WO2011162249A1 - Method for manufacturing a monosaccharide, a disaccharide, and/or an oligosaccharide - Google Patents
Method for manufacturing a monosaccharide, a disaccharide, and/or an oligosaccharide Download PDFInfo
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- WO2011162249A1 WO2011162249A1 PCT/JP2011/064151 JP2011064151W WO2011162249A1 WO 2011162249 A1 WO2011162249 A1 WO 2011162249A1 JP 2011064151 W JP2011064151 W JP 2011064151W WO 2011162249 A1 WO2011162249 A1 WO 2011162249A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/12—Disaccharides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P2201/00—Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
Definitions
- the present invention relates to a method for producing polysaccharides, particularly monosaccharides, disaccharides, and / or oligosaccharides (hereinafter collectively referred to as “decomposed sugars”) from biomass.
- a method of pretreating biomass in advance prior to the saccharification step has been studied.
- the pretreatment method for example, a sulfuric acid method, an organic solvent method, a hydrothermal treatment method, mechanical crushing, and the like are known. Also, a method for efficiently producing an ethanol raw material by combining these methods has been studied.
- a coarse pulverization step for coarsely pulverizing a raw material containing lignocellulose a fine pulverization step for finely pulverizing a coarsely pulverized product
- fine pulverization A method (Patent Document 1) including a hydrothermal treatment step of hydrothermally treating an object and a dehydration step of dehydrating the hydrothermal treatment product has been reported.
- the present invention relates to the following 1) to 12). 1) Next steps (1) to (3): (1) A step of applying a compressive shear stress to a polysaccharide to pulverize the polysaccharide to obtain a pulverized polysaccharide; (2) a step of heat-treating the pulverized polysaccharide at 185 to 230 ° C. in the presence of water; (3) A step of obtaining a solid part by solid-liquid separation from the reaction product after the heat treatment, and allowing a hydrolase to act on the solid part, A process for producing monosaccharides, disaccharides and / or oligosaccharides.
- Patent Document 1 describes that when the finely pulverized product obtained in the pulverization step is hydrothermally treated, about 0.98 MPa (about 180 ° C. or less as saturated steam) is preferable in order to suppress excessive decomposition of sugar. is there.
- the Patent Document 1 does not disclose the implementation of hydrothermal treatment, and the present inventor performed the pulverization step of cellulose-based biomass and the hydrothermal treatment step according to the method described in Patent Literature 1, and then the actual enzymatic saccharification step.
- the yield of cellulose after the hydrothermal treatment step is high, it has been found that the reaction is difficult to proceed in the enzymatic saccharification step, and the amount of decomposition sugar produced as a whole may be reduced.
- the present invention relates to providing a method capable of improving the saccharification rate and efficiently producing a decomposed sugar from a polysaccharide.
- the inventors of the present invention have intensively studied in view of the above problems.
- the temperature can be reduced as in Patent Document 1.
- the yield of cellulose after heat treatment is lower than when heat-treated, the subsequent enzymatic saccharification step unexpectedly improves the saccharification rate significantly and ultimately increases the yield of decomposed sugar. I found it.
- the saccharification rate can be improved, and the decomposing sugar can be produced efficiently.
- the method of the present invention can also be applied to cellulosic biomass, and can be expected as a technique for improving the production efficiency of useful substances such as ethanol from biomass.
- Step (1) is a step of obtaining a pulverized polysaccharide by pulverizing the polysaccharide by applying compressive shear stress.
- the polysaccharide used in the present invention include cellulose, hemicellulose, xyloglucan, pectin, starch, mannan, glucomannan, galactomannan, chitin, chitosan, inulin, alginic acid, agar, fucoidan, laminarin, ⁇ -glucan, pullulan. Natural polysaccharides such as these or derivatives thereof.
- Examples of the derivative include esters in which at least a part of the hydroxyl group is esterified, ethers in which at least a part of the hydroxyl group is etherified, and the like. These can be used alone or in combination of two or more. Of these, cellulose, hemicellulose, chitin, and chitosan are preferable, and cellulose and hemicellulose are more preferable because they are inexpensive and can be converted into useful substances by fermentation production after decomposition.
- the molecular weight of the polysaccharide used in the present invention is not particularly limited, but generally it is preferably 1,000 or more and 5,000,000 or less.
- the raw material containing the said polysaccharide for example, biomass
- Biomass is an organic resource derived from living organisms, excluding fossil resources.
- the biomass include cellulose-based, starch-based, and saccharide-based biomass, and these can be used alone or in combination of two or more.
- Cellulose biomass is mainly composed of cellulose, hemicellulose, and lignin.
- cellulose hemicellulose
- lignin For example, cotton, wood pulp, kenaf, hemp, small-diameter wood, thinned wood, sawdust, wood waste, waste paper, newspaper, wrapping paper, tissue paper Woody materials such as toilet paper and cardboard; plant biomass such as bagasse, switchgrass, elephant grass, rice straw and wheat straw.
- starch-based biomass include rice, wheat, corn, and potato
- examples of the saccharide-based biomass include sugar-based biomass such as sugar cane, sugar beet, seaweed, shrimp shell, and crab shell.
- a compression shearing pulverizer In order to pulverize the polysaccharide by applying a compression shear stress, a compression shearing pulverizer can be used.
- the compression shearing type pulverizer is a machine that can apply both compressive stress and shear stress, and examples thereof include a vibrating rod mill and a vibrating ball mill. Among these, a vibrating rod mill is preferable from the viewpoint of saccharification rate and production efficiency.
- the rod is not particularly limited, but preferably has an outer diameter of 0.1 to 100 mm, more preferably 0.5 to 50 mm. Further, the filling rate of the rod (apparent volume of the agitating part of the vibration mill) varies depending on the model, but is preferably 10 to 97%, more preferably 15 to 95%.
- the pulverization conditions such as the pulverization time and the rotational speed of the pulverizer may be set as appropriate in order to form a desired pulverized product.
- the average particle size of the pulverized product is 0.002 to 0.3 mm, further 0.005 to 0.2 mm, further 0.01 to 0.05 mm, and further 0.01 to 0.04 mm. It is preferable from the viewpoint of efficiency.
- an average particle diameter means the average value calculated
- the polysaccharide may be coarsely pulverized in advance before being pulverized by applying compressive shear stress.
- the method for coarse pulverization is not particularly limited, and for example, as a pulverizer, a cutter pulverizer such as a grinder or roll cutter, an impact pulverizer such as a hammer mill, a grinding pulverizer such as a colloid mill, or the like may be used. it can.
- the coarse pulverization is preferably performed so that the average particle size of the polysaccharide is 1 to 15 cm, and more preferably 3 to 5 cm.
- Step (2) is a step in which the pulverized product obtained in step (1) is heated at 185 to 230 ° C. in the presence of water. It does not restrict
- the pulverized product is preferably used in the form of a slurry, and the content of polysaccharide in the slurry is 1 to 400 g / L, more preferably 5 to 300 g / L, and more preferably 8 to 200 g / L from the viewpoint of fluidity. Is preferred. Examples of the slurry include water and various buffer solutions.
- the temperature of the heat treatment is in the range of 185 to 230 ° C., but 185 to 220 ° C., further 190 to 210 ° C., and further 185 to 200 ° C. is preferable from the viewpoint of saccharification rate and production efficiency.
- the heating means include water vapor and electricity.
- the heat treatment time in the range of 185 ° C. to 230 ° C. varies depending on the reaction method and the type of polysaccharide, but is preferably 10 minutes to 90 minutes, more preferably 15 minutes to 85 minutes, and further from the viewpoint of the decomposition rate and production efficiency. It is preferably 20 minutes to 80 minutes, more preferably 30 minutes to 65 minutes.
- the heat treatment time means the total time in a state included in the specified temperature range, and the temperature may change as long as the temperature is included in the range. Therefore, the temperature raising and lowering times are included in the heat treatment time as long as they are included in the temperature range. If the temperature is intermittently included in the temperature range as a result of the temperature change, the total time included in the temperature range is set as the time.
- the pressure during the heat treatment is preferably set to the saturated vapor pressure of water or higher, and further 1.1 to 10.0 MPa, further 1.2 to 8.0 MPa, and further 1.3 to 6.0 MPa. Further, 1.6 to 2.6 MPa is preferable.
- a gas may be used, and examples of the gas used include inert gas, water vapor, nitrogen gas, and helium gas.
- the pressure may be set by restricting the outlet channel with a valve or the like.
- Step (3) is a step of obtaining a solid part by solid-liquid separation from the reaction product after the heat treatment, and allowing a hydrolase to act on the solid part.
- the method for obtaining the solid part from the reaction product after the heat treatment by solid-liquid separation is not particularly limited, and can be performed by filtration, centrifugation, and sedimentation while cooling as necessary.
- the cooling temperature is 100 ° C. or lower, preferably 80 ° C. or lower.
- the hydrolase used in the present invention is not particularly limited as long as it has an activity of hydrolyzing polysaccharides.
- amylase, glucoamylase, cellulase, dextranase, glucanase, glucosidase, galactosidase, Mannosidase, agarase, lactose, mutanase, chitinase, chitosanase and the like can be mentioned.
- the origin of the hydrolase is not limited, and it may be an artificial enzyme by gene recombination technique, partial hydrolysis or the like.
- the form of the hydrolase is not particularly limited, and dried enzyme protein, particles containing enzyme protein, liquid containing enzyme protein, and the like can be used.
- the amount of hydrolase used varies depending on the conditions of the hydrolysis reaction, the type of polysaccharide, etc. For example, in the case of cellulase, 1 to 200 FPU per gram of the solid part is preferable, and 5 to 50 FPU is more preferable.
- the FPU activity is measured by the IUPAC method shown below. First, 50 mg of filter paper (Whatman No. 1) is used as a substrate, 0.5 mL of enzyme solution and 1.0 mL of 0.05 M citrate buffer (pH 4.8) are added thereto, and the enzyme reaction is carried out at 50 ° C. for 1.0 hour. Then, 3.0 mL of dinitrosalicylic acid reagent is added, and the mixture is heated at 100 ° C.
- the temperature and pH can be selected according to the characteristics of the enzyme to be used.
- the pH should be 3 to 8, more preferably 4 to 7.
- the temperature is preferably 10 to 80 ° C., more preferably 20 to 60 ° C.
- the reaction time varies depending on the type of polysaccharide and the like, but is preferably 1 to 240 hours, more preferably 2 to 200 hours, and further preferably 3 to 140 hours from the viewpoint of saccharification rate and production efficiency.
- examples of the monosaccharide obtained by the hydrolysis reaction include glucose, fructose, mannose, galactose, xylose, arabinose and the like.
- examples of the disaccharide include cellobiose, maltose, di-N-acetylchitobiose, and the like.
- Oligosaccharides include those having 3 to 10 monosaccharide units.
- a decomposed sugar can be produced from a polysaccharide at a saccharification rate of 60 to 100%, further 70 to 100%, and further 80 to 100%.
- the saccharification rate was obtained by dividing the total mass of water-soluble sugars (monosaccharides and disaccharides typified by glucose) produced by the hydrolysis reaction by the mass of polysaccharides contained in the raw material subjected to the hydrolysis reaction. Say the value.
- the yield (yield) of the decomposition sugar through all the processes is 60% or more, Furthermore, 65% or more, Furthermore, 70% or more.
- polysaccharides include many examples including cellulose, and cellulose includes ⁇ -cellulose having high crystallinity, modified ⁇ -cellulose and ⁇ -classified as hemicellulose. There is also cellulose.
- the yield of polysaccharides, the saccharification rate, and the yield of decomposing sugars are based on ⁇ -cellulose, which is generally used as the cellulose purity in biomass. The method for measuring the amount of ⁇ -cellulose is described in the Examples.
- the obtained decomposed sugar can be used to produce useful substances such as ethanol, polylactic acid, amino acids, xylitol, and erythritol by performing microbial fermentation or chemical conversion using these decomposed sugars as a sugar source.
- ⁇ Measurement of ⁇ -cellulose amount> Distilled water (150 mL), sodium chlorite (1.0 g) and acetic acid (0.2 mL) are added to about 2.5 g of the degreased product obtained by Sosley extraction, and heated on a hot water bath at 80 ° C. Addition of sodium chlorite and acetic acid and heating for 1 hour after the addition were further performed twice. The contents were filtered, and the filtration residue was washed with water and acetone and then dried. 1 g of the obtained dried product was added to 25 mL of 17.5% aqueous sodium hydroxide solution and left for 30 minutes. After 30 minutes, 25 mL of water was added, stirred for 1 minute, and allowed to stand for 5 minutes. The contents were filtered, and the filter residue was washed with 10% aqueous acetic acid and boiling water and then dried. The weight of the obtained dry residue was defined as the amount of ⁇ -cellulose.
- ⁇ -cellulose yield (%) ⁇ mass of ⁇ -cellulose contained in the solid part obtained by solid-liquid separation of the heat-treated reaction product obtained in step (2) ⁇ / ⁇ in step (2) Mass of ⁇ -cellulose contained in pulverized raw material polysaccharide ⁇ ⁇ 100 (i)
- Saccharification rate (%) ⁇ (water-soluble sugar mass after reaction) ⁇ (water-soluble sugar mass before reaction) ⁇ / mass of ⁇ -cellulose ⁇ 100 (ii)
- Example 1 100 g of dried rice straw was pre-ground with a grinder so that the volume-based average particle size was 3 to 5 cm.
- a vibrating rod mill (MB-1 manufactured by Chuo Kako Co., Ltd., total volume 3.5L) was filled with 13 rods with an outer diameter of 30 mm (filling rate 74%), and 100 g of pre-ground rice straw was charged, 1,200c By shaking for 10 minutes at / min, ground rice straw was obtained.
- the volume standard average particle size was 0.04 mm.
- a slurry obtained by mixing 10 g of this ground rice straw ( ⁇ -cellulose content 32% by mass) and 90 g of water is placed in a batch hydrothermal apparatus (Start 200 New Quick, volume 180 mL, manufactured by Nitto Koatsu Co., Ltd.).
- the temperature rising rate was 3.4 ° C./min, and after reaching 200 ° C., it was immediately cooled. The time at 185 ° C. to 230 ° C. was 30 minutes.
- a solid part solid residue was obtained by filtration.
- the residue was fractionated so that the dry mass was 5% by mass, and 0.1 M citrate buffer solution having a pH of 5.0 was added to make a total of 10 g. 62.5 ⁇ L of cellulase (Novozymes Cell Crust 1.5 L) was added thereto for saccharification (addition of residue per 1 g of dry weight was 8.75 FPU).
- the saccharification reaction was carried out in a thermostatic bath at 50 ° C. for 48 hours while shaking at 110 r / min. After completion of the reaction, the liquid part was filtered with a 0.2 ⁇ m filter, and the water-soluble sugar concentration was measured. Cellobiose and glucose were measured as water-soluble sugars.
- Example 2 A water-soluble sugar was obtained in the same manner as in Example 1 except that after reaching 200 ° C. in hydrothermal treatment, the mixture was cooled at 200 ° C. for 30 minutes and then cooled. The time spent at 185 ° C. to 230 ° C. was 65 minutes.
- Example 3 A water-soluble sugar was obtained in the same manner as in Example 1 except that the batch hydrothermal apparatus was previously pressurized to 1 MPa with nitrogen gas.
- Comparative Example 1 In the hydrothermal treatment, a water-soluble sugar was obtained in the same manner as in Example 1 except that it was immediately cooled after reaching 180 ° C.
- Example 2 The ground rice straw obtained in Example 1 was enzymatically saccharified without hydrothermal treatment.
- Comparative Example 3 A crushed rice straw was obtained by charging 40 g of dried rice straw into a grinder manufactured by Iwatani Corporation (Lab Miller 800DG, volume 260 mL) and stirring at 20,000 r / min for 10 minutes. The volume standard average particle diameter was 0.27 mm. 10 g of this ground rice straw ( ⁇ -cellulose content 32 mass%) and 90 g of water were mixed, and then heat treatment and enzymatic saccharification treatment were carried out in the same manner as in Example 2 to obtain a water-soluble sugar.
- Comparative Example 4 A ground rice straw was obtained in the same manner as in Comparative Example 3 except that stirring was performed at 20,000 r / min for 120 minutes. The volume standard average particle diameter was 0.05 mm. 10 g of this ground rice straw ( ⁇ -cellulose content 32 mass%) and 90 g of water were mixed, and then heat treatment and enzymatic saccharification treatment were carried out in the same manner as in Example 2 to obtain a water-soluble sugar. Table 1 shows the conditions and results of each Example and Comparative Example.
Abstract
The disclosed method for manufacturing a monosaccharide, a disaccharide, and/or an oligosaccharide includes: a step (1) in which a polysaccharide is pulverized via the application of compression shear stress, yielding a pulverized polysaccharide; a step (2) in which the pulverized polysaccharide is heat-treated at 185-230°C in the presence of water; and a step (3) in which the solid portion of the heat-treated reactant is obtained via solid-liquid separation and acted on by a hydrolase.
Description
本発明は、多糖類、特にバイオマスから単糖類、二糖類、及び／又はオリゴ糖（以下、総称して「分解糖」ともいう）を製造する方法に関する。
The present invention relates to a method for producing polysaccharides, particularly monosaccharides, disaccharides, and / or oligosaccharides (hereinafter collectively referred to as “decomposed sugars”) from biomass.
近年、化石資源に代えてセルロース系、デンプン系、或いは糖質系のバイオマスからエタノールや乳酸等の有用物質を製造する技術の開発が望まれている。とりわけ、非食糧資源であるセルロース系バイオマスを有効利用する技術への関心が高まっている。
バイオマスからのエタノール等の製造は、バイオマスを糖化工程においてグルコース等の糖に分解した後、これを発酵工程においてエタノール等に変換することにより行うことができる。糖化は、硫酸等を用いる酸糖化と酵素糖化に大別される。
酵素糖化は、酸糖化に比して緩和な条件で加水分解できる等の利点があるが、特にセルロース系バイオマスを利用する場合、反応速度が非常に遅く、糖化率の向上が大きな課題になっている。 In recent years, development of technology for producing useful substances such as ethanol and lactic acid from cellulose-based, starch-based, or saccharide-based biomass in place of fossil resources has been desired. In particular, there is a growing interest in technologies that make effective use of cellulosic biomass, which is a non-food resource.
Production of ethanol or the like from biomass can be performed by decomposing biomass into sugars such as glucose in the saccharification step, and then converting this to ethanol or the like in the fermentation step. Saccharification is roughly divided into acid saccharification using sulfuric acid or the like and enzyme saccharification.
Enzymatic saccharification has the advantage that it can be hydrolyzed under mild conditions compared to acid saccharification, but especially when cellulosic biomass is used, the reaction rate is very slow and increasing the saccharification rate is a major issue. Yes.
バイオマスからのエタノール等の製造は、バイオマスを糖化工程においてグルコース等の糖に分解した後、これを発酵工程においてエタノール等に変換することにより行うことができる。糖化は、硫酸等を用いる酸糖化と酵素糖化に大別される。
酵素糖化は、酸糖化に比して緩和な条件で加水分解できる等の利点があるが、特にセルロース系バイオマスを利用する場合、反応速度が非常に遅く、糖化率の向上が大きな課題になっている。 In recent years, development of technology for producing useful substances such as ethanol and lactic acid from cellulose-based, starch-based, or saccharide-based biomass in place of fossil resources has been desired. In particular, there is a growing interest in technologies that make effective use of cellulosic biomass, which is a non-food resource.
Production of ethanol or the like from biomass can be performed by decomposing biomass into sugars such as glucose in the saccharification step, and then converting this to ethanol or the like in the fermentation step. Saccharification is roughly divided into acid saccharification using sulfuric acid or the like and enzyme saccharification.
Enzymatic saccharification has the advantage that it can be hydrolyzed under mild conditions compared to acid saccharification, but especially when cellulosic biomass is used, the reaction rate is very slow and increasing the saccharification rate is a major issue. Yes.
糖化率向上のための技術として、糖化工程に先立って予めバイオマスを前処理する方法が検討されている。前処理方法としては、例えば、硫酸法、有機溶媒法、水熱処理法、機械的破砕等が知られている。
また、これらの方法を組み合わせてエタノール原料を効率的に製造する方法も検討され、例えば、リグノセルロースを含有する原料を粗粉砕する粗粉砕工程、粗粉砕物を微粉砕する微粉砕工程、微粉砕物を水熱処理する水熱処理工程、及び水熱処理物を脱水する脱水工程を包含する方法（特許文献１）が報告されている。 As a technique for improving the saccharification rate, a method of pretreating biomass in advance prior to the saccharification step has been studied. As the pretreatment method, for example, a sulfuric acid method, an organic solvent method, a hydrothermal treatment method, mechanical crushing, and the like are known.
Also, a method for efficiently producing an ethanol raw material by combining these methods has been studied. For example, a coarse pulverization step for coarsely pulverizing a raw material containing lignocellulose, a fine pulverization step for finely pulverizing a coarsely pulverized product, and fine pulverization A method (Patent Document 1) including a hydrothermal treatment step of hydrothermally treating an object and a dehydration step of dehydrating the hydrothermal treatment product has been reported.
また、これらの方法を組み合わせてエタノール原料を効率的に製造する方法も検討され、例えば、リグノセルロースを含有する原料を粗粉砕する粗粉砕工程、粗粉砕物を微粉砕する微粉砕工程、微粉砕物を水熱処理する水熱処理工程、及び水熱処理物を脱水する脱水工程を包含する方法（特許文献１）が報告されている。 As a technique for improving the saccharification rate, a method of pretreating biomass in advance prior to the saccharification step has been studied. As the pretreatment method, for example, a sulfuric acid method, an organic solvent method, a hydrothermal treatment method, mechanical crushing, and the like are known.
Also, a method for efficiently producing an ethanol raw material by combining these methods has been studied. For example, a coarse pulverization step for coarsely pulverizing a raw material containing lignocellulose, a fine pulverization step for finely pulverizing a coarsely pulverized product, and fine pulverization A method (Patent Document 1) including a hydrothermal treatment step of hydrothermally treating an object and a dehydration step of dehydrating the hydrothermal treatment product has been reported.
本発明は、次の１）～１２）に関する。
１）次の工程（１）～（３）：
（１）多糖類に圧縮せん断応力を加えて粉砕し、多糖類の粉砕物を得る工程、
（２）多糖類の粉砕物を水の存在下、１８５～２３０℃で加熱処理する工程、
（３）加熱処理後の反応物から固液分離により固体部を得、これに加水分解酵素を作用させる工程、
を包含する、単糖類、二糖類、及び／又はオリゴ糖の製造方法。
２）１８５～２３０℃で加熱処理する時間が１０分～９０分である、上記１）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。
３）１８５～２３０℃で加熱処理する時間が１５分～８５分である、上記１）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。
４）１８５～２３０℃で加熱処理する時間が２０分～８０分である、上記１）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。
５）１８５～２３０℃で加熱処理する時間が３０分～６５分である、上記１）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。
６）振動ロッドミルを用いて多糖類に圧縮せん断応力を加えるものである、上記１）～５）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。
７）加熱処理を水の飽和蒸気圧以上の圧力で行う、上記１）～６）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。
８）加熱処理を１．１～１０．０ＭＰａの圧力で行う、上記１）～６）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。
９）加熱処理を１．２～８．０ＭＰａの圧力で行う、上記１）～６）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。
１０）加熱処理を１．３～６．０ＭＰａの圧力で行う、上記１）～６）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。
１１）加熱処理を１．６～２．６ＭＰａの圧力で行う、上記１）～６）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。
１２）多糖類としてセルロース含有バイオマスを用いる、上記１）～１１）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。 The present invention relates to the following 1) to 12).
1) Next steps (1) to (3):
(1) A step of applying a compressive shear stress to a polysaccharide to pulverize the polysaccharide to obtain a pulverized polysaccharide;
(2) a step of heat-treating the pulverized polysaccharide at 185 to 230 ° C. in the presence of water;
(3) A step of obtaining a solid part by solid-liquid separation from the reaction product after the heat treatment, and allowing a hydrolase to act on the solid part,
A process for producing monosaccharides, disaccharides and / or oligosaccharides.
2) The method for producing monosaccharides, disaccharides and / or oligosaccharides of 1) above, wherein the heat treatment time at 185 to 230 ° C. is 10 to 90 minutes.
3) The method for producing monosaccharides, disaccharides and / or oligosaccharides of 1) above, wherein the heat treatment time at 185 to 230 ° C. is 15 to 85 minutes.
4) The method for producing monosaccharides, disaccharides and / or oligosaccharides of 1) above, wherein the heat treatment time at 185 to 230 ° C. is 20 minutes to 80 minutes.
5) The method for producing monosaccharides, disaccharides and / or oligosaccharides of 1) above, wherein the time for heat treatment at 185 to 230 ° C. is 30 minutes to 65 minutes.
6) The method for producing monosaccharides, disaccharides and / or oligosaccharides according to 1) to 5) above, wherein a compressive shear stress is applied to the polysaccharides using a vibrating rod mill.
7) The method for producing monosaccharides, disaccharides and / or oligosaccharides according to the above 1) to 6), wherein the heat treatment is performed at a pressure equal to or higher than the saturated vapor pressure of water.
8) The method for producing monosaccharides, disaccharides and / or oligosaccharides of 1) to 6) above, wherein the heat treatment is performed at a pressure of 1.1 to 10.0 MPa.
9) The method for producing monosaccharides, disaccharides and / or oligosaccharides of 1) to 6) above, wherein the heat treatment is performed at a pressure of 1.2 to 8.0 MPa.
10) The method for producing monosaccharides, disaccharides and / or oligosaccharides of 1) to 6) above, wherein the heat treatment is performed at a pressure of 1.3 to 6.0 MPa.
11) The method for producing monosaccharides, disaccharides and / or oligosaccharides of 1) to 6) above, wherein the heat treatment is performed at a pressure of 1.6 to 2.6 MPa.
12) The method for producing monosaccharides, disaccharides and / or oligosaccharides of 1) to 11) above, wherein cellulose-containing biomass is used as the polysaccharide.
１）次の工程（１）～（３）：
（１）多糖類に圧縮せん断応力を加えて粉砕し、多糖類の粉砕物を得る工程、
（２）多糖類の粉砕物を水の存在下、１８５～２３０℃で加熱処理する工程、
（３）加熱処理後の反応物から固液分離により固体部を得、これに加水分解酵素を作用させる工程、
を包含する、単糖類、二糖類、及び／又はオリゴ糖の製造方法。
２）１８５～２３０℃で加熱処理する時間が１０分～９０分である、上記１）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。
３）１８５～２３０℃で加熱処理する時間が１５分～８５分である、上記１）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。
４）１８５～２３０℃で加熱処理する時間が２０分～８０分である、上記１）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。
５）１８５～２３０℃で加熱処理する時間が３０分～６５分である、上記１）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。
６）振動ロッドミルを用いて多糖類に圧縮せん断応力を加えるものである、上記１）～５）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。
７）加熱処理を水の飽和蒸気圧以上の圧力で行う、上記１）～６）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。
８）加熱処理を１．１～１０．０ＭＰａの圧力で行う、上記１）～６）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。
９）加熱処理を１．２～８．０ＭＰａの圧力で行う、上記１）～６）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。
１０）加熱処理を１．３～６．０ＭＰａの圧力で行う、上記１）～６）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。
１１）加熱処理を１．６～２．６ＭＰａの圧力で行う、上記１）～６）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。
１２）多糖類としてセルロース含有バイオマスを用いる、上記１）～１１）の単糖類、二糖類、及び／又はオリゴ糖の製造方法。 The present invention relates to the following 1) to 12).
1) Next steps (1) to (3):
(1) A step of applying a compressive shear stress to a polysaccharide to pulverize the polysaccharide to obtain a pulverized polysaccharide;
(2) a step of heat-treating the pulverized polysaccharide at 185 to 230 ° C. in the presence of water;
(3) A step of obtaining a solid part by solid-liquid separation from the reaction product after the heat treatment, and allowing a hydrolase to act on the solid part,
A process for producing monosaccharides, disaccharides and / or oligosaccharides.
2) The method for producing monosaccharides, disaccharides and / or oligosaccharides of 1) above, wherein the heat treatment time at 185 to 230 ° C. is 10 to 90 minutes.
3) The method for producing monosaccharides, disaccharides and / or oligosaccharides of 1) above, wherein the heat treatment time at 185 to 230 ° C. is 15 to 85 minutes.
4) The method for producing monosaccharides, disaccharides and / or oligosaccharides of 1) above, wherein the heat treatment time at 185 to 230 ° C. is 20 minutes to 80 minutes.
5) The method for producing monosaccharides, disaccharides and / or oligosaccharides of 1) above, wherein the time for heat treatment at 185 to 230 ° C. is 30 minutes to 65 minutes.
6) The method for producing monosaccharides, disaccharides and / or oligosaccharides according to 1) to 5) above, wherein a compressive shear stress is applied to the polysaccharides using a vibrating rod mill.
7) The method for producing monosaccharides, disaccharides and / or oligosaccharides according to the above 1) to 6), wherein the heat treatment is performed at a pressure equal to or higher than the saturated vapor pressure of water.
8) The method for producing monosaccharides, disaccharides and / or oligosaccharides of 1) to 6) above, wherein the heat treatment is performed at a pressure of 1.1 to 10.0 MPa.
9) The method for producing monosaccharides, disaccharides and / or oligosaccharides of 1) to 6) above, wherein the heat treatment is performed at a pressure of 1.2 to 8.0 MPa.
10) The method for producing monosaccharides, disaccharides and / or oligosaccharides of 1) to 6) above, wherein the heat treatment is performed at a pressure of 1.3 to 6.0 MPa.
11) The method for producing monosaccharides, disaccharides and / or oligosaccharides of 1) to 6) above, wherein the heat treatment is performed at a pressure of 1.6 to 2.6 MPa.
12) The method for producing monosaccharides, disaccharides and / or oligosaccharides of 1) to 11) above, wherein cellulose-containing biomass is used as the polysaccharide.
前記特許文献１には、微粉砕工程で得た微粉砕物を水熱処理する際、糖の過分解を抑えるため、約０．９８ＭＰａ（飽和水蒸気として約１８０℃以下）以下が好ましい旨の記載がある。しかしながら、前記特許文献１には水熱処理の実施について開示がなく、本発明者が特許文献１記載の方法に従ってセルロース系バイオマスの粉砕工程、水熱処理工程を行い、次いで実際に酵素糖化工程を行ったところ、水熱処理工程後のセルロース収率は高いものの、酵素糖化工程で反応が進みにくく、全体として分解糖の生成量が少なくなる場合があることが判明した。一方、セルロース系バイオマスを単に機械的破砕した後に酵素糖化に供しても、経時的に反応速度が低下し、十分な糖化率が得られない場合がある。
従って、本発明は、糖化率を向上させ、効率よく多糖類から分解糖を製造することのできる方法を提供することに関する。 Patent Document 1 describes that when the finely pulverized product obtained in the pulverization step is hydrothermally treated, about 0.98 MPa (about 180 ° C. or less as saturated steam) is preferable in order to suppress excessive decomposition of sugar. is there. However, the Patent Document 1 does not disclose the implementation of hydrothermal treatment, and the present inventor performed the pulverization step of cellulose-based biomass and the hydrothermal treatment step according to the method described in Patent Literature 1, and then the actual enzymatic saccharification step. However, although the yield of cellulose after the hydrothermal treatment step is high, it has been found that the reaction is difficult to proceed in the enzymatic saccharification step, and the amount of decomposition sugar produced as a whole may be reduced. On the other hand, even if cellulosic biomass is simply mechanically crushed and then subjected to enzymatic saccharification, the reaction rate decreases with time, and a sufficient saccharification rate may not be obtained.
Therefore, the present invention relates to providing a method capable of improving the saccharification rate and efficiently producing a decomposed sugar from a polysaccharide.
従って、本発明は、糖化率を向上させ、効率よく多糖類から分解糖を製造することのできる方法を提供することに関する。 Patent Document 1 describes that when the finely pulverized product obtained in the pulverization step is hydrothermally treated, about 0.98 MPa (about 180 ° C. or less as saturated steam) is preferable in order to suppress excessive decomposition of sugar. is there. However, the Patent Document 1 does not disclose the implementation of hydrothermal treatment, and the present inventor performed the pulverization step of cellulose-based biomass and the hydrothermal treatment step according to the method described in Patent Literature 1, and then the actual enzymatic saccharification step. However, although the yield of cellulose after the hydrothermal treatment step is high, it has been found that the reaction is difficult to proceed in the enzymatic saccharification step, and the amount of decomposition sugar produced as a whole may be reduced. On the other hand, even if cellulosic biomass is simply mechanically crushed and then subjected to enzymatic saccharification, the reaction rate decreases with time, and a sufficient saccharification rate may not be obtained.
Therefore, the present invention relates to providing a method capable of improving the saccharification rate and efficiently producing a decomposed sugar from a polysaccharide.
本発明者らは、上記課題に鑑み鋭意検討したところ、多糖類を特定の方法で粉砕した後に、水の存在下、１８５～２３０℃で加熱処理すれば、特許文献１のような低い温度で加熱処理した場合より加熱処理後のセルロース収率は低いにもかかわらず、その後の酵素糖化工程では全く意外にも糖化率が顕著に向上し、最終的に分解糖の収率が高くなることを見出した。
The inventors of the present invention have intensively studied in view of the above problems. As a result, if the polysaccharide is pulverized by a specific method and then heat-treated at 185 to 230 ° C. in the presence of water, the temperature can be reduced as in Patent Document 1. Although the yield of cellulose after heat treatment is lower than when heat-treated, the subsequent enzymatic saccharification step unexpectedly improves the saccharification rate significantly and ultimately increases the yield of decomposed sugar. I found it.
本発明によれば、糖化率を向上させることができ、分解糖を効率よく製造することができる。本発明の方法は、セルロース系のバイオマスにも適用可能であり、バイオマスからのエタノ－ル等有用物質の製造を高効率化する技術として期待できる。
According to the present invention, the saccharification rate can be improved, and the decomposing sugar can be produced efficiently. The method of the present invention can also be applied to cellulosic biomass, and can be expected as a technique for improving the production efficiency of useful substances such as ethanol from biomass.
工程（１）は、多糖類に圧縮せん断応力を加えて粉砕して、多糖類の粉砕物を得る工程である。
本発明で用いられる多糖類としては、例えば、セルロース、ヘミセルロース、キシログルカン、ペクチン、澱粉、マンナン、グルコマンナン、ガラクトマンナン、キチン、キトサン、イヌリン、アルギン酸、寒天、フコイダン、ラミナリン、β－グルカン、プルラン等の天然多糖又はこれらの誘導体が挙げられる。誘導体としては、例えば、水酸基の少なくとも一部がエステル化されたエステル類、水酸基の少なくとも一部がエーテル化されたエーテル類等が例示される。これらは単独で又は２種以上を組み合わせて用いることができる。なかでも、安価かつ分解後の発酵生産により有用物質に変換可能である点から、セルロース、ヘミセルロース、キチン、キトサンが好ましく、更にセルロース、ヘミセルロースが好ましい。本発明で用いられる多糖類の分子量は、特に限定されないが、一般的には１，０００以上５，０００，０００以下であることが好ましい。 Step (1) is a step of obtaining a pulverized polysaccharide by pulverizing the polysaccharide by applying compressive shear stress.
Examples of the polysaccharide used in the present invention include cellulose, hemicellulose, xyloglucan, pectin, starch, mannan, glucomannan, galactomannan, chitin, chitosan, inulin, alginic acid, agar, fucoidan, laminarin, β-glucan, pullulan. Natural polysaccharides such as these or derivatives thereof. Examples of the derivative include esters in which at least a part of the hydroxyl group is esterified, ethers in which at least a part of the hydroxyl group is etherified, and the like. These can be used alone or in combination of two or more. Of these, cellulose, hemicellulose, chitin, and chitosan are preferable, and cellulose and hemicellulose are more preferable because they are inexpensive and can be converted into useful substances by fermentation production after decomposition. The molecular weight of the polysaccharide used in the present invention is not particularly limited, but generally it is preferably 1,000 or more and 5,000,000 or less.
本発明で用いられる多糖類としては、例えば、セルロース、ヘミセルロース、キシログルカン、ペクチン、澱粉、マンナン、グルコマンナン、ガラクトマンナン、キチン、キトサン、イヌリン、アルギン酸、寒天、フコイダン、ラミナリン、β－グルカン、プルラン等の天然多糖又はこれらの誘導体が挙げられる。誘導体としては、例えば、水酸基の少なくとも一部がエステル化されたエステル類、水酸基の少なくとも一部がエーテル化されたエーテル類等が例示される。これらは単独で又は２種以上を組み合わせて用いることができる。なかでも、安価かつ分解後の発酵生産により有用物質に変換可能である点から、セルロース、ヘミセルロース、キチン、キトサンが好ましく、更にセルロース、ヘミセルロースが好ましい。本発明で用いられる多糖類の分子量は、特に限定されないが、一般的には１，０００以上５，０００，０００以下であることが好ましい。 Step (1) is a step of obtaining a pulverized polysaccharide by pulverizing the polysaccharide by applying compressive shear stress.
Examples of the polysaccharide used in the present invention include cellulose, hemicellulose, xyloglucan, pectin, starch, mannan, glucomannan, galactomannan, chitin, chitosan, inulin, alginic acid, agar, fucoidan, laminarin, β-glucan, pullulan. Natural polysaccharides such as these or derivatives thereof. Examples of the derivative include esters in which at least a part of the hydroxyl group is esterified, ethers in which at least a part of the hydroxyl group is etherified, and the like. These can be used alone or in combination of two or more. Of these, cellulose, hemicellulose, chitin, and chitosan are preferable, and cellulose and hemicellulose are more preferable because they are inexpensive and can be converted into useful substances by fermentation production after decomposition. The molecular weight of the polysaccharide used in the present invention is not particularly limited, but generally it is preferably 1,000 or more and 5,000,000 or less.
また、本発明における多糖類として、前記多糖類を含む原料、例えば、バイオマスを用いることもできる。バイオマスとは、生物由来の有機資源で、化石資源を除いたものである。バイオマスとしては、セルロース系、デンプン系、或いは糖質系のバイオマスが挙げられ、これらは単独で又は２種以上を組み合わせて用いることができる。
なかでも、資源の有効活用の点から、セルロースを含有するセルロース系バイオマスを用いることが好ましい。 Moreover, the raw material containing the said polysaccharide, for example, biomass, can also be used as a polysaccharide in this invention. Biomass is an organic resource derived from living organisms, excluding fossil resources. Examples of the biomass include cellulose-based, starch-based, and saccharide-based biomass, and these can be used alone or in combination of two or more.
Especially, it is preferable to use the cellulose biomass containing a cellulose from the point of effective utilization of resources.
なかでも、資源の有効活用の点から、セルロースを含有するセルロース系バイオマスを用いることが好ましい。 Moreover, the raw material containing the said polysaccharide, for example, biomass, can also be used as a polysaccharide in this invention. Biomass is an organic resource derived from living organisms, excluding fossil resources. Examples of the biomass include cellulose-based, starch-based, and saccharide-based biomass, and these can be used alone or in combination of two or more.
Especially, it is preferable to use the cellulose biomass containing a cellulose from the point of effective utilization of resources.
セルロース系バイオマスは、セルロース、ヘミセルロース及びリグニンを主成分とするものであり、例えば、綿、木材系パルプ、ケナフ、麻、小径木、間伐材、おが屑、木屑、古紙、新聞紙、包装紙、ティッシュペーパー、トイレットペーパー、ダンボール等の木質系；バガス、スイッチグラス、エレファントグラス、稲ワラ、ムギワラ等の草木系のバイオマスが挙げられる。また、デンプン系バイオマスとしては、例えば、米、麦、トウモロコシ、イモ等が挙げられ、糖質系バイオマスとしては、サトウキビ、テンサイ、海藻、エビ殻、カニ殻等の糖質系バイオマスが挙げられる。
Cellulose biomass is mainly composed of cellulose, hemicellulose, and lignin. For example, cotton, wood pulp, kenaf, hemp, small-diameter wood, thinned wood, sawdust, wood waste, waste paper, newspaper, wrapping paper, tissue paper Woody materials such as toilet paper and cardboard; plant biomass such as bagasse, switchgrass, elephant grass, rice straw and wheat straw. Examples of the starch-based biomass include rice, wheat, corn, and potato, and examples of the saccharide-based biomass include sugar-based biomass such as sugar cane, sugar beet, seaweed, shrimp shell, and crab shell.
多糖類に圧縮せん断応力を加えて粉砕するには、圧縮せん断式粉砕機を用いることができる。圧縮せん断式粉砕機は、圧縮応力とせん断応力の両方を付加できる機械で、例えば、振動ロッドミル、振動ボールミル等が挙げられる。なかでも、糖化率及び生産効率の点から、振動ロッドミルが好ましい。ロッドは、特に制限されないが、外径が０．１～１００ｍｍ、さらに０．５～５０ｍｍのものが好ましい。また、ロッドの充填率（振動ミルの攪拌部の容積に対するロッドの見かけの体積）は、機種により異なるが、１０～９７％が好ましく、１５～９５％がより好ましい。
粉砕時間、粉砕機の回転数等の粉砕条件は、所望の粉砕物を形成するために適宜設定すればよい。 In order to pulverize the polysaccharide by applying a compression shear stress, a compression shearing pulverizer can be used. The compression shearing type pulverizer is a machine that can apply both compressive stress and shear stress, and examples thereof include a vibrating rod mill and a vibrating ball mill. Among these, a vibrating rod mill is preferable from the viewpoint of saccharification rate and production efficiency. The rod is not particularly limited, but preferably has an outer diameter of 0.1 to 100 mm, more preferably 0.5 to 50 mm. Further, the filling rate of the rod (apparent volume of the agitating part of the vibration mill) varies depending on the model, but is preferably 10 to 97%, more preferably 15 to 95%.
The pulverization conditions such as the pulverization time and the rotational speed of the pulverizer may be set as appropriate in order to form a desired pulverized product.
粉砕時間、粉砕機の回転数等の粉砕条件は、所望の粉砕物を形成するために適宜設定すればよい。 In order to pulverize the polysaccharide by applying a compression shear stress, a compression shearing pulverizer can be used. The compression shearing type pulverizer is a machine that can apply both compressive stress and shear stress, and examples thereof include a vibrating rod mill and a vibrating ball mill. Among these, a vibrating rod mill is preferable from the viewpoint of saccharification rate and production efficiency. The rod is not particularly limited, but preferably has an outer diameter of 0.1 to 100 mm, more preferably 0.5 to 50 mm. Further, the filling rate of the rod (apparent volume of the agitating part of the vibration mill) varies depending on the model, but is preferably 10 to 97%, more preferably 15 to 95%.
The pulverization conditions such as the pulverization time and the rotational speed of the pulverizer may be set as appropriate in order to form a desired pulverized product.
粉砕物の平均粒径は、０．００２～０．３ｍｍ、さらに０．００５～０．２ｍｍ、更に０．０１～０．０５ｍｍ、更に０．０１～０．０４ｍｍであることが糖化率及び生産効率の点から好ましい。なお、本明細書において平均粒径とは、レーザー回折散乱法により体積基準に従って求められる平均値をいう。
The average particle size of the pulverized product is 0.002 to 0.3 mm, further 0.005 to 0.2 mm, further 0.01 to 0.05 mm, and further 0.01 to 0.04 mm. It is preferable from the viewpoint of efficiency. In addition, in this specification, an average particle diameter means the average value calculated | required according to a volume reference | standard by the laser diffraction scattering method.
また、多糖類は、圧縮せん断応力を加えて粉砕する前に、予め粗粉砕しておいてもよい。粗粉砕する方法は特に制限されず、例えば、粉砕機として、グラインダー・ロールカッター等のカッター式粉砕機、ハンマーミル等の衝撃式粉砕機、コロイドミル等の摩砕式粉砕機等を用いることができる。
粗粉砕は、多糖類の平均粒径が、１～１５ｃｍ、更に３～５ｃｍとなるように行うのが好ましい。 In addition, the polysaccharide may be coarsely pulverized in advance before being pulverized by applying compressive shear stress. The method for coarse pulverization is not particularly limited, and for example, as a pulverizer, a cutter pulverizer such as a grinder or roll cutter, an impact pulverizer such as a hammer mill, a grinding pulverizer such as a colloid mill, or the like may be used. it can.
The coarse pulverization is preferably performed so that the average particle size of the polysaccharide is 1 to 15 cm, and more preferably 3 to 5 cm.
粗粉砕は、多糖類の平均粒径が、１～１５ｃｍ、更に３～５ｃｍとなるように行うのが好ましい。 In addition, the polysaccharide may be coarsely pulverized in advance before being pulverized by applying compressive shear stress. The method for coarse pulverization is not particularly limited, and for example, as a pulverizer, a cutter pulverizer such as a grinder or roll cutter, an impact pulverizer such as a hammer mill, a grinding pulverizer such as a colloid mill, or the like may be used. it can.
The coarse pulverization is preferably performed so that the average particle size of the polysaccharide is 1 to 15 cm, and more preferably 3 to 5 cm.
工程（２）は、工程（１）より得られた粉砕物を水の存在下、１８５～２３０℃で加熱処理する工程である。加熱処理方法としては、特に制限されず、公知の方法を適用できる。バッチ攪拌式でも連続流通式でもよい。加熱処理の際に用いる水は、例えば、水道水、蒸留水、イオン交換水、精製水等が例示される。
粉砕物はスラリー状にして用いるのが好ましく、スラリー中の多糖類の含有量は、流動性の点から、１～４００ｇ／Ｌ、更に５～３００ｇ／Ｌ、更に８～２００ｇ／Ｌとするのが好ましい。スラリーとしては、水、各種緩衝液等が挙げられる。 Step (2) is a step in which the pulverized product obtained in step (1) is heated at 185 to 230 ° C. in the presence of water. It does not restrict | limit especially as a heat processing method, A well-known method is applicable. Batch stirring type or continuous flow type may be used. Examples of water used in the heat treatment include tap water, distilled water, ion exchange water, purified water, and the like.
The pulverized product is preferably used in the form of a slurry, and the content of polysaccharide in the slurry is 1 to 400 g / L, more preferably 5 to 300 g / L, and more preferably 8 to 200 g / L from the viewpoint of fluidity. Is preferred. Examples of the slurry include water and various buffer solutions.
粉砕物はスラリー状にして用いるのが好ましく、スラリー中の多糖類の含有量は、流動性の点から、１～４００ｇ／Ｌ、更に５～３００ｇ／Ｌ、更に８～２００ｇ／Ｌとするのが好ましい。スラリーとしては、水、各種緩衝液等が挙げられる。 Step (2) is a step in which the pulverized product obtained in step (1) is heated at 185 to 230 ° C. in the presence of water. It does not restrict | limit especially as a heat processing method, A well-known method is applicable. Batch stirring type or continuous flow type may be used. Examples of water used in the heat treatment include tap water, distilled water, ion exchange water, purified water, and the like.
The pulverized product is preferably used in the form of a slurry, and the content of polysaccharide in the slurry is 1 to 400 g / L, more preferably 5 to 300 g / L, and more preferably 8 to 200 g / L from the viewpoint of fluidity. Is preferred. Examples of the slurry include water and various buffer solutions.
加熱処理の温度は、１８５～２３０℃の範囲であるが、更に１８５～２２０℃、更に１９０～２１０℃、更に１８５～２００℃であるのが糖化率及び生産効率の点から好ましい。加熱の手段は、例えば、水蒸気、電気が挙げられる。
The temperature of the heat treatment is in the range of 185 to 230 ° C., but 185 to 220 ° C., further 190 to 210 ° C., and further 185 to 200 ° C. is preferable from the viewpoint of saccharification rate and production efficiency. Examples of the heating means include water vapor and electricity.
１８５℃～２３０℃の範囲で加熱処理する時間は、反応方法や多糖類の種類によって異なるが、分解率及び生産効率の点から、１０分～９０分が好ましく、更に１５分～８５分、更に２０分～８０分、更に３０分～６５分が好ましい。なお、加熱処理する時間とは、規定されている温度範囲に含まれる状態の時間の合計を意味し、温度が当該範囲に含まれている限り、温度が変化しても構わない。従って、昇温及び降温の時間も、当該温度範囲に含まれる限り加熱処理する時間に含むものとする。また、温度が変化した結果、断続的に当該温度範囲に含まれる場合は、当該温度範囲に含まれる時間の総計をもって当該時間とする。
The heat treatment time in the range of 185 ° C. to 230 ° C. varies depending on the reaction method and the type of polysaccharide, but is preferably 10 minutes to 90 minutes, more preferably 15 minutes to 85 minutes, and further from the viewpoint of the decomposition rate and production efficiency. It is preferably 20 minutes to 80 minutes, more preferably 30 minutes to 65 minutes. Note that the heat treatment time means the total time in a state included in the specified temperature range, and the temperature may change as long as the temperature is included in the range. Therefore, the temperature raising and lowering times are included in the heat treatment time as long as they are included in the temperature range. If the temperature is intermittently included in the temperature range as a result of the temperature change, the total time included in the temperature range is set as the time.
また、加熱処理時の圧力は、水の飽和蒸気圧又はそれ以上に設定するのが好ましく、更に１．１～１０．０ＭＰａ、更に１．２～８．０ＭＰａ、更に１．３～６．０ＭＰａ、更に１．６～２．６ＭＰａが好ましい。加圧する場合はガスを用いてもよく、用いられるガスは、例えば、不活性ガス、水蒸気、窒素ガス、ヘリウムガス等が挙げられる。連続流通式の場合はバルブなどで出口流路を絞ることにより圧力を設定してもよい。
The pressure during the heat treatment is preferably set to the saturated vapor pressure of water or higher, and further 1.1 to 10.0 MPa, further 1.2 to 8.0 MPa, and further 1.3 to 6.0 MPa. Further, 1.6 to 2.6 MPa is preferable. In the case of pressurization, a gas may be used, and examples of the gas used include inert gas, water vapor, nitrogen gas, and helium gas. In the case of a continuous flow type, the pressure may be set by restricting the outlet channel with a valve or the like.
工程（３）は、加熱処理後の反応物から固液分離により固体部を得、これに加水分解酵素を作用させる工程である。反応物から液体部を除去することで、過分解物を除去することができ、後の発酵工程で糖過分解物による発酵阻害を防ぐことができる。
加熱処理後の反応物から固液分離により固体部を得る方法としては、特に制限されず、必要に応じ冷却しながら、ろ過、遠心分離、沈降分離により行うことができる。冷却温度は、１００℃以下、好ましくは８０℃以下が好ましい。 Step (3) is a step of obtaining a solid part by solid-liquid separation from the reaction product after the heat treatment, and allowing a hydrolase to act on the solid part. By removing the liquid part from the reaction product, it is possible to remove the overlysate, and to prevent fermentation inhibition by the sugar overlysate in the subsequent fermentation step.
The method for obtaining the solid part from the reaction product after the heat treatment by solid-liquid separation is not particularly limited, and can be performed by filtration, centrifugation, and sedimentation while cooling as necessary. The cooling temperature is 100 ° C. or lower, preferably 80 ° C. or lower.
加熱処理後の反応物から固液分離により固体部を得る方法としては、特に制限されず、必要に応じ冷却しながら、ろ過、遠心分離、沈降分離により行うことができる。冷却温度は、１００℃以下、好ましくは８０℃以下が好ましい。 Step (3) is a step of obtaining a solid part by solid-liquid separation from the reaction product after the heat treatment, and allowing a hydrolase to act on the solid part. By removing the liquid part from the reaction product, it is possible to remove the overlysate, and to prevent fermentation inhibition by the sugar overlysate in the subsequent fermentation step.
The method for obtaining the solid part from the reaction product after the heat treatment by solid-liquid separation is not particularly limited, and can be performed by filtration, centrifugation, and sedimentation while cooling as necessary. The cooling temperature is 100 ° C. or lower, preferably 80 ° C. or lower.
本発明で用いられる加水分解酵素としては、多糖類に対して加水分解活性を有する酵素であれば特に制限はないが、例えば、アミラーゼ、グルコアミラーゼ、セルラーゼ、デキストラナーゼ、グルカナーゼ、グルコシダーゼ、ガラクトシダーゼ、マンノシダーゼ、アガラーゼ、ラクトーゼ、ムタナーゼ、キチナーゼ、キトサナーゼ等が挙げられる。
加水分解酵素は、その起源に限定はなく、さらに、遺伝子組み換え技術、部分加水分解等による人工酵素であってもよい。
また、加水分解酵素の形態は特に限定されず、酵素蛋白質の乾燥物、酵素蛋白質を含む粒子、及び酵素蛋白質を含む液体等を用いることができる。 The hydrolase used in the present invention is not particularly limited as long as it has an activity of hydrolyzing polysaccharides. For example, amylase, glucoamylase, cellulase, dextranase, glucanase, glucosidase, galactosidase, Mannosidase, agarase, lactose, mutanase, chitinase, chitosanase and the like can be mentioned.
The origin of the hydrolase is not limited, and it may be an artificial enzyme by gene recombination technique, partial hydrolysis or the like.
The form of the hydrolase is not particularly limited, and dried enzyme protein, particles containing enzyme protein, liquid containing enzyme protein, and the like can be used.
加水分解酵素は、その起源に限定はなく、さらに、遺伝子組み換え技術、部分加水分解等による人工酵素であってもよい。
また、加水分解酵素の形態は特に限定されず、酵素蛋白質の乾燥物、酵素蛋白質を含む粒子、及び酵素蛋白質を含む液体等を用いることができる。 The hydrolase used in the present invention is not particularly limited as long as it has an activity of hydrolyzing polysaccharides. For example, amylase, glucoamylase, cellulase, dextranase, glucanase, glucosidase, galactosidase, Mannosidase, agarase, lactose, mutanase, chitinase, chitosanase and the like can be mentioned.
The origin of the hydrolase is not limited, and it may be an artificial enzyme by gene recombination technique, partial hydrolysis or the like.
The form of the hydrolase is not particularly limited, and dried enzyme protein, particles containing enzyme protein, liquid containing enzyme protein, and the like can be used.
加水分解酵素の使用量は、加水分解反応の条件、多糖類の種類等によって異なるが、例えば、セルラーゼの場合、固体部１ｇあたり１～２００ＦＰＵが好ましく、更に５～５０ＦＰＵが好ましい。ここでＦＰＵ活性は以下に示すＩＵＰＡＣ法により測定される。まず、濾紙（ワットマンＮｏ．１）５０ｍｇを基質とし、これに酵素液０．５ｍＬと０．０５Ｍクエン酸緩衝液（ｐＨ４．８）１．０ｍＬを加え、５０℃で１．０時間酵素反応を行った後、ジニトロサリチル酸試薬３．０ｍＬを加え、１００℃で５．０分間加熱し発色させる。冷却後、これにイオン交換水２０ｍＬを加え５４０ｎｍの波長で比色定量する。１分間に１μｍｏｌのグルコースに相当する還元糖を生成する酵素量を１ユニット（ＦＰＵ）とする。
The amount of hydrolase used varies depending on the conditions of the hydrolysis reaction, the type of polysaccharide, etc. For example, in the case of cellulase, 1 to 200 FPU per gram of the solid part is preferable, and 5 to 50 FPU is more preferable. Here, the FPU activity is measured by the IUPAC method shown below. First, 50 mg of filter paper (Whatman No. 1) is used as a substrate, 0.5 mL of enzyme solution and 1.0 mL of 0.05 M citrate buffer (pH 4.8) are added thereto, and the enzyme reaction is carried out at 50 ° C. for 1.0 hour. Then, 3.0 mL of dinitrosalicylic acid reagent is added, and the mixture is heated at 100 ° C. for 5.0 minutes for color development. After cooling, 20 mL of ion-exchanged water is added thereto, and colorimetric determination is performed at a wavelength of 540 nm. The amount of enzyme that produces a reducing sugar corresponding to 1 μmol of glucose per minute is defined as 1 unit (FPU).
多糖類と加水分解酵素との反応は、使用する酵素の特性に合わせて温度やｐＨを選択することが可能であるが、例えば、セルラーゼを用いる場合、ｐＨ３～８、更にｐＨ４～７とすることが好ましく、温度は１０～８０℃、更に２０～６０℃とすることが好ましい。
For the reaction between the polysaccharide and the hydrolase, the temperature and pH can be selected according to the characteristics of the enzyme to be used. For example, when cellulase is used, the pH should be 3 to 8, more preferably 4 to 7. The temperature is preferably 10 to 80 ° C., more preferably 20 to 60 ° C.
反応時間は、多糖類の種類等によって異なるが、糖化率及び生産効率の点から、例えば、１～２４０時間が好ましく、更に２～２００時間、更に３～１４０時間が好ましい。
The reaction time varies depending on the type of polysaccharide and the like, but is preferably 1 to 240 hours, more preferably 2 to 200 hours, and further preferably 3 to 140 hours from the viewpoint of saccharification rate and production efficiency.
かくして、加水分解反応により得られる単糖類としては、例えば、グルコース、フルクトース、マンノース、ガラクトース、キシロース、アラビノース等が挙げられる。また、二糖類としては、セロビオース、マルトース、ジ－Ｎ－アセチルキトビオース等が挙げられ、オリゴ糖としては３～１０の単糖単位を有するものをいい、例えば、セロトリオース、セロテトラオース、セロペンタオース、セロヘキサオース、マルトトリオース、マルトテトラオース、マルトペンタオース、マルトヘキサオース、トリ－Ｎ－アセチルキトトリオース、テトラ－Ｎ－アセチルキトテトラオース、ペンタ－Ｎ－アセチルキトペンタオース、ヘキサ－Ｎ－アセチルキトヘキサオース等が挙げられる。
Thus, examples of the monosaccharide obtained by the hydrolysis reaction include glucose, fructose, mannose, galactose, xylose, arabinose and the like. Examples of the disaccharide include cellobiose, maltose, di-N-acetylchitobiose, and the like. Oligosaccharides include those having 3 to 10 monosaccharide units. For example, cellotriose, cellotetraose, cello Pentaose, cellohexaose, maltotriose, maltotetraose, maltopentaose, maltohexaose, tri-N-acetylchitotriose, tetra-N-acetylchitotetraose, penta-N-acetylchitopentaose, Examples include hexa-N-acetylchitohexaose.
本発明方法によれば、６０～１００％、更に７０～１００％、更に８０～１００％の糖化率で多糖類から分解糖を製造することができる。なお、糖化率とは、加水分解反応により生成した水溶性糖（グルコースに代表される単糖類及び二糖類）の合計質量を、加水分解反応に供した原料に含まれる多糖類の質量で割った値を云う。
また、全工程を通した分解糖の収率（歩留まり）は６０％以上、更に６５％以上、更に７０％以上であることが製造効率の点から好ましい。
なお、前述したように、多糖類にはセルロースをはじめとして多くのものが例示され、さらにセルロースには結晶性の高いα-セルロースのほかに、ヘミセルロースとして分類される変性したβ-セルロースとγ-セルロースもある。本願では、多糖類の収率、糖化率、及び分解糖収率について、一般的にバイオマス中のセルロース純度として用いられるα-セルロースを基準とする。α-セルロース量の測定方法は実施例に記載した。 According to the method of the present invention, a decomposed sugar can be produced from a polysaccharide at a saccharification rate of 60 to 100%, further 70 to 100%, and further 80 to 100%. The saccharification rate was obtained by dividing the total mass of water-soluble sugars (monosaccharides and disaccharides typified by glucose) produced by the hydrolysis reaction by the mass of polysaccharides contained in the raw material subjected to the hydrolysis reaction. Say the value.
Moreover, it is preferable from the point of manufacturing efficiency that the yield (yield) of the decomposition sugar through all the processes is 60% or more, Furthermore, 65% or more, Furthermore, 70% or more.
As described above, polysaccharides include many examples including cellulose, and cellulose includes α-cellulose having high crystallinity, modified β-cellulose and γ-classified as hemicellulose. There is also cellulose. In this application, the yield of polysaccharides, the saccharification rate, and the yield of decomposing sugars are based on α-cellulose, which is generally used as the cellulose purity in biomass. The method for measuring the amount of α-cellulose is described in the Examples.
また、全工程を通した分解糖の収率（歩留まり）は６０％以上、更に６５％以上、更に７０％以上であることが製造効率の点から好ましい。
なお、前述したように、多糖類にはセルロースをはじめとして多くのものが例示され、さらにセルロースには結晶性の高いα-セルロースのほかに、ヘミセルロースとして分類される変性したβ-セルロースとγ-セルロースもある。本願では、多糖類の収率、糖化率、及び分解糖収率について、一般的にバイオマス中のセルロース純度として用いられるα-セルロースを基準とする。α-セルロース量の測定方法は実施例に記載した。 According to the method of the present invention, a decomposed sugar can be produced from a polysaccharide at a saccharification rate of 60 to 100%, further 70 to 100%, and further 80 to 100%. The saccharification rate was obtained by dividing the total mass of water-soluble sugars (monosaccharides and disaccharides typified by glucose) produced by the hydrolysis reaction by the mass of polysaccharides contained in the raw material subjected to the hydrolysis reaction. Say the value.
Moreover, it is preferable from the point of manufacturing efficiency that the yield (yield) of the decomposition sugar through all the processes is 60% or more, Furthermore, 65% or more, Furthermore, 70% or more.
As described above, polysaccharides include many examples including cellulose, and cellulose includes α-cellulose having high crystallinity, modified β-cellulose and γ-classified as hemicellulose. There is also cellulose. In this application, the yield of polysaccharides, the saccharification rate, and the yield of decomposing sugars are based on α-cellulose, which is generally used as the cellulose purity in biomass. The method for measuring the amount of α-cellulose is described in the Examples.
得られる分解糖は、これら分解糖を糖源として、微生物発酵を行ったり、化学変換したりすることにより、エタノール、ポリ乳酸、アミノ酸、キシリトールやエリスリトール等の有用物質を製造することができる。
The obtained decomposed sugar can be used to produce useful substances such as ethanol, polylactic acid, amino acids, xylitol, and erythritol by performing microbial fermentation or chemical conversion using these decomposed sugars as a sugar source.
＜α－セルロース量の測定＞
ソッスレー抽出により得られる脱脂品約２．５ｇに蒸留水１５０ｍＬ、亜塩素酸ナトリウム１．０ｇ、酢酸０．２ｍＬを加え、８０℃の湯浴上で加熱する。亜塩素酸ナトリウム、酢酸の添加と添加後の１時間加熱を、さらに２回行った。内容物をろ過し、ろ過残渣は水、アセトンで洗浄後、乾燥させた。得られた乾燥品１ｇを１７．５％水酸化ナトリウム水溶液２５ｍＬに加え、３０分間放置した。３０分後、水２５ｍＬを加え、１分間攪拌後、５分間静置した。内容物をろ過し、ろ過残渣を１０％酢酸水および煮沸水で洗浄後、乾燥させた。得られた乾燥残渣の重量をα－セルロース量とした。 <Measurement of α-cellulose amount>
Distilled water (150 mL), sodium chlorite (1.0 g) and acetic acid (0.2 mL) are added to about 2.5 g of the degreased product obtained by Sosley extraction, and heated on a hot water bath at 80 ° C. Addition of sodium chlorite and acetic acid and heating for 1 hour after the addition were further performed twice. The contents were filtered, and the filtration residue was washed with water and acetone and then dried. 1 g of the obtained dried product was added to 25 mL of 17.5% aqueous sodium hydroxide solution and left for 30 minutes. After 30 minutes, 25 mL of water was added, stirred for 1 minute, and allowed to stand for 5 minutes. The contents were filtered, and the filter residue was washed with 10% aqueous acetic acid and boiling water and then dried. The weight of the obtained dry residue was defined as the amount of α-cellulose.
ソッスレー抽出により得られる脱脂品約２．５ｇに蒸留水１５０ｍＬ、亜塩素酸ナトリウム１．０ｇ、酢酸０．２ｍＬを加え、８０℃の湯浴上で加熱する。亜塩素酸ナトリウム、酢酸の添加と添加後の１時間加熱を、さらに２回行った。内容物をろ過し、ろ過残渣は水、アセトンで洗浄後、乾燥させた。得られた乾燥品１ｇを１７．５％水酸化ナトリウム水溶液２５ｍＬに加え、３０分間放置した。３０分後、水２５ｍＬを加え、１分間攪拌後、５分間静置した。内容物をろ過し、ろ過残渣を１０％酢酸水および煮沸水で洗浄後、乾燥させた。得られた乾燥残渣の重量をα－セルロース量とした。 <Measurement of α-cellulose amount>
Distilled water (150 mL), sodium chlorite (1.0 g) and acetic acid (0.2 mL) are added to about 2.5 g of the degreased product obtained by Sosley extraction, and heated on a hot water bath at 80 ° C. Addition of sodium chlorite and acetic acid and heating for 1 hour after the addition were further performed twice. The contents were filtered, and the filtration residue was washed with water and acetone and then dried. 1 g of the obtained dried product was added to 25 mL of 17.5% aqueous sodium hydroxide solution and left for 30 minutes. After 30 minutes, 25 mL of water was added, stirred for 1 minute, and allowed to stand for 5 minutes. The contents were filtered, and the filter residue was washed with 10% aqueous acetic acid and boiling water and then dried. The weight of the obtained dry residue was defined as the amount of α-cellulose.
＜水溶性糖濃度（グルコース濃度、セロビオース濃度）の測定＞
日立製作所製高速液体クロマトグラフを用い、昭和電工製カラムＡｓａｈｉｐａｋ ＮＨ２Ｐ－５０ ４Ｅ （４．５ｍｍφ×２５０ｍ）を装着し、カラム温度２０℃でグラジエント法により行った。移動相Ａ液はアセトニトリル、Ｂ液は３０％メタノール水とし、１．００ｍＬ／分で送液した。グラジエント条件は以下のとおりである。
時間（分） Ａ液（％） Ｂ液（％）
０ ２０ ８０
４５ ５０ ５０
４５．１ ２０ ８０
５５ ２０ ８０
試料注入量は５μＬ、検出はＥＳＡ Ｂｉｏｓｃｉｅｎｃｅｓ社製コロナＣＡＤ検出器を用いた。 <Measurement of water-soluble sugar concentration (glucose concentration, cellobiose concentration)>
Using a high performance liquid chromatograph manufactured by Hitachi, Ltd., a column Asahipak NH2P-50 4E (4.5 mmφ × 250 m) manufactured by Showa Denko was installed, and a gradient method was performed at a column temperature of 20 ° C. The mobile phase A solution was acetonitrile, and the B solution was 30% methanol water, and the solution was sent at 1.00 mL / min. The gradient conditions are as follows.
Time (min) A liquid (%) B liquid (%)
0 20 80
45 50 50
45.1 20 80
55 20 80
The sample injection amount was 5 μL, and detection was performed using a corona CAD detector manufactured by ESA Biosciences.
日立製作所製高速液体クロマトグラフを用い、昭和電工製カラムＡｓａｈｉｐａｋ ＮＨ２Ｐ－５０ ４Ｅ （４．５ｍｍφ×２５０ｍ）を装着し、カラム温度２０℃でグラジエント法により行った。移動相Ａ液はアセトニトリル、Ｂ液は３０％メタノール水とし、１．００ｍＬ／分で送液した。グラジエント条件は以下のとおりである。
時間（分） Ａ液（％） Ｂ液（％）
０ ２０ ８０
４５ ５０ ５０
４５．１ ２０ ８０
５５ ２０ ８０
試料注入量は５μＬ、検出はＥＳＡ Ｂｉｏｓｃｉｅｎｃｅｓ社製コロナＣＡＤ検出器を用いた。 <Measurement of water-soluble sugar concentration (glucose concentration, cellobiose concentration)>
Using a high performance liquid chromatograph manufactured by Hitachi, Ltd., a column Asahipak NH2P-50 4E (4.5 mmφ × 250 m) manufactured by Showa Denko was installed, and a gradient method was performed at a column temperature of 20 ° C. The mobile phase A solution was acetonitrile, and the B solution was 30% methanol water, and the solution was sent at 1.00 mL / min. The gradient conditions are as follows.
Time (min) A liquid (%) B liquid (%)
0 20 80
45 50 50
45.1 20 80
55 20 80
The sample injection amount was 5 μL, and detection was performed using a corona CAD detector manufactured by ESA Biosciences.
＜体積基準平均粒径の測定＞
レーザー回折式粒度分布計（ベックマン・コールター社製、ＬＳ １３ ３２０）により測定した。 <Measurement of volume-based average particle size>
It measured with the laser diffraction type particle size distribution analyzer (the Beckman Coulter company make, LS13320).
レーザー回折式粒度分布計（ベックマン・コールター社製、ＬＳ １３ ３２０）により測定した。 <Measurement of volume-based average particle size>
It measured with the laser diffraction type particle size distribution analyzer (the Beckman Coulter company make, LS13320).
＜α－セルロース収率の算出＞
α－セルロース収率は次式（ｉ）により算出した。
α－セルロース収率（％）＝｛工程（２）で得られた加熱処理後の反応物を固液分離して得た固体部に含まれるα－セルロースの質量｝／｛工程（２）に供した原料多糖類の粉砕物に含まれるα－セルロースの質量｝×１００ （ｉ） <Calculation of α-cellulose yield>
The α-cellulose yield was calculated by the following formula (i).
α-cellulose yield (%) = {mass of α-cellulose contained in the solid part obtained by solid-liquid separation of the heat-treated reaction product obtained in step (2)} / {in step (2) Mass of α-cellulose contained in pulverized raw material polysaccharide} × 100 (i)
α－セルロース収率は次式（ｉ）により算出した。
α－セルロース収率（％）＝｛工程（２）で得られた加熱処理後の反応物を固液分離して得た固体部に含まれるα－セルロースの質量｝／｛工程（２）に供した原料多糖類の粉砕物に含まれるα－セルロースの質量｝×１００ （ｉ） <Calculation of α-cellulose yield>
The α-cellulose yield was calculated by the following formula (i).
α-cellulose yield (%) = {mass of α-cellulose contained in the solid part obtained by solid-liquid separation of the heat-treated reaction product obtained in step (2)} / {in step (2) Mass of α-cellulose contained in pulverized raw material polysaccharide} × 100 (i)
＜糖化率の算出＞
糖化率は次式（ｉｉ）により算出した。
糖化率（％）＝｛（反応後の水溶性糖質量）－（反応前の水溶性糖質量）｝／α－セルロースの質量×１００ （ｉｉ） <Calculation of saccharification rate>
The saccharification rate was calculated by the following formula (ii).
Saccharification rate (%) = {(water-soluble sugar mass after reaction) − (water-soluble sugar mass before reaction)} / mass of α-cellulose × 100 (ii)
糖化率は次式（ｉｉ）により算出した。
糖化率（％）＝｛（反応後の水溶性糖質量）－（反応前の水溶性糖質量）｝／α－セルロースの質量×１００ （ｉｉ） <Calculation of saccharification rate>
The saccharification rate was calculated by the following formula (ii).
Saccharification rate (%) = {(water-soluble sugar mass after reaction) − (water-soluble sugar mass before reaction)} / mass of α-cellulose × 100 (ii)
＜分解糖収率の算出＞
上記α－セルロース収率と糖化率を乗じることで分解糖収率（％）を算出した。 <Calculation of decomposition sugar yield>
The degradation sugar yield (%) was calculated by multiplying the α-cellulose yield and the saccharification rate.
上記α－セルロース収率と糖化率を乗じることで分解糖収率（％）を算出した。 <Calculation of decomposition sugar yield>
The degradation sugar yield (%) was calculated by multiplying the α-cellulose yield and the saccharification rate.
実施例１
乾燥した稲ワラ１００ｇをグラインダーで体積基準平均粒径が３～５ｃｍとなるように予備粉砕した。振動ロッドミル（（株）中央化工機製ＭＢ－１、容器全容積３．５Ｌ）に外径３０ｍｍのロッドを１３本充填し（充填率７４％）、予備粉砕した稲ワラ１００ｇを仕込み、１，２００ｃ／ｍｉｎで１０分間振動することにより、粉砕稲ワラを得た。体積基準平均粒径は０．０４ｍｍであった。
この粉砕稲ワラ（α－セルロース含有量３２質量％）１０ｇと水９０ｇを混合したスラリーを、バッチ式水熱処理装置（日東高圧製Ｓｔａｒｔ２００Ｎｅｗ Ｑｕｉｃｋ、容積１８０ｍＬ）に入れ、上部空間を窒素置換し、加熱した。昇温速度は３．４℃／分とし、２００℃に到達後、直ちに冷却した。１８５℃～２３０℃にあった時間は３０分であった。室温まで冷却後、ろ過により固体部（固形分残渣）を得た。残渣の乾燥質量が５質量％になるように分取し、ｐＨ５．０の０．１Ｍクエン酸緩衝溶液を加えて全体を１０ｇにした。これにセルラーゼ（ノボザイムズ製セルクラスト１．５Ｌ）を６２．５μＬ添加して糖化した（残渣の乾燥重量1ｇあたりの添加は８．７５ＦＰＵ）。糖化反応は５０℃の恒温槽中、１１０ｒ／ｍｉｎで振とうしながら４８時間行った。反応終了後、液部を０．２μｍのフィルターでろ過し、水溶性糖濃度を測定した。水溶性糖としては、セロビオース、グルコースが測定された。 Example 1
100 g of dried rice straw was pre-ground with a grinder so that the volume-based average particle size was 3 to 5 cm. A vibrating rod mill (MB-1 manufactured by Chuo Kako Co., Ltd., total volume 3.5L) was filled with 13 rods with an outer diameter of 30 mm (filling rate 74%), and 100 g of pre-ground rice straw was charged, 1,200c By shaking for 10 minutes at / min, ground rice straw was obtained. The volume standard average particle size was 0.04 mm.
A slurry obtained by mixing 10 g of this ground rice straw (α-cellulose content 32% by mass) and 90 g of water is placed in a batch hydrothermal apparatus (Start 200 New Quick, volume 180 mL, manufactured by Nitto Koatsu Co., Ltd.). did. The temperature rising rate was 3.4 ° C./min, and after reaching 200 ° C., it was immediately cooled. The time at 185 ° C. to 230 ° C. was 30 minutes. After cooling to room temperature, a solid part (solid residue) was obtained by filtration. The residue was fractionated so that the dry mass was 5% by mass, and 0.1 M citrate buffer solution having a pH of 5.0 was added to make a total of 10 g. 62.5 μL of cellulase (Novozymes Cell Crust 1.5 L) was added thereto for saccharification (addition of residue per 1 g of dry weight was 8.75 FPU). The saccharification reaction was carried out in a thermostatic bath at 50 ° C. for 48 hours while shaking at 110 r / min. After completion of the reaction, the liquid part was filtered with a 0.2 μm filter, and the water-soluble sugar concentration was measured. Cellobiose and glucose were measured as water-soluble sugars.
乾燥した稲ワラ１００ｇをグラインダーで体積基準平均粒径が３～５ｃｍとなるように予備粉砕した。振動ロッドミル（（株）中央化工機製ＭＢ－１、容器全容積３．５Ｌ）に外径３０ｍｍのロッドを１３本充填し（充填率７４％）、予備粉砕した稲ワラ１００ｇを仕込み、１，２００ｃ／ｍｉｎで１０分間振動することにより、粉砕稲ワラを得た。体積基準平均粒径は０．０４ｍｍであった。
この粉砕稲ワラ（α－セルロース含有量３２質量％）１０ｇと水９０ｇを混合したスラリーを、バッチ式水熱処理装置（日東高圧製Ｓｔａｒｔ２００Ｎｅｗ Ｑｕｉｃｋ、容積１８０ｍＬ）に入れ、上部空間を窒素置換し、加熱した。昇温速度は３．４℃／分とし、２００℃に到達後、直ちに冷却した。１８５℃～２３０℃にあった時間は３０分であった。室温まで冷却後、ろ過により固体部（固形分残渣）を得た。残渣の乾燥質量が５質量％になるように分取し、ｐＨ５．０の０．１Ｍクエン酸緩衝溶液を加えて全体を１０ｇにした。これにセルラーゼ（ノボザイムズ製セルクラスト１．５Ｌ）を６２．５μＬ添加して糖化した（残渣の乾燥重量1ｇあたりの添加は８．７５ＦＰＵ）。糖化反応は５０℃の恒温槽中、１１０ｒ／ｍｉｎで振とうしながら４８時間行った。反応終了後、液部を０．２μｍのフィルターでろ過し、水溶性糖濃度を測定した。水溶性糖としては、セロビオース、グルコースが測定された。 Example 1
100 g of dried rice straw was pre-ground with a grinder so that the volume-based average particle size was 3 to 5 cm. A vibrating rod mill (MB-1 manufactured by Chuo Kako Co., Ltd., total volume 3.5L) was filled with 13 rods with an outer diameter of 30 mm (filling rate 74%), and 100 g of pre-ground rice straw was charged, 1,200c By shaking for 10 minutes at / min, ground rice straw was obtained. The volume standard average particle size was 0.04 mm.
A slurry obtained by mixing 10 g of this ground rice straw (α-cellulose content 32% by mass) and 90 g of water is placed in a batch hydrothermal apparatus (Start 200 New Quick, volume 180 mL, manufactured by Nitto Koatsu Co., Ltd.). did. The temperature rising rate was 3.4 ° C./min, and after reaching 200 ° C., it was immediately cooled. The time at 185 ° C. to 230 ° C. was 30 minutes. After cooling to room temperature, a solid part (solid residue) was obtained by filtration. The residue was fractionated so that the dry mass was 5% by mass, and 0.1 M citrate buffer solution having a pH of 5.0 was added to make a total of 10 g. 62.5 μL of cellulase (Novozymes Cell Crust 1.5 L) was added thereto for saccharification (addition of residue per 1 g of dry weight was 8.75 FPU). The saccharification reaction was carried out in a thermostatic bath at 50 ° C. for 48 hours while shaking at 110 r / min. After completion of the reaction, the liquid part was filtered with a 0.2 μm filter, and the water-soluble sugar concentration was measured. Cellobiose and glucose were measured as water-soluble sugars.
実施例２
水熱処理において２００℃に到達後、２００℃で３０分保持した後に冷却した以外は実施例１と同様にして水溶性糖を得た。１８５℃～２３０℃にあった時間は６５分であった。 Example 2
A water-soluble sugar was obtained in the same manner as in Example 1 except that after reaching 200 ° C. in hydrothermal treatment, the mixture was cooled at 200 ° C. for 30 minutes and then cooled. The time spent at 185 ° C. to 230 ° C. was 65 minutes.
水熱処理において２００℃に到達後、２００℃で３０分保持した後に冷却した以外は実施例１と同様にして水溶性糖を得た。１８５℃～２３０℃にあった時間は６５分であった。 Example 2
A water-soluble sugar was obtained in the same manner as in Example 1 except that after reaching 200 ° C. in hydrothermal treatment, the mixture was cooled at 200 ° C. for 30 minutes and then cooled. The time spent at 185 ° C. to 230 ° C. was 65 minutes.
実施例３
バッチ式水熱処理装置をあらかじめ窒素ガスで１ＭＰａに加圧した以外は実施例１と同様にして水溶性糖を得た。 Example 3
A water-soluble sugar was obtained in the same manner as in Example 1 except that the batch hydrothermal apparatus was previously pressurized to 1 MPa with nitrogen gas.
バッチ式水熱処理装置をあらかじめ窒素ガスで１ＭＰａに加圧した以外は実施例１と同様にして水溶性糖を得た。 Example 3
A water-soluble sugar was obtained in the same manner as in Example 1 except that the batch hydrothermal apparatus was previously pressurized to 1 MPa with nitrogen gas.
比較例１
水熱処理において、１８０℃に到達後、直ちに冷却した以外は実施例１と同様にして水溶性糖を得た。 Comparative Example 1
In the hydrothermal treatment, a water-soluble sugar was obtained in the same manner as in Example 1 except that it was immediately cooled after reaching 180 ° C.
水熱処理において、１８０℃に到達後、直ちに冷却した以外は実施例１と同様にして水溶性糖を得た。 Comparative Example 1
In the hydrothermal treatment, a water-soluble sugar was obtained in the same manner as in Example 1 except that it was immediately cooled after reaching 180 ° C.
比較例２
実施例１で得た粉砕稲ワラを、水熱処理せずに酵素糖化した。 Comparative Example 2
The ground rice straw obtained in Example 1 was enzymatically saccharified without hydrothermal treatment.
実施例１で得た粉砕稲ワラを、水熱処理せずに酵素糖化した。 Comparative Example 2
The ground rice straw obtained in Example 1 was enzymatically saccharified without hydrothermal treatment.
比較例３
岩谷産業（株）製グラインダー（ラボミルサー８００ＤＧ、容積２６０ｍＬ）に乾燥した稲ワラ４０ｇを仕込んで２０，０００ｒ／ｍｉｎで１０分間攪拌することにより、粉砕稲ワラを得た。体積基準平均粒径は０．２７ｍｍであった。この粉砕稲ワラ（α－セルロース含有量３２質量％）１０ｇと水９０ｇを混合し、その後実施例２と同様にして加熱処理と酵素糖化処理を行い、水溶性糖を得た。 Comparative Example 3
A crushed rice straw was obtained by charging 40 g of dried rice straw into a grinder manufactured by Iwatani Corporation (Lab Miller 800DG, volume 260 mL) and stirring at 20,000 r / min for 10 minutes. The volume standard average particle diameter was 0.27 mm. 10 g of this ground rice straw (α-cellulose content 32 mass%) and 90 g of water were mixed, and then heat treatment and enzymatic saccharification treatment were carried out in the same manner as in Example 2 to obtain a water-soluble sugar.
岩谷産業（株）製グラインダー（ラボミルサー８００ＤＧ、容積２６０ｍＬ）に乾燥した稲ワラ４０ｇを仕込んで２０，０００ｒ／ｍｉｎで１０分間攪拌することにより、粉砕稲ワラを得た。体積基準平均粒径は０．２７ｍｍであった。この粉砕稲ワラ（α－セルロース含有量３２質量％）１０ｇと水９０ｇを混合し、その後実施例２と同様にして加熱処理と酵素糖化処理を行い、水溶性糖を得た。 Comparative Example 3
A crushed rice straw was obtained by charging 40 g of dried rice straw into a grinder manufactured by Iwatani Corporation (Lab Miller 800DG, volume 260 mL) and stirring at 20,000 r / min for 10 minutes. The volume standard average particle diameter was 0.27 mm. 10 g of this ground rice straw (α-cellulose content 32 mass%) and 90 g of water were mixed, and then heat treatment and enzymatic saccharification treatment were carried out in the same manner as in Example 2 to obtain a water-soluble sugar.
比較例４
２０，０００ｒ／ｍｉｎで１２０分間攪拌した以外は比較例３と同様にして粉砕稲ワラを得た。体積基準平均粒径は０．０５ｍｍであった。この粉砕稲ワラ（α－セルロース含有量３２質量％）１０ｇと水９０ｇを混合し、その後実施例２と同様にして加熱処理と酵素糖化処理を行い、水溶性糖を得た。
各実施例及び比較例の条件と結果を表１に示す。 Comparative Example 4
A ground rice straw was obtained in the same manner as in Comparative Example 3 except that stirring was performed at 20,000 r / min for 120 minutes. The volume standard average particle diameter was 0.05 mm. 10 g of this ground rice straw (α-cellulose content 32 mass%) and 90 g of water were mixed, and then heat treatment and enzymatic saccharification treatment were carried out in the same manner as in Example 2 to obtain a water-soluble sugar.
Table 1 shows the conditions and results of each Example and Comparative Example.
２０，０００ｒ／ｍｉｎで１２０分間攪拌した以外は比較例３と同様にして粉砕稲ワラを得た。体積基準平均粒径は０．０５ｍｍであった。この粉砕稲ワラ（α－セルロース含有量３２質量％）１０ｇと水９０ｇを混合し、その後実施例２と同様にして加熱処理と酵素糖化処理を行い、水溶性糖を得た。
各実施例及び比較例の条件と結果を表１に示す。 Comparative Example 4
A ground rice straw was obtained in the same manner as in Comparative Example 3 except that stirring was performed at 20,000 r / min for 120 minutes. The volume standard average particle diameter was 0.05 mm. 10 g of this ground rice straw (α-cellulose content 32 mass%) and 90 g of water were mixed, and then heat treatment and enzymatic saccharification treatment were carried out in the same manner as in Example 2 to obtain a water-soluble sugar.
Table 1 shows the conditions and results of each Example and Comparative Example.
表１から明らかなように、セルロースを振動ロッドミルで粉砕後、１８５～２３０℃で加熱処理した実施例１～３では、いずれも糖化率が高く、全工程を通した分解糖の収率は高かった。これに対し、１８０℃未満で加熱処理した比較例１、及び振動ロッドミルの代わりにグラインダーを用いて粉砕した比較例３及び４では、加熱処理後のα－セルロース収率が高いものの、糖化反応後の糖化率が低く、全工程を通した分解糖の収率は低いという結果であった。また、加熱処理をしなかった比較例２は糖化率が低く、分解糖の収率も低いという結果であった。
As is apparent from Table 1, in Examples 1 to 3, where cellulose was pulverized with a vibrating rod mill and then heat-treated at 185 to 230 ° C., all had a high saccharification rate and a high yield of decomposing sugar throughout the entire process. It was. In contrast, in Comparative Example 1 that was heat-treated at less than 180 ° C. and Comparative Examples 3 and 4 that were ground using a grinder instead of a vibrating rod mill, the α-cellulose yield after the heat treatment was high, but after the saccharification reaction As a result, the saccharification rate was low and the yield of decomposing sugar throughout the entire process was low. Moreover, the comparative example 2 which did not heat-process was a result that the saccharification rate is low and the yield of decomposition sugar is also low.
Claims (12)
- 次の工程（１）～（３）：
（１）多糖類に圧縮せん断応力を加えて粉砕し、多糖類の粉砕物を得る工程、
（２）多糖類の粉砕物を水の存在下、１８５～２３０℃で加熱処理する工程、
（３）加熱処理後の反応物から固液分離により固体部を得、これに加水分解酵素を作用させる工程、
を包含する、単糖類、二糖類、及び／又はオリゴ糖の製造方法。 Next steps (1) to (3):
(1) A step of applying a compressive shear stress to a polysaccharide to pulverize the polysaccharide to obtain a pulverized polysaccharide;
(2) a step of heat-treating the pulverized polysaccharide at 185 to 230 ° C. in the presence of water;
(3) A step of obtaining a solid part by solid-liquid separation from the reaction product after the heat treatment, and allowing a hydrolase to act on the solid part,
A process for producing monosaccharides, disaccharides and / or oligosaccharides. - １８５～２３０℃で加熱処理する時間が１０分～９０分である、請求項１記載の単糖類、二糖類、及び／又はオリゴ糖の製造方法。 The method for producing monosaccharides, disaccharides and / or oligosaccharides according to claim 1, wherein the time for heat treatment at 185 to 230 ° C is 10 minutes to 90 minutes.
- １８５～２３０℃で加熱処理する時間が１５分～８５分である、請求項１記載の単糖類、二糖類、及び／又はオリゴ糖の製造方法。 The method for producing monosaccharides, disaccharides and / or oligosaccharides according to claim 1, wherein the heat treatment time at 185 to 230 ° C is 15 minutes to 85 minutes.
- １８５～２３０℃で加熱処理する時間が２０分～８０分である、請求項１記載の単糖類、二糖類、及び／又はオリゴ糖の製造方法。 The method for producing monosaccharides, disaccharides and / or oligosaccharides according to claim 1, wherein the heat treatment time at 185 to 230 ° C is 20 minutes to 80 minutes.
- １８５～２３０℃で加熱処理する時間が３０分～６５分である、請求項１記載の単糖類、二糖類、及び／又はオリゴ糖の製造方法。 The method for producing monosaccharides, disaccharides and / or oligosaccharides according to claim 1, wherein the heat treatment time at 185 to 230 ° C is 30 minutes to 65 minutes.
- 振動ロッドミルを用いて多糖類に圧縮せん断応力を加えるものである、請求項１～５のいずれか１項記載の単糖類、二糖類、及び／又はオリゴ糖の製造方法。 The method for producing monosaccharides, disaccharides and / or oligosaccharides according to any one of claims 1 to 5, wherein a compressive shear stress is applied to the polysaccharides using a vibration rod mill.
- 加熱処理を水の飽和蒸気圧以上の圧力で行う、請求項１～６のいずれか１記載の単糖類、二糖類、及び／又はオリゴ糖の製造方法。 The method for producing monosaccharides, disaccharides and / or oligosaccharides according to any one of claims 1 to 6, wherein the heat treatment is performed at a pressure equal to or higher than a saturated vapor pressure of water.
- 加熱処理を１．１～１０．０ＭＰａの圧力で行う、請求項１～６のいずれか１記載の単糖類、二糖類、及び／又はオリゴ糖の製造方法。 The method for producing monosaccharides, disaccharides and / or oligosaccharides according to any one of claims 1 to 6, wherein the heat treatment is performed at a pressure of 1.1 to 10.0 MPa.
- 加熱処理を１．２～８．０ＭＰａの圧力で行う、請求項１～６のいずれか１記載の単糖類、二糖類、及び／又はオリゴ糖の製造方法。 The method for producing monosaccharides, disaccharides and / or oligosaccharides according to any one of claims 1 to 6, wherein the heat treatment is performed at a pressure of 1.2 to 8.0 MPa.
- 加熱処理を１．３～６．０ＭＰａの圧力で行う、請求項１～６のいずれか１記載の単糖類、二糖類、及び／又はオリゴ糖の製造方法。 The method for producing monosaccharides, disaccharides and / or oligosaccharides according to any one of claims 1 to 6, wherein the heat treatment is performed at a pressure of 1.3 to 6.0 MPa.
- 加熱処理を１．６～２．６ＭＰａの圧力で行う、請求項１～６のいずれか１記載の単糖類、二糖類、及び／又はオリゴ糖の製造方法。 The method for producing monosaccharides, disaccharides and / or oligosaccharides according to any one of claims 1 to 6, wherein the heat treatment is performed at a pressure of 1.6 to 2.6 MPa.
- 多糖類としてセルロース含有バイオマスを用いる、請求項１～１１のいずれか１項記載の単糖類、二糖類、及び／又はオリゴ糖の製造方法。 The method for producing monosaccharides, disaccharides and / or oligosaccharides according to any one of claims 1 to 11, wherein cellulose-containing biomass is used as the polysaccharide.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2010-141534 | 2010-06-22 | ||
| JP2010141534A JP2012005359A (en) | 2010-06-22 | 2010-06-22 | Method for manufacturing monosaccharide, disaccharide, and/or oligosaccharide |
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| WO2011162249A1 true WO2011162249A1 (en) | 2011-12-29 |
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| PCT/JP2011/064151 WO2011162249A1 (en) | 2010-06-22 | 2011-06-21 | Method for manufacturing a monosaccharide, a disaccharide, and/or an oligosaccharide |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10327900A (en) * | 1997-06-02 | 1998-12-15 | Agency Of Ind Science & Technol | Production of water-soluble oligosaccharides and monosaccharides |
| JP2004089016A (en) * | 2002-08-29 | 2004-03-25 | Tsukishima Kikai Co Ltd | Method for treating waste of building material |
| JP2006136263A (en) * | 2004-11-12 | 2006-06-01 | National Institute Of Advanced Industrial & Technology | Method for treating lignocellulosic biomass |
| JP2008271962A (en) * | 2007-04-04 | 2008-11-13 | National Institute Of Advanced Industrial & Technology | Method for producing saccharide |
| JP2009124973A (en) * | 2007-11-21 | 2009-06-11 | National Institute Of Advanced Industrial & Technology | Method for producing ethanol raw material and ethanol from lignocellulosic biomass |
| JP2009171951A (en) * | 2007-12-27 | 2009-08-06 | Kao Corp | Method for producing sugar |
| JP2009284867A (en) * | 2008-05-30 | 2009-12-10 | Toshiba Corp | Cellulose saccharification system, ethanol production system and method thereof |
-
2010
- 2010-06-22 JP JP2010141534A patent/JP2012005359A/en not_active Withdrawn
-
2011
- 2011-06-21 WO PCT/JP2011/064151 patent/WO2011162249A1/en active Application Filing
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10327900A (en) * | 1997-06-02 | 1998-12-15 | Agency Of Ind Science & Technol | Production of water-soluble oligosaccharides and monosaccharides |
| JP2004089016A (en) * | 2002-08-29 | 2004-03-25 | Tsukishima Kikai Co Ltd | Method for treating waste of building material |
| JP2006136263A (en) * | 2004-11-12 | 2006-06-01 | National Institute Of Advanced Industrial & Technology | Method for treating lignocellulosic biomass |
| JP2008271962A (en) * | 2007-04-04 | 2008-11-13 | National Institute Of Advanced Industrial & Technology | Method for producing saccharide |
| JP2009124973A (en) * | 2007-11-21 | 2009-06-11 | National Institute Of Advanced Industrial & Technology | Method for producing ethanol raw material and ethanol from lignocellulosic biomass |
| JP2009171951A (en) * | 2007-12-27 | 2009-08-06 | Kao Corp | Method for producing sugar |
| JP2009284867A (en) * | 2008-05-30 | 2009-12-10 | Toshiba Corp | Cellulose saccharification system, ethanol production system and method thereof |
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