sampled, centrifuged, and filtered through a 0.22 μm filter. Culture filtrate pH measurement, HPLC analysis, and activity measurement were followed by cryopreservation. For each of the strains other than Kawaratake listed in Table 1, cultivation and sample collection were performed in the same procedure as in the case of Kawaratake.

3) HPLC analysis The culture filtrate of each strain described in Table 1 was subjected to HPLC analysis. The analysis conditions are as follows.
(HPLC analysis conditions)
Capillary column: Shodex SUGAR SP0810 or Shodex SUGAR KS-801
Capillary size: 8.0mmID × 300mmL
Flow rate: 0.6 ml / min
Column temperature: 80 ° C
Extract liquid: Degassed distilled water Sample: 10 μl

4) Cultivation using a carbon source other than glucose Instead of glucose, which is the carbon source of T medium, the same concentration of fructose, mannose, galactose, cellobiose, maltose, arabinose, xylose, sucrose, starch (Starch), CMC (carboxymethylcellulose) ), Wheat bran, rice straw, and rice straw after ball milling (BM-Rice straw), the assimilation and ethanol productivity were confirmed. The procedure is the same as 2) and 3). The ball mill treatment was performed for 4 hours (10 minutes operation, 10 minutes pause) with a planetary ball mill Gokin planet M2-3F (manufactured by Nagao System) using a zirconia container and zirconia balls.

  The results of the above 1) to 4) are shown in Table 2 and Table 3. Each numerical value is an average of the results of three experiments.

  Table 2 is a table comparing the ethanol production rate when cultivating Trametes hirsuta and Trametes versicolor with glucose as a substrate with mold (Rhizopus oryzae, Mucor corticolous) and yeast (Saccharomyces cerevisiae). . From this table, it can be seen that Aragekawaratake and Kawaratake have higher ethanol productivity than mold and yeast.

  Table 3 shows various types of carbon sources (20 g / 1 L of medium) as substrates, and the four strains described in Table 1 (Trametes hirsuta, Trametes versicolor, White-faced bamboo (Trametes suaveolens), Pleurotus ostreatus) ) Is cultivated for 5 days (g / medium 1 L) From this table, it can be seen that, for example, Aragekawaratake, Kawaratake, and White-fly, fructose, mannose, galactose, cellobiose, maltose, arabinose, xylose, sucrose By metabolizing starch (Starch), CMC (carboxymethylcellulose), wheat bran (Rice straw), and rice straw after ball milling (BM-Rice straw) It can be seen that ethanol can be produced. Leucose, mannose, cellobiose and starch showed high values.

  Moreover, Kawaratake and Aragekawaratake showed significantly higher ethanol production than other strains when xylose, wheat bran, rice straw and rice straw after ball milling were used as substrates. In particular, when ordinary mushrooms are used, it is difficult to produce ethanol in an amount exceeding 2 g / L using xylose as a carbon source, but Kawaratake is 7.2 g / L and Aragekawaratake is 3. It was a surprising result that it showed a high output of 9 g / L. Since xylose is a component that is abundantly contained in various biomasses, it is considered that ethanol can be produced from various biomasses more efficiently than conventional methods when using Kawaratake or Aragekawaratake. In addition, when the same experiment was conducted a plurality of times, the amount of ethanol produced from xylose by Kawaratake was sometimes higher than 8.0 g / L. In addition, the amount of ethanol produced from xylose, wheat bran, rice straw, and rice straw after ball milling when using Aragekawaratake was high and sometimes showed a value of about 90% of that of Kawaratake.

  In addition, when using Arakawakawatake, Kawaratake, and white mushroom, even when the rice straw is not ball milled (3.0, 3.3, 1.5), it is treated with the ball mill (3.4, 3.7). Productivity close to 1.7) was observed. Usually, the ball mill is considered to promote saccharification by destroying the skeleton of cellulose, and is said to promote ethanol productivity with twice the efficiency. On the other hand, it is considered that the productivity did not change so much before and after the ball mill treatment when using agaricus mushrooms, mushrooms and white mushrooms in this example, because these strains decomposed cellulose. Therefore, it can be said that the ethanol production method using Algae kawatake, Kawaratake, and Shiroamitake is an excellent method that can produce ethanol efficiently without performing a special crushing step such as a ball mill. In particular, since the ball mill is an operation that requires a large amount of energy, the above method is significant from the viewpoint of energy saving.

5) Microaerobic culture, aerobic culture, and anaerobic culture Microalbic, aerobic, or anaerobic culture was carried out in T medium using glucose as a carbon source. . The microaerobic condition is the same as 2) above. The aerobic condition was changed to the silicone rubber stopper in the culture procedure of 2) above, and the culture was carried out with a lid with a breathable silico stopper. Anaerobic conditions were performed in the culture procedure of 2) above by filling nitrogen gas and sealing with a silicon rubber stopper. As a result, agaricus mushroom, agaricus mushroom and white mushrooms showed similar ethanol production when cultured under microaerobic and anaerobic conditions. In addition, agaricus mushrooms and mushrooms showed an ethanol production amount of about 70% under microaerobic conditions under aerobic conditions.

6) Examination of direct ethanol productivity from biomass Whether or not Kawaratake can produce ethanol directly from biomass in a single step without saccharification, and its productivity The wheat bran medium and the rice straw medium were used as carbon sources.

  Wheat bran is a mixture of the outer skin, germ, and endosperm remaining after milling. It is generated about 30% during milling and contains hemicellulose, cellulose and lignin as insoluble dietary fibers. The composition of common wheat bran is 35.7% glucose, 17.9% xylose, 46.4% lignin and others. Most of the wheat bran is currently used as livestock feed, but due to sluggish domestic demand for feed and the implementation of the Food Recycling Law, other food residues are becoming feedstuffs. In the future, the demand for wheat bran feed will decline and the price may decline. For this reason, effective use of wheat bran other than for feed has become an important issue (for example, [Seiichi Higuchi, Manabu Takahashi, Akitoshi Yamaji, “Development of New Utilization Technology for Wheat-derived Functional Components”, Saitama Sangyo Refer to Technical Center Research Report Vol. 5 (2007)].

  Rice straw is dried rice stalk and contains hemicellulose, cellulose and lignin as insoluble dietary fibers. The general rice straw composition is 29.4% glucose, 15.6% xylose, 55% lignin and others.

  The wheat bran medium and the rice straw medium have the same basic composition except that glucose is replaced with wheat bran or rice straw medium in the T medium of 1) above.

As a result of the above 6), 5.2 g of ethanol was confirmed from 20 g of wheat bran after 4 days of culture. This is estimated to have been converted in a yield of 95% from the total amount of glucose and xylose available for fermentation contained in wheat bran, 10.7 g. The above yield is calculated assuming that the amount of ethanol is 100% when it is assumed that glucose and xylose contained in wheat bran are all used for ethanol production. The reaction when ethanol is produced from 100 g of glucose can be represented by C ₆ H ₁₂ O ₆ (100 g) → 2C ₂ H ₅ OH (51.1 g) + CO ₂ (48.9 g). The reaction when ethanol is produced from 100 g of xylose can be expressed as 3C ₅ H ₁₀ O ₅ (100 g) → 5C ₂ H ₅ OH (51.1 g) + 5CO ₂ (48.9 g). If there is 1 ton of wheat bran, 535 kg (sugar that can be used for fermentation) × 0.51 × 0.95 (conversion rate) = 259.2 kg (328 L) of ethanol can be recovered.

  In addition, 3.3 g of ethanol was confirmed after 20 days of cultivation from 20 g of rice straw. This is estimated to have been converted in a yield of 72% from 9.0 g of the total amount of glucose and xylose available for fermentation contained in rice straw. If 1 ton of rice straw is present, 450 kg (sugar usable for fermentation) × 0.51 × 0.72 (conversion rate) = 165.2 kg (209 L) of ethanol can be recovered.

  In addition, even when ethanol is produced from rice straw using a conventional genetically modified bacterium and usually pretreated with sulfuric acid or the like, the yield is less than 80%. On the other hand, it was found that if Kawaratake is used, ethanol can be produced with a yield of 72%, which is close to the conventional method, without chemical treatment such as sulfuric acid.

  Furthermore, the direct productivity of ethanol from wheat bran or rice straw was investigated in the same manner by changing the moth bamboo to the moth moth bamboo. As a result, the yield was close to that obtained when Kawaratake was used.

7) Examination of direct ethanol productivity from raw garbage Using the raw garbage as a carbon source, the ethanol production ability of Aragekawaratake, Kawaratake, and Shiroamitake was examined. The garbage that was disposed of as garbage was obtained on the same day at the cafeteria in the national university corporation Tottori University (4-1, Koyamacho, Tottori City, Tottori Prefecture, 101-101). Thereafter, the garbage was stored at 4 ° C. This raw garbage contains vegetables, fruits, meat, fish, cooked rice, corn and the like. Next, water was added to the garbage to prepare a garbage medium (pH not adjusted). The preparation of the garbage medium was performed within 24 hours after the garbage was obtained from the cafeteria. The amount of garbage per liter of the garbage medium is 385 g. In addition, the total sugar amount of raw garbage was estimated to be about 6% (23.1 g).

  In the above procedures 2) to 3), the ethanol production was evaluated by changing the T medium to a garbage medium. The results are shown in Table 4. In addition, the value in a table | surface shows ethanol production amount (g / L). Each numerical value is an average of three experiments.

  As shown in Table 4, high concentrations of ethanol could be produced from raw garbage in a single step without using a separate saccharification treatment by using Kawaratake, Aragekawaratake and Shiroamitake. Specifically, in the case of Kawaratake, 8.4 g of ethanol was confirmed from 385 g of garbage after 5 days of culture. This is estimated that ethanol was converted in a yield of 71% from the total sugar amount of 23.1 g (assuming about 6% content) contained in the garbage.

  It is known that the average water content of raw garbage is 77 ± 4% and the total sugar content is 9 ± 4%. Accordingly, if there is 1 ton of garbage, 90 kg (total sugar amount) × 0.51 (based on glucose) × 0.71 (conversion rate) = 33 kg (41 L) of ethanol can be recovered.

  In Japan, about 20 million tons of garbage is discharged annually, of which about 17 million tons are not recycled. Emissions from cities with a population of 300,000 to 400,000 are estimated to be about 60 tons per day. Normally, the incineration cost is more than 10,000 yen per ton. Even if ethanol production from garbage is attempted by a conventional method, saccharification and fermentation cannot be performed in a single step, and there is a problem that costs, energy, and work time increase. On the other hand, it was found that saccharification and fermentation can be performed in a single step in the production of ethanol from garbage by using Kawaratake, Aragekawaratake, or Shiroamitake as described above. Moreover, the raw garbage to be used only needs to be mixed with water, and no special pretreatment such as pH adjustment was required. Therefore, the ethanol production method from raw garbage using Kawaratake, Aragekawaratake or Shiroamitake can be said to be an unprecedented low cost and low environmental impact type ethanol production method.

8) Examination of direct ethanol productivity from commercially available starch-based foods Ethanol-producing ability of Kawaratake was examined using commercially available starch-based foods as a carbon source. First, water was added to commercially available udon (raw noodles), Chinese noodles (raw noodles), or cooked rice to prepare three types of commercially available starch food media (pH not adjusted). In any medium, the commercially available starch-based food content per liter of the medium is 235 g. The total sugar amount was estimated as about 24% (57.1 g) for udon, about 28% (66.4 g) for Chinese soba noodles, and about 37% (88.1 g) for cooked rice. In addition, as for the commercially available starch-type food used above, all used what passed about 2 days from the expiration date.

  In the above procedures 2) to 3), the ethanol production was evaluated by changing the T medium to three types of commercially available starch-based food medium. The results are shown in Table 5. In addition, the value in a table | surface shows ethanol production amount (g / L). Each numerical value is an average of three experiments.

  As shown in Table 5, by using Kawaratake, a high concentration of ethanol could be produced from a commercially available starch-based food in a single step without any additional saccharification treatment. Specifically, from 238 g of udon, 28.8 g of ethanol was confirmed after 21 days of culture. In addition, production of 34.8 g of ethanol was confirmed from 238 g of Chinese buckwheat after 21 days of culture. In addition, 37.6 g of ethanol was confirmed after culturing 21 days from 238 g of cooked rice. All of these results indicate that the conversion rate to ethanol was 80% or more with respect to the contained sugar. As described above, by using Kawaratake, it was possible to efficiently produce ethanol without wasting food that had lost its value as a product. Moreover, the food to be used did not require special pretreatment such as pH adjustment.

9) Examination of direct ethanol productivity from milk We examined the ethanol production ability of Kawaratake using milk as a carbon source. First, water was added to commercially available milk at a volume ratio of 1: 1 to prepare a milk medium (pH not adjusted). The amount of lactose per liter of this milk medium is 23 g. The milk used was about 2 days after the expiration date.

  In the procedures 2) to 3), the T medium was changed to a milk medium, and the ethanol production was evaluated. The results are shown in Table 6. In addition, the value in a table | surface shows ethanol production amount (g / L). Each numerical value is an average value of two experiments. The numbers in parentheses indicate the remaining amount of lactose (g / L) in the medium.

  As shown in Table 6, by using Kawaratake, a high concentration of ethanol could be produced from milk in a single step without separate saccharification treatment. Specifically, 9.1 g of ethanol was confirmed after 8 days from 1 L of milk medium. This is estimated that ethanol was converted at a yield of 70% or more from 23 g of lactose contained in milk. As described above, by using Kawaratake, it was possible to efficiently produce ethanol without wasting milk that was no longer valuable as a product. Moreover, the milk to be used did not require special pretreatment such as pH adjustment or milk fat removal.

<Consideration of results>
Considering the above results. According to the alcohol production method of the present example, ethanol is efficiently produced in a single process directly from biomass with a single basidiomycete without performing a process of hydrolyzing biomass to produce sugar (saccharification). Can be produced. In addition, unlike the conventional method, this production method does not require saccharification treatment with an acid or the like, so that the environmental, facility, work, and cost burdens associated with this are reduced. Further, according to this production method, ethanol productivity close to that obtained when the ball mill treatment is performed can be obtained without performing the ball mill treatment. Therefore, energy required for a crushing process such as ball milling can be reduced.

  Moreover, according to the alcohol production method using Kawaratake and Aragekawaratake, xylose can be efficiently used as a carbon source. This makes it possible to produce ethanol from biomass more efficiently than the conventional method.

  In the above, this invention was demonstrated based on the Example. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are within the scope of the present invention.

  For example, in the above-described embodiment, a medium containing T medium, wheat bran medium, rice straw medium, or the like is used as the carbon source, but other carbon sources can be used as well. Specifically, woody biomass can be used as a carbon source.

  As described above, the basidiomycete used in the present invention is superior in alcohol production efficiency and carbon source assimilation, so that alcohol production efficiency is improved, an alcohol production method, an alcohol beverage production method, and an alcohol-containing food production method. It is useful as an inoculum for them.

200 Inoculum 202 Container 204 Lid 206 Hypha 208 Sawdust 300 Inoculum 302 Container 304 Wood chip 306 Hypha 400 Inoculum 402 Container 404 Lid 406 Hypha 408 Liquid medium

Claims (23)

  A method for producing alcohol, wherein a basidiomycete belonging to the genus Trametes is used to produce alcohol from a carbon source.   The alcohol production method according to claim 1, wherein the basidiomycete comprises one or more basidiomycetes selected from the group consisting of Trametes hirsuta, Trametes versicolor, and Trametes suaveolens.   The alcohol production method of Claim 1 or 2 which produces | generates alcohol by fermenting the said carbon source using the said basidiomycete.   The method for producing alcohol according to claim 1, wherein the carbon source contains sugar.   The carbon source is bran, rice straw, cellulose, wood, sawdust, paper, pulp, straw, rice husk, raw garbage, food, milk beverage, waste milk beverage, agricultural residue, bagasse, cut grass, and weed The alcohol production method according to any one of claims 1 to 4, comprising one or more selected from the group consisting of catechols.   The alcohol production method according to claim 1, wherein the carbon source is raw garbage.   The alcohol production method according to claim 1, wherein the carbon source is a waste milk-based beverage. The basidiomycete saccharifies the carbon source to produce sugar;
The basidiomycete produces alcohol by fermenting the sugar produced by saccharification;
The method for producing alcohol according to claim 1, comprising:   The alcohol production method according to claim 8, wherein the saccharification and the fermentation are performed in parallel in the same container.   The alcohol production method according to any one of claims 1 to 7, wherein the carbon source contains one or more sugars selected from the group consisting of glucose, fructose, mannose, galactose, cellobiose, maltose, arabinose, xylose, and sucrose.   The alcohol production method according to any one of claims 1 to 10, wherein alcohol is produced by an integrated bioprocess.   The alcohol production method according to claim 11, wherein the integrated bioprocess includes a step of degrading lignin contained in the carbon source using the basidiomycete.   The alcohol production method according to claim 11 or 12, wherein the integrated bioprocess includes a step of decomposing cellulose and hemicellulose contained in the carbon source using the basidiomycete. The integrated bioprocess is:
Decomposing lignin, cellulose, and hemicellulose using the basidiomycetes to produce monosaccharides or oligosaccharides;
Fermenting the monosaccharide or the oligosaccharide using the basidiomycete,
The method for producing alcohol according to claim 11, comprising: The monosaccharide comprises hexose or pentose;
The oligosaccharide includes one or more sugars selected from the group consisting of cellobiose, maltose, and sucrose;
The alcohol production method according to claim 14.   The alcohol production method according to claim 15, wherein the monosaccharide includes one or more sugars selected from the group consisting of glucose, fructose, mannose, galactose, arabinose, and xylose.   The alcohol production method according to any one of claims 1 to 16, wherein the basidiomycete is Aragekawaratake or Kawaratake.   The alcohol production method of Claim 17 which produces | generates alcohol from a pentose using the said Alage Kawaratake or Kawaratake.   The alcohol production method according to claim 18, wherein the pentose is xylose.   The alcohol production method according to claim 1, wherein the alcohol includes ethanol.   A method for producing an alcoholic beverage, comprising using a basidiomycete basidiomycete to produce a liquid containing alcohol from a carbon source.   A method for producing an alcohol-containing food, comprising using a basidiomycete basidiomycete to produce a composition containing alcohol from a carbon source. Hyphae of basidiomycetes belonging to the genus Shiroamitake,
A carrier carrying mycelia;
An inoculum for producing alcohol from a carbon source using a basidiomycete basidiomycete, comprising: